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1
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Brown GE, Bodke VV, Ware BR, Khetani SR. Liver portal fibroblasts induce the functions of primary human hepatocytes in vitro. Commun Biol 2025; 8:721. [PMID: 40346200 PMCID: PMC12064700 DOI: 10.1038/s42003-025-08135-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2024] [Accepted: 04/25/2025] [Indexed: 05/11/2025] Open
Abstract
In vitro human liver models are critical to mitigate species-specific differences observed for toxicology, disease modeling, and regenerative medicine. Interactions with mesenchyme (i.e., fibroblasts) can promote phenotypic functions of primary human hepatocytes (PHHs) in culture; however, using liver-derived fibroblasts remains elusive. Portal fibroblasts (PFs) around the portal triad influence bile duct formation during development, but their role in regulating homeostatic hepatic functions remains unknown. Here, we show that human liver PFs induce long-term phenotypic functions in PHHs at higher levels than activated hepatic stellate cells across 2-dimensional and 3-dimensional culture formats. While PF-conditioned media induces some hepatic functions, partly via insulin-like growth factor binding protein-5 signaling, direct contact is necessary to induce optimal functional levels. Inhibiting Notch signaling reduces progenitor-like characteristics of PHHs and further enhances functionality. Overall, this work demonstrates a unique role for PFs in modulating hepatic functions and provides all-human and all-liver coculture strategies for downstream applications.
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Affiliation(s)
- Grace E Brown
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, IL, USA
| | - Vedant V Bodke
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, IL, USA
| | - Brenton R Ware
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, IL, USA
- School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA
| | - Salman R Khetani
- Department of Biomedical Engineering, University of Illinois Chicago, Chicago, IL, USA.
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2
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Johnston EK, Fang Z, Soto-Gutierrez A, Taner CB, Cook KE, Yang L, Abbott RD. Engineering a three-dimensional liver steatosis model. Biochim Biophys Acta Mol Basis Dis 2025; 1871:167888. [PMID: 40328412 DOI: 10.1016/j.bbadis.2025.167888] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2024] [Revised: 04/28/2025] [Accepted: 05/02/2025] [Indexed: 05/08/2025]
Abstract
Liver transplantation is the key treatment for liver failure, yet organ scarcity, exacerbated by high discard rates of steatotic livers, leads to high waitlist mortality. Preclinical models of steatosis are necessary to understand the pathophysiology of the disease and to develop pharmacological interventions to decrease disease burden and liver discard rate. In this paper, we develop an expedited 3D steatotic organoid model containing primary human hepatocytes and non-parenchymal cells. We present our iterative approach as we transition from 2D to 3D models and from immortalized to primary cells to optimize conditions for the development of a 3D human steatosis model. Both primary cell aggregation and steatosis induction time were reduced from the standard, 5-7 days, to 2 days. Our 3D model incorporates human primary hepatocytes from discarded liver tissues, which have not been used in organoids previously due to their rapid loss of phenotype in culture. After optimizing our steatosis induction media there was a mix of macro- and micro-steatosis in these primary hepatocytes which is consistent with the human pathology. Our approach achieves a model reflective of the liver pathology, preserving cellular phenotypes and viability while exhibiting markers of oxidative stress, a key factor contributing to complications in the transplantation of steatotic livers.
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Affiliation(s)
- Elizabeth K Johnston
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Zhou Fang
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | | | - C Burcin Taner
- Department of Transplantation, Mayo Clinic, Jacksonville, FL 32224, USA
| | - Keith E Cook
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA
| | - Liu Yang
- Department of Transplantation, Mayo Clinic, Jacksonville, FL 32224, USA
| | - Rosalyn D Abbott
- Department of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA.
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3
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Bhatt S S, Krishna Kumar J, Laya S, Thakur G, Nune M. Scaffold-mediated liver regeneration: A comprehensive exploration of current advances. J Tissue Eng 2024; 15:20417314241286092. [PMID: 39411269 PMCID: PMC11475092 DOI: 10.1177/20417314241286092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2024] [Accepted: 09/08/2024] [Indexed: 10/19/2024] Open
Abstract
The liver coordinates over 500 biochemical processes crucial for maintaining homeostasis, detoxification, and metabolism. Its specialized cells, arranged in hexagonal lobules, enable it to function as a highly efficient metabolic engine. However, diseases such as cirrhosis, fatty liver disease, and hepatitis present significant global health challenges. Traditional drug development is expensive and often ineffective at predicting human responses, driving interest in advanced in vitro liver models utilizing 3D bioprinting and microfluidics. These models strive to mimic the liver's complex microenvironment, improving drug screening and disease research. Despite its resilience, the liver is vulnerable to chronic illnesses, injuries, and cancers, leading to millions of deaths annually. Organ shortages hinder liver transplantation, highlighting the need for alternative treatments. Tissue engineering, employing polymer-based scaffolds and 3D bioprinting, shows promise. This review examines these innovative strategies, including liver organoids and liver tissue-on-chip technologies, to address the challenges of liver diseases.
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Affiliation(s)
- Supriya Bhatt S
- Manipal Institute of Regenerative Medicine, Bengaluru, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Jayanthi Krishna Kumar
- Manipal Institute of Regenerative Medicine, Bengaluru, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Shurthi Laya
- Manipal Institute of Regenerative Medicine, Bengaluru, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
- Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Goutam Thakur
- Department of Biomedical Engineering, Manipal Institute of Technology, Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Manasa Nune
- Manipal Institute of Regenerative Medicine, Bengaluru, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
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Froghi S, de Andrade MO, Hadi LM, Gelat P, Rashidi H, Quaglia A, Fuller B, Saffari N, Davidson B. Liver Ultrasound Histotripsy: Novel Analysis of the Histotripsy Site Cell Constituents with Implications for Histotripsy Application in Cell Transplantation and Cancer Therapy. Bioengineering (Basel) 2023; 10:bioengineering10020276. [PMID: 36829770 PMCID: PMC9952788 DOI: 10.3390/bioengineering10020276] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2022] [Revised: 01/14/2023] [Accepted: 02/09/2023] [Indexed: 02/25/2023] Open
Abstract
Introduction: Allogenic hepatocyte transplantation is an attractive alternative to whole-organ transplantation, particularly for the treatment of metabolic disorders and acute liver failure. However, the shortage of human donor organs for cell isolation, the low cell yield from decellularisation regimes, and low engraftment rates from portal administration of donor cells have restricted its clinical application. Using ultrasound histotripsy to provide a nidus in the liver for direct cell transplantation offers a new approach to overcoming key limitations in current cell therapy. We have analysed the liver cavity constituents to assess their potential as a site for cell delivery and implantation. Methods: Using human organ retrieval techniques, pig livers were collected from the abattoir and transported in ice-cold storage to the laboratory. Following 2 h of cold storage, the livers were flushed with organ preservation solution and placed on an organ perfusion circuit to maintain viability. Organs were perfused with Soltran™ organ preservation solution via the portal vein at a temperature of 24-30 °C. The perfusion circuit was oxygenated through equilibration with room air. Perfused livers (n=5) were subjected to ultrasound histotripsy, producing a total of 130 lesions. Lesions were generated by applying 50 pulses at 1 Hz pulse repetition frequency and 1% duty cycle using a single element 2 MHz bowl-shaped transducer (Sonic Concepts, H-148). Following histotripsy, a focal liver lesion was produced, which had a liquid centre. The fluid from each lesion was aspirated and cultured in medium (RPMI) at 37 °C in an incubator. Cell cultures were analysed at 1 and 7 days for cell viability and a live-dead assay was performed. The histotripsy sites were excised following aspiration and H&E staining was used to characterise the liver lesions. Cell morphology was determined by histology. Results: Histotripsy created a subcapsular lesion (~5 mm below the liver capsule; size ranging from 3 to 5 mm), which contained a suspension of cells. On average, 61×104 cells per mL were isolated. Hepatocytes were present in the aspirate, were viable at 24 h post isolation and remained viable in culture for up to 1 week, as determined by phalloidin/DAPI cell viability stains. Cultures up to 21 days revealed metabolically active live hepatocyte. Live-dead assays confirmed hepatocyte viability at 1 week (Day 1: 12% to Day 7: 45% live cells; p < 0.0001), which retained metabolic activity and morphology, confirmed on assay and microscopy. Cell Titre-GloTM showed a peak metabolic activity at 1 week (average luminescence 24.6 RLU; p < 0.0001) post-culture compared with the control (culture medium alone), reduced to 1/3 of peak level (7.85 RLU) by day 21. Conclusions: Histotripsy of the liver allows isolation and culture of hepatocytes with a high rate of viability after 1 week in culture. Reproducing these findings using human livers may lead to wide clinical applications in cell therapy.
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Affiliation(s)
- Saied Froghi
- Department of HPB & Liver Transplantation Surgery, Royal Free London NHS Foundation Trust, Pond Street, Hampstead, London NW3 2QG, UK
- Centre for Surgical Innovation, Organ Regeneration and Transplantation, UCL Division of Surgery & Interventional Sciences, Royal Free Hospital Campus, Pond Street, Hampstead, London NW3 2QG, UK
- Correspondence: or
| | - Matheus Oliveira de Andrade
- Ultrasonics Group, Department of Mechanical Engineering, Roberts Engineering Building, University College London, Torrington Place, London WC1E 7JE, UK
| | - Layla Mohammad Hadi
- Centre for Surgical Innovation, Organ Regeneration and Transplantation, UCL Division of Surgery & Interventional Sciences, Royal Free Hospital Campus, Pond Street, Hampstead, London NW3 2QG, UK
| | - Pierre Gelat
- Centre for Surgical Innovation, Organ Regeneration and Transplantation, UCL Division of Surgery & Interventional Sciences, Royal Free Hospital Campus, Pond Street, Hampstead, London NW3 2QG, UK
| | - Hassan Rashidi
- Stem Cell & Regenerative Medicine Section, UCL Great Ormond Street Institute of Child Health, London WC1N 1EH, UK
| | - Alberto Quaglia
- Department of Cellular Pathology, Royal Free London NHS Foundation Trust, Pond Street, Hampstead, London NW3 2QG, UK
| | - Barry Fuller
- Centre for Surgical Innovation, Organ Regeneration and Transplantation, UCL Division of Surgery & Interventional Sciences, Royal Free Hospital Campus, Pond Street, Hampstead, London NW3 2QG, UK
| | - Nader Saffari
- Ultrasonics Group, Department of Mechanical Engineering, Roberts Engineering Building, University College London, Torrington Place, London WC1E 7JE, UK
| | - Brian Davidson
- Department of HPB & Liver Transplantation Surgery, Royal Free London NHS Foundation Trust, Pond Street, Hampstead, London NW3 2QG, UK
- Centre for Surgical Innovation, Organ Regeneration and Transplantation, UCL Division of Surgery & Interventional Sciences, Royal Free Hospital Campus, Pond Street, Hampstead, London NW3 2QG, UK
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Kaur I, Vasudevan A, Rawal P, Tripathi DM, Ramakrishna S, Kaur S, Sarin SK. Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements. Bioengineering (Basel) 2023; 10:131. [PMID: 36829625 PMCID: PMC9952008 DOI: 10.3390/bioengineering10020131] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 01/11/2023] [Accepted: 01/13/2023] [Indexed: 01/20/2023] Open
Abstract
Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.
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Affiliation(s)
- Impreet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Ashwini Vasudevan
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Preety Rawal
- School of Biotechnology, Gautam Buddha University, Greater Noida 201312, India
| | - Dinesh M. Tripathi
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Seeram Ramakrishna
- Department of Mechanical Engineering, National University of Singapore, Singapore 117581, Singapore
| | - Savneet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Shiv K. Sarin
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
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Establishment and Characterization of an HBV Viral Spread and Infectious System following Long-Term Passage of an HBV Clinical Isolate in the Primary Human Hepatocyte and Fibroblast Coculture System. J Virol 2022; 96:e0084922. [PMID: 36037476 PMCID: PMC9517703 DOI: 10.1128/jvi.00849-22] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The existing cell culture-based methods to study hepatitis B virus (HBV) have limitations and do not allow for viral long-term passage. The aim of this study was to develop a robust in vitro long-term viral passage system with optimized cell culture conditions and a viral isolate with the ability to spread and passage. An HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized primary human hepatocyte (PHH)/human fibroblast coculture system. The passaged UV-treated virus was sequenced and further characterized. In addition, a panel of mutant viruses containing different combinations of mutations observed in this virus was investigated. The clinical isolate was passaged for 20 rounds with 21 days per round in an optimized PHH/human fibroblast coculture system while subject to UV mutagenesis. This passaged UV-mutated isolate harbored four mutations: G225A (sR24K) in the S gene, A2062T in the core gene, and two mutations G1764A and C1766T (xV131I) in the basal core promoter (BCP) region. In vitro characterization of the four mutations suggested that the two BCP mutations G1764A and C1766T contributed to the increased viral replication and viral infectivity. A robust in vitro long-term HBV viral passage system has been established by passaging a UV-treated clinical isolate in an optimized PHH/fibroblast coculture system. The two BCP mutations played a key role in the virus's ability to passage. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization. IMPORTANCE The existing cell culture-based methods to study HBV have limitations and do not allow for viral long-term passage. In this study, an HBV genotype A clinical isolate was subjected to multiple rounds of UV treatment and passaged in an optimized PHH/human fibroblast coculture system. This passaged UV-mutated isolate carried four mutations across the HBV genome, and in vitro characterization of the four mutations suggested that the two basal core promoter (BCP) mutations G1764A and C1766T played a key role in the virus's ability to passage. In summary, we have developed a robust in vitro long-term HBV viral passage system by passaging an UV-treated HBV genotype A clinical isolate in an optimized PHH/human fibroblast coculture system. This passage system can be used for studying the entire life cycle of HBV and has the potential for in vitro drug-resistance selection upon further optimization.
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7
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Xiao J, Li X, Zhou Z, Guan S, Zhuo L, Gao B. Development of an in vitro insulin resistance dissociated model of hepatic steatosis by co-culture system. Biosci Trends 2022; 16:257-266. [PMID: 35965099 DOI: 10.5582/bst.2022.01242] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Abstract
The evidence shows that there is an associated relationship between hepatosteatosis and insulin resistance. While some existing genetic induction animal and patient models challenge this relationship, indicating that hepatosteatosis is dissociated from insulin resistance. However, the molecular mechanisms of this dissociation remain poorly understood due to a lack of available, reliable, and simplistic setup models. Currently, we used primary rat hepatocytes (rHPCs), co-cultured with rat hepatic stellate cells (HSC-T6) or human foreskin fibroblast cells (HFF-1) in stimulation with high insulin and glucose, to develop a model of steatosis charactered as dissociated lipid accumulation from insulin resistance. Oil-Red staining significantly showed intracellular lipid accumulated in the developed model. Gene expression of sterol regulatory element-binding protein 1c (SREBP1c) and elongase of very-long-chain fatty acids 6 (ELOVL6), key genes responsible for lipogenesis, were detected and obviously increased in this model. Inversely, the insulin resistance related genes expression included phosphoenolpyruvate carboxykinase 1 (PCK1), pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), and glucose-6-phosphatase (G6pase) were decreased, suggesting a dissociation relationship between steatosis and insulin resistance in the developed model. As well, the drug metabolism of this developed model was investigated and showed up-regulation of cytochrome P450 3A (CYP3A) and down-regulation of cytochrome P450 2E1 (CYP2E1) and cytochrome P450 1A2 (CYP1A2). Taken together, those results demonstrate that the in vitro model of dissociated steatosis from insulin resistance was successfully created by our co-cultured cells in high insulin and glucose medium, which will be a potential model for investigating the mechanism of insulin resistance dissociated steatosis, and discovering a novel drug for its treatment.
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Affiliation(s)
- Jiangwei Xiao
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.,National Engineering Research Center for Healthcare Devices, Guangzhou, China.,Guangdong Key Lab of Medical Electronic Instruments and Polymer Material Products, Guangzhou, China
| | - Xiang Li
- Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Zongbao Zhou
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.,National Engineering Research Center for Healthcare Devices, Guangzhou, China.,Guangdong Key Lab of Medical Electronic Instruments and Polymer Material Products, Guangzhou, China
| | - Shuwen Guan
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.,National Engineering Research Center for Healthcare Devices, Guangzhou, China.,Guangdong Key Lab of Medical Electronic Instruments and Polymer Material Products, Guangzhou, China
| | - Lingjian Zhuo
- Department of Emergency Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, China
| | - Botao Gao
- Institute of Biological and Medical Engineering, Guangdong Academy of Sciences, Guangzhou, China.,National Engineering Research Center for Healthcare Devices, Guangzhou, China.,Guangdong Key Lab of Medical Electronic Instruments and Polymer Material Products, Guangzhou, China
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8
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de Hoyos-Vega JM, Hong HJ, Stybayeva G, Revzin A. Hepatocyte cultures: From collagen gel sandwiches to microfluidic devices with integrated biosensors. APL Bioeng 2021; 5:041504. [PMID: 34703968 PMCID: PMC8519630 DOI: 10.1063/5.0058798] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 09/21/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatocytes are parenchymal cells of the liver responsible for drug detoxification, urea and bile production, serum protein synthesis, and glucose homeostasis. Hepatocytes are widely used for drug toxicity studies in bioartificial liver devices and for cell-based liver therapies. Because hepatocytes are highly differentiated cells residing in a complex microenvironment in vivo, they tend to lose hepatic phenotype and function in vitro. This paper first reviews traditional culture approaches used to rescue hepatic function in vitro and then discusses the benefits of emerging microfluidic-based culture approaches. We conclude by reviewing integration of hepatocyte cultures with bioanalytical or sensing approaches.
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Affiliation(s)
- Jose M. de Hoyos-Vega
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Hye Jin Hong
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
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9
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Pluta KD, Ciezkowska M, Wisniewska M, Wencel A, Pijanowska DG. Cell-based clinical and experimental methods for assisting the function of impaired livers – Present and future of liver support systems. Biocybern Biomed Eng 2021. [DOI: 10.1016/j.bbe.2021.06.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
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10
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Yao T, Zhang Y, Lv M, Zang G, Ng SS, Chen X. Advances in 3D cell culture for liver preclinical studies. Acta Biochim Biophys Sin (Shanghai) 2021; 53:643-651. [PMID: 33973620 DOI: 10.1093/abbs/gmab046] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/27/2020] [Indexed: 11/13/2022] Open
Abstract
The 3D cell culture model is an indispensable tool in the study of liver biology in the field of health and disease and the development of clinically relevant products for liver therapies. The 3D culture model captures critical factors of the microenvironmental niche required by hepatocytes for exhibiting optimal phenotypes, thus enabling the pursuit of a range of preclinical studies that are not entirely feasible in conventional 2D cell models. In this review, we highlight the major attributes associated with and the components needed for the development of a functional 3D liver culture model for a range of applications.
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Affiliation(s)
- Ting Yao
- Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
| | - Yi Zhang
- Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
| | - Mengjiao Lv
- Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
| | - Guoqing Zang
- Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
| | - Soon Seng Ng
- Department of Metabolism, Digestion and Reproduction, Faculty of Medicine, Imperial College London, London W2 1PG, UK
| | - Xiaohua Chen
- Department of Infectious Diseases, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China
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11
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Yang Q, Humphreys SC, Lade JM, Li AP. Prolonged cultured human hepatocytes as an in vitro experimental system for the evaluation of potency and duration of activity of RNA therapeutics: Demonstration of prolonged duration of gene silencing effects of a GalNAc-conjugated human hypoxanthine phosphoribosyl transferase (HPRT1) siRNA. Biochem Pharmacol 2020; 189:114374. [PMID: 33358826 DOI: 10.1016/j.bcp.2020.114374] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2020] [Revised: 12/14/2020] [Accepted: 12/15/2020] [Indexed: 12/18/2022]
Abstract
We report here the evaluation of a novel in vitro experimental model, prolonged cultured human hepatocytes (PCHC), as an experimental system to evaluate the potency and duration of effects of oligonucleotide therapeutics. A novel observation was made on the redifferentiation of PCHC upon prolonged culturing based on mRNA profiling of characteristic hepatic differentiation marker genes albumin, transferrin, and transthyretin. Consistent with the known de-differentiation of cultured human hepatocytes, decreases in marker gene expression were observed upon culturing of the hepatocytes for 2 days. A novel observation of re-differentiation was observed on day 7 as demonstrated by an increase in expression of the marker genes to levels similar to that observed on the first day of culture. The expression of the differentiation marker genes was highest on day 7, followed by a gradual decrease but remained higher than that on day 2 for up to the longest culture duration evaluated of 41 days. The redifferentiation phenomenon suggests that PCHC may be useful for the evaluation of the duration of effects of oligonucleotide therapeutics on gene expression in human hepatocytes. A proof of concept study was thereby conducted with PCHC with a GalNAc-conjugated siRNA targeting human hypoxanthine phosphoribosyl transferase1 (HPRT1). HPRT1 mRNA expression in siRNA-treated cultures decreased to 21% of that in untreated hepatocytes on day 1, <10% from days 2 to 12, <20% from days 16 to 33, and eventually recovered to 64% by day 41. Our results suggest that PCHC represent a clinically-relevant cost- and time-efficient experimental tool to aid in the evaluation of GalNAc-siRNA silencing activity, providing information on both efficacy and duration of efficacy. PCHC may be applicable in the drug development setting as a species- and cell type-relevant experimental tool to aid the development of oligonucleotide therapeutics.
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Affiliation(s)
- Qian Yang
- In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States
| | - Sara C Humphreys
- In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States
| | - Julie M Lade
- In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States
| | - Albert P Li
- In Vitro ADMET Laboratories Inc., Columbia, MD (QY, APL) and Amgen Inc., San Francisco, CA (SCH, JML), United States.
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12
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The fibrotic response of primary liver spheroids recapitulates in vivo hepatic stellate cell activation. Biomaterials 2020; 261:120335. [PMID: 32891040 DOI: 10.1016/j.biomaterials.2020.120335] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2020] [Revised: 07/06/2020] [Accepted: 08/15/2020] [Indexed: 02/07/2023]
Abstract
A major obstacle in the development of efficient therapies for progressive liver fibrosis is the lack of representative in vitro models of liver fibrosis to aid in understanding the mechanisms of the disease and to promote the development of pharmaceuticals. Our aim was to develop a relevant in vitro mouse liver fibrosis model, based on the central hypothesis that liver fibrosis in vitro cannot be studied using only hepatic stellate cells (HSCs)-the main producer of scar tissue during fibrosis-, but requires cultures in which at least hepatocytes are integrated. We established robust methods to generate co-culture spheroids from freshly isolated mouse hepatocytes and HSCs. Characteristics and functionality of these spheroids were analyzed by qPCR of cell-type specific markers, CYP induction and immunohistochemistry. Compound toxicity was determined by ATP-assays. Hepatocytes and HSCs maintained their cell-type specific marker expression over a 15-day culture period without major hepatocyte dedifferentiation or HSC activation. Exposure of spheroids to TGFβ can directly activate HSCs, while acetaminophen exposure mounts a hepatocyte damage dependent activation of HSCs. Pharmaceuticals with known anti-fibrotic properties, such as Valproic acid and Verteporfin, reduce HSC activation in response to hepatocyte damage in these cultures. A comparison between the fibrotic response of the spheroid co-cultures and in vivo activated HSCs showed that these 3D co-cultures are more representative than the commonly used 2D HSC monocultures. Finally, we showed that the 3D cultures can be integrated in microfluidic chips. We conclude that our hepatocyte-stellate cell-spheroid cultures are a robust in vitro model of liver fibrosis. This model could be used to further unravel the mechanism of HSC activation and facilitate the discovery of, or testing for novel anti-fibrotic compounds, as these spheroids better reproduce HSC in vivo activation compared to the more traditional 2D mono-culture models.
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The application of omics-based human liver platforms for investigating the mechanism of drug-induced hepatotoxicity in vitro. Arch Toxicol 2019; 93:3067-3098. [PMID: 31586243 DOI: 10.1007/s00204-019-02585-5] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2019] [Accepted: 09/25/2019] [Indexed: 12/13/2022]
Abstract
Drug-induced liver injury (DILI) complicates safety assessment for new drugs and poses major threats to both patient health and drug development in the pharmaceutical industry. A number of human liver cell-based in vitro models combined with toxicogenomics methods have been developed as an alternative to animal testing for studying human DILI mechanisms. In this review, we discuss the in vitro human liver systems and their applications in omics-based drug-induced hepatotoxicity studies. We furthermore present bioinformatic approaches that are useful for analyzing toxicogenomic data generated from these models and discuss their current and potential contributions to the understanding of mechanisms of DILI. Human pluripotent stem cells, carrying donor-specific genetic information, hold great potential for advancing the study of individual-specific toxicological responses. When co-cultured with other liver-derived non-parenchymal cells in a microfluidic device, the resulting dynamic platform enables us to study immune-mediated drug hypersensitivity and accelerates personalized drug toxicology studies. A flexible microfluidic platform would also support the assembly of a more advanced organs-on-a-chip device, further bridging gap between in vitro and in vivo conditions. The standard transcriptomic analysis of these cell systems can be complemented with causality-inferring approaches to improve the understanding of DILI mechanisms. These approaches involve statistical techniques capable of elucidating regulatory interactions in parts of these mechanisms. The use of more elaborated human liver models, in harmony with causality-inferring bioinformatic approaches will pave the way for establishing a powerful methodology to systematically assess DILI mechanisms across a wide range of conditions.
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14
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Díaz-Bello B, Monroy-Romero AX, Pérez-Calixto D, Zamarrón-Hernández D, Serna-Marquez N, Vázquez-Victorio G, Hautefeuille M. Method for the Direct Fabrication of Polyacrylamide Hydrogels with Controlled Stiffness in Polystyrene Multiwell Plates for Mechanobiology Assays. ACS Biomater Sci Eng 2019; 5:4219-4227. [DOI: 10.1021/acsbiomaterials.9b00988] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Affiliation(s)
- Beatriz Díaz-Bello
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Ana Ximena Monroy-Romero
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Daniel Pérez-Calixto
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Diego Zamarrón-Hernández
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Nathalia Serna-Marquez
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Genaro Vázquez-Victorio
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
| | - Mathieu Hautefeuille
- Departamento de Física, Facultad de Ciencias, Universidad Nacional Autónoma de México, Circuito Exterior S/N, Ciudad Universitaria, Coyoacan 04510, Mexico
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15
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Brovold M, Almeida JI, Pla-Palacín I, Sainz-Arnal P, Sánchez-Romero N, Rivas JJ, Almeida H, Dachary PR, Serrano-Aulló T, Soker S, Baptista PM. Naturally-Derived Biomaterials for Tissue Engineering Applications. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2019; 1077:421-449. [PMID: 30357702 PMCID: PMC7526297 DOI: 10.1007/978-981-13-0947-2_23] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Naturally-derived biomaterials have been used for decades in multiple regenerative medicine applications. From the simplest cell microcarriers made of collagen or alginate, to highly complex decellularized whole-organ scaffolds, these biomaterials represent a class of substances that is usually first in choice at the time of electing a functional and useful biomaterial. Hence, in this chapter we describe the several naturally-derived biomaterials used in tissue engineering applications and their classification, based on composition. We will also describe some of the present uses of the generated tissues like drug discovery, developmental biology, bioprinting and transplantation.
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Affiliation(s)
- Matthew Brovold
- Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA
| | - Joana I Almeida
- Health Research Institute of Aragón (IIS Aragón), Zaragoza, Spain
| | - Iris Pla-Palacín
- Health Research Institute of Aragón (IIS Aragón), Zaragoza, Spain
| | - Pilar Sainz-Arnal
- Health Research Institute of Aragón (IIS Aragón), Zaragoza, Spain
- Aragon Health Sciences Institute (IACS), Zaragoza, Spain
| | | | - Jesus J Rivas
- Health Research Institute of Aragón (IIS Aragón), Zaragoza, Spain
| | - Helen Almeida
- Health Research Institute of Aragón (IIS Aragón), Zaragoza, Spain
| | - Pablo Royo Dachary
- Instituto de Investigación Sanitária de Aragón (IIS Aragón), Zaragoza, Spain
- Liver Transplant Unit, Gastroenterology Department, Lozano Blesa University Hospital, Zaragoza, Spain
| | - Trinidad Serrano-Aulló
- Instituto de Investigación Sanitária de Aragón (IIS Aragón), Zaragoza, Spain
- Liver Transplant Unit, Gastroenterology Department, Lozano Blesa University Hospital, Zaragoza, Spain
| | - Shay Soker
- Wake Forest Institute for Regenerative Medicine, Winston-Salem, NC, USA.
| | - Pedro M Baptista
- Instituto de Investigación Sanitária de Aragón (IIS Aragón), Zaragoza, Spain.
- Center for Biomedical Research Network Liver and Digestive Diseases (CIBERehd), Zaragoza, Spain.
- Instituto de Investigación Sanitaria de la Fundación Jiménez Díaz, Madrid, Spain.
- Biomedical and Aerospace Engineering Department, Universidad Carlos III de Madrid, Madrid, Spain.
- Fundación ARAID, Zaragoza, Spain.
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16
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Pettinato G, Lehoux S, Ramanathan R, Salem MM, He LX, Muse O, Flaumenhaft R, Thompson MT, Rouse EA, Cummings RD, Wen X, Fisher RA. Generation of fully functional hepatocyte-like organoids from human induced pluripotent stem cells mixed with Endothelial Cells. Sci Rep 2019; 9:8920. [PMID: 31222080 PMCID: PMC6586904 DOI: 10.1038/s41598-019-45514-3] [Citation(s) in RCA: 105] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2019] [Accepted: 06/10/2019] [Indexed: 12/16/2022] Open
Abstract
Despite advances in stem cell research, cell transplantation therapy for liver failure is impeded by a shortage of human primary hepatocytes (HPH), along with current differentiation protocol limitations. Several studies have examined the concept of co-culture of human induced pluripotent cells (hiPSCs) with various types of supporting non-parenchymal cells to attain a higher differentiation yield and to improve hepatocyte-like cell functions both in vitro and in vivo. Co-culturing hiPSCs with human endothelial cells (hECs) is a relatively new technique that requires more detailed studies. Using our 3D human embryoid bodies (hEBs) formation technology, we interlaced Human Adipose Microvascular Endothelial Cells (HAMEC) with hiPSCs, leading to a higher differentiation yield and notable improvements across a wide range of hepatic functions. We conducted a comprehensive gene and protein secretion analysis of our HLCs coagulation factors profile, showing promising results in comparison with HPH. Furthermore, a stage-specific glycomic analysis revealed that the differentiated hepatocyte-like clusters (HLCs) resemble the glycan features of a mature tissue rather than cells in culture. We tested our HLCs in animal models, where the presence of HAMEC in the clusters showed a consistently better performance compared to the hiPSCs only group in regard to persistent albumin secretion post-transplantation.
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Affiliation(s)
- Giuseppe Pettinato
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
| | - Sylvain Lehoux
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
- Glycomics Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Rajesh Ramanathan
- Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA
| | - Mohamed M Salem
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
- Neurosurgical Service, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Li-Xia He
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Oluwatoyosi Muse
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Robert Flaumenhaft
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Melissa T Thompson
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Emily A Rouse
- Glycomics Core, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Richard D Cummings
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Xuejun Wen
- Department of Chemical and Life Science Engineering, Virginia Commonwealth University, Richmond, VA, USA
| | - Robert A Fisher
- Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
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Sivakumar P, Kitson C, Jarai G. Modeling and measuring extracellular matrix alterations in fibrosis: challenges and perspectives for antifibrotic drug discovery. Connect Tissue Res 2019; 60:62-70. [PMID: 30071759 DOI: 10.1080/03008207.2018.1500557] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
An imbalance of extracellular matrix (ECM) deposition and turnover is a hallmark of fibrotic pathologies as opposed to normal repair response to injury across several organs. Antifibrotic approaches to date have targeted multiple mechanisms and pathways involved in inflammation, angiogenesis, injury, wound repair, ECM biosynthesis, assembly, crosslinking and degradation. Many of these approaches have been unsuccessful which may in part be due to suboptimal models and the lack of validated functional ECM end points relevant to fibrosis. In addition, drug discovery and development for fibrotic diseases has been challenging due to the lack of translatability from in vivo models to the clinic. Targeting growth factor signaling pathways such as transforming growth factor beta (TGFβ), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF) are possible in simple recombinant cell models and the approval of the tyrosine kinase inhibitor, nintedanib (Ofev) is testament to the approach. However, drug targets directly impacting ECM synthesis, assembly or degradation have proven clinically intractable to date. The reasons for a lack of progress are many and include; non-traditional drug targets, lack of suitable high throughput screening assays and translational models, incomplete understanding of the role of the target. Here, we review the role of ECM in fibrosis, the challenges of ECM-targeted antifibrotic approaches, progress in the development of functional and biomarker-related ECM assays and where new translational models of fibrotic ECM remodeling could support drug discovery for fibrotic diseases.
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Affiliation(s)
- Pitchumani Sivakumar
- a Fibrosis Translational Research and Development , Bristol-Myers Squibb , Pennington , NJ , USA
| | - Christopher Kitson
- b Fibrosis Discovery Biology , Bristol-Myers Squibb , Pennington , NJ , USA
| | - Gabor Jarai
- a Fibrosis Translational Research and Development , Bristol-Myers Squibb , Pennington , NJ , USA
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18
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Green CJ, Parry SA, Gunn PJ, Ceresa CDL, Rosqvist F, Piché ME, Hodson L. Studying non-alcoholic fatty liver disease: the ins and outs of in vivo, ex vivo and in vitro human models. Horm Mol Biol Clin Investig 2018; 41:/j/hmbci.ahead-of-print/hmbci-2018-0038/hmbci-2018-0038.xml. [PMID: 30098284 DOI: 10.1515/hmbci-2018-0038] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2018] [Accepted: 06/22/2018] [Indexed: 02/07/2023]
Abstract
The prevalence of non-alcoholic fatty liver disease (NAFLD) is increasing. Determining the pathogenesis and pathophysiology of human NAFLD will allow for evidence-based prevention strategies, and more targeted mechanistic investigations. Various in vivo, ex situ and in vitro models may be utilised to study NAFLD; but all come with their own specific caveats. Here, we review the human-based models and discuss their advantages and limitations in regards to studying the development and progression of NAFLD. Overall, in vivo whole-body human studies are advantageous in that they allow for investigation within the physiological setting, however, limited accessibility to the liver makes direct investigations challenging. Non-invasive imaging techniques are able to somewhat overcome this challenge, whilst the use of stable-isotope tracers enables mechanistic insight to be obtained. Recent technological advances (i.e. normothermic machine perfusion) have opened new opportunities to investigate whole-organ metabolism, thus ex situ livers can be investigated directly. Therefore, investigations that cannot be performed in vivo in humans have the potential to be undertaken. In vitro models offer the ability to perform investigations at a cellular level, aiding in elucidating the molecular mechanisms of NAFLD. However, a number of current models do not closely resemble the human condition and work is ongoing to optimise culturing parameters in order to recapitulate this. In summary, no single model currently provides insight into the development, pathophysiology and progression across the NAFLD spectrum, each experimental model has limitations, which need to be taken into consideration to ensure appropriate conclusions and extrapolation of findings are made.
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Affiliation(s)
- Charlotte J Green
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
| | - Siôn A Parry
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
| | - Pippa J Gunn
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
| | - Carlo D L Ceresa
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
- Nuffield Department of Surgical Sciences, University of Oxford, Oxford, UK
| | - Fredrik Rosqvist
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
- Department of Public Health and Caring Sciences, Clinical Nutrition and Metabolism, Uppsala University, Uppsala, Sweden
| | - Marie-Eve Piché
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford, UK
- Quebec Heart and Lung Institute, Laval University, Quebec, Canada
| | - Leanne Hodson
- University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, Churchill Hospital,Old Road Headington, Oxford OX3 7LE, United Kingdom of Great Britain and Northern Ireland
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19
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Davidson MD, Kukla DA, Khetani SR. Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development. Integr Biol (Camb) 2018; 9:662-677. [PMID: 28702667 DOI: 10.1039/c7ib00027h] [Citation(s) in RCA: 42] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
In non-alcoholic steatohepatitis (NASH), hepatic stellate cells (HSC) differentiate into myofibroblast-like cells that cause fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. Thus, modeling interactions between activated HSCs and hepatocytes in vitro can aid in the development of anti-NASH/fibrosis therapeutics and lead to a better understanding of disease progression. Species-specific differences in drug metabolism and disease pathways now necessitate the supplementation of animal studies with data acquired using human liver models; however, current models do not adequately model the negative effects of primary human activated HSCs on the phenotype of otherwise well-differentiated primary human hepatocytes (PHHs) as in vivo. Therefore, here we first determined the long-term effects of primary human activated HSCs on PHH phenotype in a micropatterned co-culture (MPCC) platform while using 3T3-J2 murine embryonic fibroblasts as the control cell type since it has been shown previously to stabilize PHH functions for 4-6 weeks. We found that HSCs were not able to stabilize the PHH phenotype to the same magnitude and longevity as the fibroblasts, which subsequently inspired the development of a micropatterned tri-culture (MPTC) platform in which (a) micropatterned PHHs were functionally stabilized using fibroblasts, and (b) the PHH phenotype was modulated by culturing HSCs within the fibroblast monolayer at physiologically-relevant ratios with PHHs. Transwell inserts containing HSCs were placed atop MPCCs containing fibroblasts to confirm the effects of paracrine signaling between PHHs and HSCs. We found that while albumin and urea secretions were relatively similar in MPTCs and MPCCs (suggesting well-differentiated PHHs), increasing HSC numbers within MPTCs downregulated hepatic cytochrome-P450 (2A6, 3A4) and transporter activities, and caused steatosis over 2 weeks. Furthermore, MPTCs secreted higher levels of pro-inflammatory interleukin-6 (IL-6) cytokine and C-reactive protein (CRP) than MPCCs. Treatment of MPCCs with HSC-conditioned culture medium confirmed that HSC secretions mediate the altered phenotype of PHHs observed in MPTCs, partly via IL-6 signaling. Lastly, we found that NADPH oxidase (NOX) inhibition and farnesoid X receptor (FXR) activation using clinically relevant drugs alleviated hepatic dysfunctions in MPTCs. In conclusion, MPTCs recapitulate symptoms of NASH- and early fibrosis-like dysfunctions in PHHs and have utility for drug discovery in this space.
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Affiliation(s)
- Matthew D Davidson
- School of Biomedical Engineering, Colorado State University, Fort Collins, CO, USA
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20
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Beckwitt CH, Clark AM, Wheeler S, Taylor DL, Stolz DB, Griffith L, Wells A. Liver 'organ on a chip'. Exp Cell Res 2018; 363:15-25. [PMID: 29291400 PMCID: PMC5944300 DOI: 10.1016/j.yexcr.2017.12.023] [Citation(s) in RCA: 144] [Impact Index Per Article: 20.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2017] [Revised: 12/21/2017] [Accepted: 12/27/2017] [Indexed: 12/14/2022]
Abstract
The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems.
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Affiliation(s)
- Colin H Beckwitt
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Research and Development Service, VA Pittsburgh Health System, Pittsburgh, PA 15240, USA
| | - Amanda M Clark
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Sarah Wheeler
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - D Lansing Taylor
- Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Donna B Stolz
- Cell Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Linda Griffith
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Alan Wells
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA; Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Research and Development Service, VA Pittsburgh Health System, Pittsburgh, PA 15240, USA.
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21
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Oseini AM, Cole BK, Issa D, Feaver RE, Sanyal AJ. Translating scientific discovery: the need for preclinical models of nonalcoholic steatohepatitis. Hepatol Int 2018; 12:6-16. [PMID: 29299759 PMCID: PMC5815925 DOI: 10.1007/s12072-017-9838-6] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/14/2017] [Accepted: 11/24/2017] [Indexed: 12/29/2022]
Abstract
Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western world, affecting about 1/3 of the US general population and remaining as a significant cause of morbidity and mortality. The hallmark of the disease is the excessive accumulation of fat within the liver cells (hepatocytes), which eventually paves the way to cellular stress, injury and apoptosis. NAFLD is strongly associated with components of the metabolic syndrome and is fast emerging as a leading cause of liver transplant in the USA. Based on clinico-pathologic classification, NAFLD may present as isolated lipid collection (steatosis) within the hepatocytes (referred to as non-alcoholic fatty liver; NAFL); or as the more aggressive phenotype (known as non-alcoholic steatohepatitis; NASH). There are currently no regulatory agency- approved medication for NAFLD, despite the enormous work and resources that have gone into the study of this condition. Therefore, there remains a huge unmet need in developing and utilizing pre-clinical models that will recapitulate the disease condition in humans. In line with progress being made in developing appropriate disease models, this review highlights the cutting-edge preclinical in vitro and animal models that try to recapitulate the human disease pathophysiology and/or clinical manifestations.
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Affiliation(s)
- Abdul M. Oseini
- Division of Gastroenterology, Department of Medicine, VCU School of Medicine, MCV Box 980341, Richmond, VA 23298-0341, USA
| | - Banumathi K. Cole
- HemoShear Therapeutics, 501 Locust Ave, Suite 301, Charlottesville, VA 22902, USA
| | - Danny Issa
- Division of Gastroenterology, Department of Medicine, VCU School of Medicine, MCV Box 980341, Richmond, VA 23298-0341, USA
| | - Ryan E. Feaver
- HemoShear Therapeutics, 501 Locust Ave, Suite 301, Charlottesville, VA 22902, USA
| | - Arun J. Sanyal
- Division of Gastroenterology, Department of Medicine, VCU School of Medicine, MCV Box 980341, Richmond, VA 23298-0341, USA
- Physiology and Molecular Pathology, MCV Box 980341, Richmond, VA 23298-0341, USA
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22
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Cole BK, Feaver RE, Wamhoff BR, Dash A. Non-alcoholic fatty liver disease (NAFLD) models in drug discovery. Expert Opin Drug Discov 2017; 13:193-205. [PMID: 29190166 DOI: 10.1080/17460441.2018.1410135] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
INTRODUCTION The progressive disease spectrum of non-alcoholic fatty liver disease (NAFLD), which includes non-alcoholic steatohepatitis (NASH), is a rapidly emerging public health crisis with no approved therapy. The diversity of various therapies under development highlights the lack of consensus around the most effective target, underscoring the need for better translatable preclinical models to study the complex progressive disease and effective therapies. Areas covered: This article reviews published literature of various mouse models of NASH used in preclinical studies, as well as complex organotypic in vitro and ex vivo liver models being developed. It discusses translational challenges associated with both kinds of models, and describes some of the studies that validate their application in NAFLD. Expert opinion: Animal models offer advantages of understanding drug distribution and effects in a whole body context, but are limited by important species differences. Human organotypic in vitro and ex vivo models with physiological relevance and translatability need to be used in a tiered manner with simpler screens. Leveraging newer technologies, like metabolomics, proteomics, and transcriptomics, and the future development of validated disease biomarkers will allow us to fully utilize the value of these models to understand disease and evaluate novel drugs in isolation or combination.
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Affiliation(s)
| | | | | | - Ajit Dash
- b Early Development Safety , Genentech Inc , South San Francisco , CA , USA
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23
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3D in vitro models of liver fibrosis. Adv Drug Deliv Rev 2017; 121:133-146. [PMID: 28697953 DOI: 10.1016/j.addr.2017.07.004] [Citation(s) in RCA: 89] [Impact Index Per Article: 11.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2017] [Revised: 06/27/2017] [Accepted: 07/06/2017] [Indexed: 02/07/2023]
Abstract
Animal testing is still the most popular preclinical assessment model for liver fibrosis. To develop efficient anti-fibrotic therapies, robust and representative in vitro models are urgently needed. The most widely used in vitro fibrosis model is the culture-induced activation of primary rodent hepatic stellate cells. While these cultures have contributed greatly to the current understanding of hepatic stellate cell activation, they seem to be inadequate to cover the complexity of this regenerative response. This review summarizes recent progress towards the development of 3D culture models of liver fibrosis. Thus far, only a few hepatic culture systems have successfully implemented hepatic stellate cells (or other non-parenchymal cells) into hepatocyte cultures. Recent advances in bioprinting, spheroid- and precision-cut liver slice cultures and the use of microfluidic bioreactors will surely lead to valid 3D in vitro models of liver fibrosis in the near future.
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24
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Hariparsad N, Ramsden D, Palamanda J, Dekeyser JG, Fahmi OA, Kenny JR, Einolf H, Mohutsky M, Pardon M, Siu YA, Chen L, Sinz M, Jones B, Walsky R, Dallas S, Balani SK, Zhang G, Buckley D, Tweedie D. Considerations from the IQ Induction Working Group in Response to Drug-Drug Interaction Guidance from Regulatory Agencies: Focus on Downregulation, CYP2C Induction, and CYP2B6 Positive Control. Drug Metab Dispos 2017; 45:1049-1059. [PMID: 28646080 DOI: 10.1124/dmd.116.074567] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2017] [Accepted: 06/16/2017] [Indexed: 12/21/2022] Open
Abstract
The European Medicines Agency (EMA), the Pharmaceutical and Medical Devices Agency (PMDA), and the Food and Drug Administration (FDA) have issued guidelines for the conduct of drug-drug interaction studies. To examine the applicability of these regulatory recommendations specifically for induction, a group of scientists, under the auspices of the Drug Metabolism Leadership Group of the Innovation and Quality (IQ) Consortium, formed the Induction Working Group (IWG). A team of 19 scientists, from 16 of the 39 pharmaceutical companies that are members of the IQ Consortium and two Contract Research Organizations reviewed the recommendations, focusing initially on the current EMA guidelines. Questions were collated from IQ member companies as to which aspects of the guidelines require further evaluation. The EMA was then approached to provide insights into their recommendations on the following: 1) evaluation of downregulation, 2) in vitro assessment of CYP2C induction, 3) the use of CITCO as the positive control for CYP2B6 induction by CAR, 4) data interpretation (a 2-fold increase in mRNA as evidence of induction), and 5) the duration of incubation of hepatocytes with test article. The IWG conducted an anonymous survey among IQ member companies to query current practices, focusing specifically on the aforementioned key points. Responses were received from 19 companies. All data and information were blinded before being shared with the IWG. The results of the survey are presented, together with consensus recommendations on downregulation, CYP2C induction, and CYP2B6 positive control. Results and recommendations related to data interpretation and induction time course will be reported in subsequent articles.
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Affiliation(s)
- Niresh Hariparsad
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Diane Ramsden
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Jairam Palamanda
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Joshua G Dekeyser
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Odette A Fahmi
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Jane R Kenny
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Heidi Einolf
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Michael Mohutsky
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Magalie Pardon
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Y Amy Siu
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Liangfu Chen
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Michael Sinz
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Barry Jones
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Robert Walsky
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Shannon Dallas
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Suresh K Balani
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - George Zhang
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - David Buckley
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
| | - Donald Tweedie
- Vertex Pharmaceuticals, Boston, Massachusetts (N.H.); Genentech, South San Francisco, California (J.R.K.); Novartis Pharmaceuticals, Florham Park, New Jersey (H.E.); Eli Lilly and Company, Indianapolis, Indiana (M.M.); Boehringer Ingelheim, Ridgefield, Connecticut (D.R.); Merck and Co., Kenilworth, New Jersey (J.P.), Amgen Inc., Thousand Oaks, California (J.D.), Pfizer Global Research and Development, Groton, Connecticut (O.A.F.); Sanofi Pharmaceuticals, ChillyMazarin, France (M.P.); Eisai Pharmaceuticals, Andover, Massachusetts (A.Y.S.); Glaxo SmithKline, King of Prussia, Pennsylvania (L.C.); Bristol-Myers Squibb, Wallingford, Connecticut (M.S.); AstraZeneca, Mölndal, Sweden (B.J.); EMD Serono, Billerica, Massachusetts (R.W.);Janssen R&D, Spring House, Pennsylvania (S.D.); Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceuticals Co., Cambridge, Massachusetts (S.K.B.); Corning Life Sciences; Woburn, Massachusetts (G.Z.); XenoTech LLC, Lenexa, Kansas (D.B.); Merck and Co., West Point, Pennsylvania (D.T.)
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Stradiot L, Verhulst S, Roosens T, Øie C, Moya I, Halder G, Mannaerts I, van Grunsven L. Functionality based method for simultaneous isolation of rodent hepatic sinusoidal cells. Biomaterials 2017; 139:91-101. [DOI: 10.1016/j.biomaterials.2017.05.047] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2017] [Revised: 05/26/2017] [Accepted: 05/29/2017] [Indexed: 01/20/2023]
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Ahmed HMM, Salerno S, Morelli S, Giorno L, De Bartolo L. 3D liver membrane system by co-culturing human hepatocytes, sinusoidal endothelial and stellate cells. Biofabrication 2017; 9:025022. [PMID: 28548045 DOI: 10.1088/1758-5090/aa70c7] [Citation(s) in RCA: 43] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
In this study, a designed approach has been utilized for the development of a 3D liver system. This approach makes use of primary human sinusoidal endothelial cells, stellate cells and hepatocytes that are seeded sequentially on hollow fiber membranes (HF) in order to mimic the layers of cells found in vivo. To this purpose modified polyethersulfone (PES) HF membranes were used for the creation of a 3D human liver system in static and dynamic conditions. In order to verify the positive effect of non-parenchymal cells on the maintenance of hepatocyte viability and functions, homotypic cultures of hepatocytes alone on the HF membranes were further investigated. The membrane surface allowed the attachment and self-assembly of the cells, forming tissue-like structures around and between fibers. Sinusoidal cells formed tube-like structures that surrounded hepatocytes organized in cords within aggregates promoted by stellate cells. The co-culture of hepatocytes with sinusoidal endothelial and hepatic stellate cells preserved structural architecture of the construct and improved the liver-specific functions. Most importantly, cells co-cultured in a HF membrane bioreactor synthesized albumin and urea for 28 days. The liver membrane bioreactor also preserved the drug biotransformation activity with a continuous production of diazepam phase I metabolites for an extended period of time. Additionally, the cell oxygen uptake rates highlighted the maintenance of the actual oxygen concentration at a level compatible with their metabolic functions.
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Affiliation(s)
- Haysam Mohamed Magdy Ahmed
- Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, via P. Bucci cubo 17/C, I-87030 Rende (CS), Italy. Department of Chemical Engineering and Materials (DIATIC), University of Calabria, Rende, Italy
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Zhou VX, Lolas M, Chang TT. Direct orthotopic implantation of hepatic organoids. J Surg Res 2016; 211:251-260. [PMID: 28501125 DOI: 10.1016/j.jss.2016.12.028] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/07/2016] [Revised: 11/29/2016] [Accepted: 12/22/2016] [Indexed: 01/27/2023]
Abstract
BACKGROUND Liver organoids show potential for development as a tissue replacement therapy for patients with end-stage liver disease, but efficient methods for introducing organoids into host livers have not been established. In this study, we aimed to develop a surgical technique to implant hepatic organoids into the liver and assess their engraftment. METHODS Donor hepatocytes were isolated from ROSA26 C57BL/6 mice, so that engrafted cells, when implanted into wild-type mice, could be easily identified by X-gal staining. Hepatic organoids were generated by three-dimensional culture in rotating wall vessel bioreactors. We qualitatively and quantitatively compared organoid engraftment to that of single-cell hepatocyte transplants. In addition, we determined the effect of adding stellate cells to hepatocytes to form co-aggregated organoids and the effect of partial hepatectomy of the host liver on organoid engraftment. RESULTS Direct orthotopic implantation of hepatic organoids within a hepatotomy site resulted in local engraftment of exogenous hepatocytes with limited durability. Hepatocyte-stellate cell organoids produced more extracellular matrix but did not significantly improve engraftment compared with hepatocyte-alone organoids. Partial hepatectomy of the host liver led to significantly decreased engraftment of organoids. Survival of organoids was limited by the presence of apoptotic hepatocytes within organoids as early as 1 h after implantation. Organoids eventually became necrotic and elicited a chronic inflammatory giant cell reaction similar to a foreign body response. CONCLUSIONS With additional organoid and host factor optimization, direct orthotopic implantation of hepatic organoids may be an approach to introduce large numbers of exogenous hepatocytes into recipient livers.
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Affiliation(s)
- Vivian X Zhou
- Department of Surgery, University of California, San Francisco, San Francisco, California
| | - Macarena Lolas
- Department of Surgery, University of California, San Francisco, San Francisco, California
| | - Tammy T Chang
- Department of Surgery, University of California, San Francisco, San Francisco, California; Liver Center, University of California, San Francisco, San Francisco, California.
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Rogozhnikov D, Luo W, Elahipanah S, O'Brien PJ, Yousaf MN. Generation of a Scaffold-Free Three-Dimensional Liver Tissue via a Rapid Cell-to-Cell Click Assembly Process. Bioconjug Chem 2016; 27:1991-8. [PMID: 27508505 DOI: 10.1021/acs.bioconjchem.6b00187] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
There has been tremendous interest in constructing in vitro liver organ models for a range of fundamental studies of cell signaling, metabolism, and infectious diseases, and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress toward studying two-dimensional hepatic function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free three-dimensional multiple cell line coculture tissue models of liver. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts among multiple hepatic cell lines to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a three cell line coculture 3D liver tissue model by assembling hepatocytes, hepatic endothelial cells, and hepatic stellate cells via a rapid intercell click ligation process. We compare and analyze the function of the superior 3D liver tissue chips with 2D coculture monolayer by assessing mitochondrial metabolic activity and evaluating drug toxicity.
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Affiliation(s)
- Dmitry Rogozhnikov
- Department of Chemistry and Biology, York University , Toronto, Ontario M3J 1P3, Canada
| | - Wei Luo
- Department of Chemistry and Biology, York University , Toronto, Ontario M3J 1P3, Canada
| | - Sina Elahipanah
- Department of Chemistry and Biology, York University , Toronto, Ontario M3J 1P3, Canada
| | - Paul J O'Brien
- Department of Chemistry and Biology, York University , Toronto, Ontario M3J 1P3, Canada
| | - Muhammad N Yousaf
- Department of Chemistry and Biology, York University , Toronto, Ontario M3J 1P3, Canada.,OrganoLinX Inc. , Toronto, Ontario M3J 1P3, Canada
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Njayou FN, Kouam AF, Simo BFN, Tchana AN, Moundipa PF. Active chemical fractions of stem bark extract of Khaya grandifoliola C.DC and Entada africana Guill. et Perr. synergistically protect primary rat hepatocytes against paracetamol-induced damage. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2016; 16:190. [PMID: 27389400 PMCID: PMC4936298 DOI: 10.1186/s12906-016-1169-y] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/17/2015] [Accepted: 06/16/2016] [Indexed: 12/04/2022]
Abstract
Background Khaya grandifoliola (Meliaceae) and Entada africana (Fabaceae) are traditionally used in Bamun (a western tribe of Cameroon) traditional medicine for the treatment of liver related diseases. In this study, the synergistic hepatoprotective effect of respective active fractions of the plants were investigated against paracetamol-induced toxicity in primary cultures of rat hepatocytes. Methods Paracetamol conferred hepatocyte toxicity, as determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay, alanine aminotransferase (ALT), superoxide dismutase (SOD), catalase (CAT) activities, malondialdehyde (MDA) and glutathione (GSH) content assays. The crude extracts were fractionated by flash chromatography and fractions were tested for hepato-(protective and curative) activities. The most active fractions of both plants were tested individually, and in combination based on their respective half effective concentration (EC50). Results The methylene chloride/methanol fractions of K. grandifoliola (75:25 v/v) (KgF25) and E. africana (90:10 v/v) (EaF10) were found to be the most hepato-protective with EC50 values of 10.30 ± 1.66 μg/ml and 13.47 ± 2.06 μg/ml respectively, comparable with that of silymarin (13.71 ± 3.87 μg/ml). These fractions and their combination significantly (P <0.05) improved cell viability, inhibited ALT leakage and MDA formation, and restored cellular CAT, SOD activities and GSH content. The combination was more effective in restoring biochemical parameters with coefficients of drugs interaction (CDI) less than 1. Conclusion These findings demonstrate that the active fractions have synergistic action in the protection of rat hepatocytes against paracetamol-induced damage and suggest that their hepatoprotective properties may be maximized by using them in combination.
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Hannoun Z, Steichen C, Dianat N, Weber A, Dubart-Kupperschmitt A. The potential of induced pluripotent stem cell derived hepatocytes. J Hepatol 2016; 65:182-199. [PMID: 26916529 DOI: 10.1016/j.jhep.2016.02.025] [Citation(s) in RCA: 70] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/25/2015] [Revised: 01/12/2016] [Accepted: 02/09/2016] [Indexed: 12/21/2022]
Abstract
Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers.
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Affiliation(s)
- Zara Hannoun
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94807, France; UMR_S1193, Université Paris-Sud, Hôpital Paul Brousse, Villejuif F-94800, France; Département hospitalo-universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Clara Steichen
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94807, France; UMR_S1193, Université Paris-Sud, Hôpital Paul Brousse, Villejuif F-94800, France; Département hospitalo-universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Noushin Dianat
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94807, France; UMR_S1193, Université Paris-Sud, Hôpital Paul Brousse, Villejuif F-94800, France; Département hospitalo-universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Anne Weber
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94807, France; UMR_S1193, Université Paris-Sud, Hôpital Paul Brousse, Villejuif F-94800, France; Département hospitalo-universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France
| | - Anne Dubart-Kupperschmitt
- INSERM U1193, Hôpital Paul Brousse, Villejuif F-94807, France; UMR_S1193, Université Paris-Sud, Hôpital Paul Brousse, Villejuif F-94800, France; Département hospitalo-universitaire Hepatinov, Hôpital Paul Brousse, Villejuif F-94807, France.
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Yanguas SC, Cogliati B, Willebrords J, Maes M, Colle I, van den Bossche B, de Oliveira CPMS, Andraus W, Alves VAF, Leclercq I, Vinken M. Experimental models of liver fibrosis. Arch Toxicol 2016; 90:1025-1048. [PMID: 26047667 PMCID: PMC4705434 DOI: 10.1007/s00204-015-1543-4] [Citation(s) in RCA: 238] [Impact Index Per Article: 26.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2015] [Accepted: 05/28/2015] [Indexed: 02/08/2023]
Abstract
Hepatic fibrosis is a wound healing response to insults and as such affects the entire world population. In industrialized countries, the main causes of liver fibrosis include alcohol abuse, chronic hepatitis virus infection and non-alcoholic steatohepatitis. A central event in liver fibrosis is the activation of hepatic stellate cells, which is triggered by a plethora of signaling pathways. Liver fibrosis can progress into more severe stages, known as cirrhosis, when liver acini are substituted by nodules, and further to hepatocellular carcinoma. Considerable efforts are currently devoted to liver fibrosis research, not only with the goal of further elucidating the molecular mechanisms that drive this disease, but equally in view of establishing effective diagnostic and therapeutic strategies. The present paper provides a state-of-the-art overview of in vivo and in vitro models used in the field of experimental liver fibrosis research.
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Affiliation(s)
- Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo, Brazil
| | - Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
| | - Isabelle Colle
- Department of Hepato-Gastroenterology, Algemeen Stedelijk Ziekenhuis, Aalst, Belgium
| | - Bert van den Bossche
- Department of Abdominal Surgery and Hepato-Pancreatico-Biliary Surgery, Algemeen Stedelijk Ziekenhuis, Aalst, Belgium
| | | | - Wellington Andraus
- Laboratory of Medical Investigation, Department of Pathology, University of São Paulo School of Medicine, São Paulo, Brazil
| | | | - Isabelle Leclercq
- Laboratoire d’Hépato-Gastro-Entérologie, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain, Brussels, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Brussels, Belgium
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Sumii T, Nakano Y, Abe T, Nakashima K, Sera T, Kudo S. The Effect of Nitric Oxide on Ammonia Decomposition in Co-cultures of Hepatocytes and Hepatic Stellate Cells. In Vitro Cell Dev Biol Anim 2016; 52:625-31. [PMID: 26896067 DOI: 10.1007/s11626-016-9999-y] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2015] [Accepted: 01/05/2016] [Indexed: 01/22/2023]
Abstract
Hepatic functions, such as albumin secretion and ammonia metabolism, are upregulated in response to hepatocyte growth factor (HGF) produced by hepatic stellate cells (HSC), as well as nitric oxide (NO) produced by endothelial cells under shear stress. However, the simultaneous effect of HSC and NO has not been previously investigated in a tri-co-culture model containing hepatocytes with HSC and endothelial cells under shear stress. We hypothesized that NO inhibits HGF production from HSC. To test this idea, we constructed a mono-culture model of hepatocytes and a co-culture model of hepatocytes and HSC and measured ammonia decomposition and HGF production in each model under NO load. Ammonia decomposition was significantly higher in the co-culture model under 0 ppm NO load, but no significant increase was observed under NO load. In the co-culture model, HGF was produced at 1.0 ng/mL under 0 ppm NO load and 0.3 ng/mL under NO load. Ammonia decomposition was increased by 1.0 ng/mL HGF, but not by 0.3 ng/mL HGF. These results indicated that NO inhibits HGF production from HSC; consequently, the effects of NO and co-culture with HSC cannot improve hepatic function simultaneously. Instead, the simultaneous effect of 1.0 ng/mL HGF and NO may further enhance hepatic function in vitro.
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Affiliation(s)
- Tateki Sumii
- Department of Mechanical Engineering, Graduate school of Mechanical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan.
| | - Yohei Nakano
- Department of Mechanical Engineering, Graduate school of Mechanical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan
| | - Takuma Abe
- Department of Mechanical Engineering, Graduate school of Mechanical Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan
| | - Kazuhiro Nakashima
- Department of Mechanical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan
| | - Toshihiro Sera
- Department of Mechanical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan
| | - Susumu Kudo
- Department of Mechanical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan
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Barbero-Becerra VJ, Giraudi PJ, Chávez-Tapia NC, Uribe M, Tiribelli C, Rosso N. The interplay between hepatic stellate cells and hepatocytes in an in vitro model of NASH. Toxicol In Vitro 2015; 29:1753-1758. [PMID: 26187275 DOI: 10.1016/j.tiv.2015.07.010] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2015] [Revised: 06/10/2015] [Accepted: 07/13/2015] [Indexed: 01/28/2023]
Abstract
BACKGROUND & AIM A complex interplay exists between hepatocytes and hepatic stellate cells (HSC) in hepatic fibrogenesis. The activation of HSCs after liver injury leads to production of extracellular matrix (ECM). Co-culture models could be useful to mimic the liver microenvironment. This study evaluates the effect of free fatty acids (FFA) on HSC cells and the interplay with hepatocytes via both soluble-mediator and cell-cell contact. METHODS The human hepatocyte cell line (HuH7) and HSC cells (LX2) were exposed to FFA for 24 h in 3 different experimental set-ups: (A) monoculture of HSC; (B) Transwell® system (effect of soluble mediators); and (C) Simultaneous Co-Culture (SCC) (cell-to-cell connections). Intracellular FFA accumulation was assessed qualitatively (microscopy) and quantitatively (flow cytometry); the activation of HSC (alpha smooth muscle actin, α-SMA) expression of ECM components were quantified by RT-PCR. RESULTS FFA exposure induces intracellular fat accumulation in all the experimental set-up but the expression of α-SMA was significantly increased only in SCC. On the contrary, the expression of ECM was substantially decreased in the transwell® system. CONCLUSIONS The HSC activation is independent of FFA accumulation but requires cell-to-cell interaction with hepatocyte. On the contrary, the gene regulation of ECM components seems to occur through paracrine mediators.
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Affiliation(s)
- Varenka J Barbero-Becerra
- Liver Research Unit, Medica Sur Clinic & Foundation, Mexico City, Puente de Piedra 150, Col. Toriello Guerra, Tlalpan, C.P. 14050 Mexico City, Mexico; Centro Studi Fegato (CSF) - Liver Research Center, Fondazione Italiana Fegato, Bldg Q AREA Science Park, Basovizza Campus SS 14 km 163.5, 34149 Trieste, Italy.
| | - Pablo J Giraudi
- Centro Studi Fegato (CSF) - Liver Research Center, Fondazione Italiana Fegato, Bldg Q AREA Science Park, Basovizza Campus SS 14 km 163.5, 34149 Trieste, Italy.
| | - Norberto C Chávez-Tapia
- Liver Research Unit, Medica Sur Clinic & Foundation, Mexico City, Puente de Piedra 150, Col. Toriello Guerra, Tlalpan, C.P. 14050 Mexico City, Mexico.
| | - Misael Uribe
- Liver Research Unit, Medica Sur Clinic & Foundation, Mexico City, Puente de Piedra 150, Col. Toriello Guerra, Tlalpan, C.P. 14050 Mexico City, Mexico.
| | - Claudio Tiribelli
- Centro Studi Fegato (CSF) - Liver Research Center, Fondazione Italiana Fegato, Bldg Q AREA Science Park, Basovizza Campus SS 14 km 163.5, 34149 Trieste, Italy; Department of Medical Sciences, University of Trieste, 34100 Trieste, Italy.
| | - Natalia Rosso
- Centro Studi Fegato (CSF) - Liver Research Center, Fondazione Italiana Fegato, Bldg Q AREA Science Park, Basovizza Campus SS 14 km 163.5, 34149 Trieste, Italy.
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El Taghdouini A, Sørensen AL, Reiner AH, Coll M, Verhulst S, Mannaerts I, Øie CI, Smedsrød B, Najimi M, Sokal E, Luttun A, Sancho-Bru P, Collas P, van Grunsven LA. Genome-wide analysis of DNA methylation and gene expression patterns in purified, uncultured human liver cells and activated hepatic stellate cells. Oncotarget 2015; 6:26729-45. [PMID: 26353929 PMCID: PMC4694948 DOI: 10.18632/oncotarget.4925] [Citation(s) in RCA: 51] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2015] [Accepted: 08/20/2015] [Indexed: 12/15/2022] Open
Abstract
BACKGROUND & AIMS Liver fibrogenesis - scarring of the liver that can lead to cirrhosis and liver cancer - is characterized by hepatocyte impairment, capillarization of liver sinusoidal endothelial cells (LSECs) and hepatic stellate cell (HSC) activation. To date, the molecular determinants of a healthy human liver cell phenotype remain largely uncharacterized. Here, we assess the transcriptome and the genome-wide promoter methylome specific for purified, non-cultured human hepatocytes, LSECs and HSCs, and investigate the nature of epigenetic changes accompanying transcriptional changes associated with activation of HSCs. MATERIAL AND METHODS Gene expression profile and promoter methylome of purified, uncultured human liver cells and culture-activated HSCs were respectively determined using Affymetrix HG-U219 genechips and by methylated DNA immunoprecipitation coupled to promoter array hybridization. Histone modification patterns were assessed at the single-gene level by chromatin immunoprecipitation and quantitative PCR. RESULTS We unveil a DNA-methylation-based epigenetic relationship between hepatocytes, LSECs and HSCs despite their distinct ontogeny. We show that liver cell type-specific DNA methylation targets early developmental and differentiation-associated functions. Integrative analysis of promoter methylome and transcriptome reveals partial concordance between DNA methylation and transcriptional changes associated with human HSC activation. Further, we identify concordant histone methylation and acetylation changes in the promoter and putative novel enhancer elements of genes involved in liver fibrosis. CONCLUSIONS Our study provides the first epigenetic blueprint of three distinct freshly isolated, human hepatic cell types and of epigenetic changes elicited upon HSC activation.
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Affiliation(s)
- Adil El Taghdouini
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Anita L. Sørensen
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Andrew H. Reiner
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
| | - Mar Coll
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Stefaan Verhulst
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Inge Mannaerts
- Liver Cell Biology Lab, Vrije Universiteit Brussel (VUB), Brussels, Belgium
| | - Cristina I. Øie
- Department of Medical Biology, Vascular Biology Research Group, UiT, The Arctic University of Norway, Tromsø, Norway
| | - Bård Smedsrød
- Department of Medical Biology, Vascular Biology Research Group, UiT, The Arctic University of Norway, Tromsø, Norway
| | - Mustapha Najimi
- Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium
| | - Etienne Sokal
- Université Catholique de Louvain, Institut de Recherche Expérimentale et Clinique (IREC), Laboratory of Pediatric Hepatology and Cell Therapy, Brussels, Belgium
| | - Aernout Luttun
- Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium
| | - Pau Sancho-Bru
- Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain
| | - Philippe Collas
- Department of Molecular medicine, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, Oslo, Norway
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Willebrords J, Pereira IVA, Maes M, Crespo Yanguas S, Colle I, Van Den Bossche B, Da Silva TC, de Oliveira CPMS, Andraus W, Alves VA, Cogliati B, Vinken M. Strategies, models and biomarkers in experimental non-alcoholic fatty liver disease research. Prog Lipid Res 2015; 59:106-125. [PMID: 26073454 PMCID: PMC4596006 DOI: 10.1016/j.plipres.2015.05.002] [Citation(s) in RCA: 131] [Impact Index Per Article: 13.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2015] [Revised: 05/13/2015] [Accepted: 05/13/2015] [Indexed: 12/12/2022]
Abstract
Non-alcoholic fatty liver disease encompasses a spectrum of liver diseases, including simple steatosis, steatohepatitis, liver fibrosis and cirrhosis and hepatocellular carcinoma. Non-alcoholic fatty liver disease is currently the most dominant chronic liver disease in Western countries due to the fact that hepatic steatosis is associated with insulin resistance, type 2 diabetes mellitus, obesity, metabolic syndrome and drug-induced injury. A variety of chemicals, mainly drugs, and diets is known to cause hepatic steatosis in humans and rodents. Experimental non-alcoholic fatty liver disease models rely on the application of a diet or the administration of drugs to laboratory animals or the exposure of hepatic cell lines to these drugs. More recently, genetically modified rodents or zebrafish have been introduced as non-alcoholic fatty liver disease models. Considerable interest now lies in the discovery and development of novel non-invasive biomarkers of non-alcoholic fatty liver disease, with specific focus on hepatic steatosis. Experimental diagnostic biomarkers of non-alcoholic fatty liver disease, such as (epi)genetic parameters and '-omics'-based read-outs are still in their infancy, but show great promise. In this paper, the array of tools and models for the study of liver steatosis is discussed. Furthermore, the current state-of-art regarding experimental biomarkers such as epigenetic, genetic, transcriptomic, proteomic and metabonomic biomarkers will be reviewed.
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Affiliation(s)
- Joost Willebrords
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
| | - Isabel Veloso Alves Pereira
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, Brazil.
| | - Michaël Maes
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
| | - Isabelle Colle
- Department of Hepatology and Gastroenterology, Algemeen Stedelijk Ziekenhuis Campus Aalst, Merestraat 80, 9300 Aalst, Belgium.
| | - Bert Van Den Bossche
- Department of Abdominal Surgery and Hepato-Pancreatico-Biliary Surgery, Algemeen Stedelijk Ziekenhuis Campus Aalst, Merestraat 80, 9300 Aalst, Belgium.
| | - Tereza Cristina Da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, Brazil.
| | | | - Wellington Andraus
- Department of Gastroenterology, University of São Paulo School of Medicine, Av. Dr. Arnaldo, 455, São Paulo, Brazil.
| | - Venâncio Avancini Alves
- Laboratory of Medical Investigation, Department of Pathology, University of São Paulo School of Medicine, Av. Dr. Arnaldo, 455, São Paulo, Brazil.
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87, São Paulo, Brazil.
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
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Usta OB, McCarty WJ, Bale S, Hegde M, Jindal R, Bhushan A, Golberg I, Yarmush ML. Microengineered cell and tissue systems for drug screening and toxicology applications: Evolution of in-vitro liver technologies. TECHNOLOGY 2015; 3:1-26. [PMID: 26167518 PMCID: PMC4494128 DOI: 10.1142/s2339547815300012] [Citation(s) in RCA: 37] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/17/2023]
Abstract
The liver performs many key functions, the most prominent of which is serving as the metabolic hub of the body. For this reason, the liver is the focal point of many investigations aimed at understanding an organism's toxicological response to endogenous and exogenous challenges. Because so many drug failures have involved direct liver toxicity or other organ toxicity from liver generated metabolites, the pharmaceutical industry has constantly sought superior, predictive in-vitro models that can more quickly and efficiently identify problematic drug candidates before they incur major development costs, and certainly before they are released to the public. In this broad review, we present a survey and critical comparison of in-vitro liver technologies along a broad spectrum, but focus on the current renewed push to develop "organs-on-a-chip". One prominent set of conclusions from this review is that while a large body of recent work has steered the field towards an ever more comprehensive understanding of what is needed, the field remains in great need of several key advances, including establishment of standard characterization methods, enhanced technologies that mimic the in-vivo cellular environment, and better computational approaches to bridge the gap between the in-vitro and in-vivo results.
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Affiliation(s)
- O B Usta
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - W J McCarty
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - S Bale
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - M Hegde
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - R Jindal
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - A Bhushan
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - I Golberg
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA
| | - M L Yarmush
- Center for Engineering in Medicine at Massachusetts General Hospital, Harvard Medical School and Shriners Hospital for Children, 51 Blossom St., Boston, MA 02114, USA ; Department of Biomedical Engineering, Rutgers University, 599 Taylor Rd., Piscataway, NJ 08854, USA
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A multi-organ chip co-culture of neurospheres and liver equivalents for long-term substance testing. J Biotechnol 2015; 205:36-46. [PMID: 25678136 DOI: 10.1016/j.jbiotec.2015.02.002] [Citation(s) in RCA: 109] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2014] [Revised: 01/23/2015] [Accepted: 02/02/2015] [Indexed: 12/12/2022]
Abstract
Current in vitro and animal tests for drug development are failing to emulate the systemic organ complexity of the human body and, therefore, often do not accurately predict drug toxicity, leading to high attrition rates in clinical studies (Paul et al., 2010). The phylogenetic distance between humans and laboratory animals is enormous, this affects the transferability of animal data on the efficacy of neuroprotective drugs. Therefore, many neuroprotective treatments that have shown promise in animals have not been successful when transferred to humans (Dragunow, 2008; Gibbons and Dragunow, 2010). We present a multi-organ chip capable of maintaining 3D tissues derived from various cell sources in a combined media circuit which bridges the gap in systemic and human tests. A steady state co-culture of human artificial liver microtissues and human neurospheres exposed to fluid flow over two weeks in the multi-organ chip has successfully proven its long-term performance. Daily lactate dehydrogenase activity measurements of the medium and immunofluorescence end-point staining proved the viability of the tissues and the maintenance of differentiated cellular phenotypes. Moreover, the lactate production and glucose consumption values of the tissues cultured indicated that a stable steady-state was achieved after 6 days of co-cultivation. The neurospheres remained differentiated neurons over the two-week cultivation in the multi-organ chip, proven by qPCR and immunofluorescence of the neuronal markers βIII-tubulin and microtubule-associated protein-2. Additionally, a two-week toxicity assay with a repeated substance exposure to the neurotoxic 2,5-hexanedione in two different concentrations induced high apoptosis within the neurospheres and liver microtissues, as shown by a strong increase of lactate dehydrogenase activity in the medium. The principal finding of the exposure of the co-culture to 2,5-hexanedione was that not only toxicity profiles of two different doses could be discriminated, but also that the co-cultures were more sensitive to the substance compared to respective single-tissue cultures in the multi-organ-chip. Thus, we provide here a new in vitro tool which might be utilized to predict the safety and efficacy of substances in clinical studies more accurately in the future.
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Green CJ, Pramfalk C, Morten KJ, Hodson L. From whole body to cellular models of hepatic triglyceride metabolism: man has got to know his limitations. Am J Physiol Endocrinol Metab 2015; 308:E1-20. [PMID: 25352434 PMCID: PMC4281685 DOI: 10.1152/ajpendo.00192.2014] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
The liver is a main metabolic organ in the human body and carries out a vital role in lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases, encompassing a spectrum of conditions from simple fatty liver (hepatic steatosis) through to cirrhosis. Although obesity is a known risk factor for hepatic steatosis, it remains unclear what factor(s) is/are responsible for the primary event leading to retention of intrahepatocellular fat. Studying hepatic processes and the etiology and progression of disease in vivo in humans is challenging, not least as NAFLD may take years to develop. We present here a review of experimental models and approaches that have been used to assess liver triglyceride metabolism and discuss their usefulness in helping to understand the aetiology and development of NAFLD.
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Affiliation(s)
- Charlotte J Green
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford United Kingdom; and
| | - Camilla Pramfalk
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford United Kingdom; and
| | - Karl J Morten
- Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
| | - Leanne Hodson
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Churchill Hospital, Oxford United Kingdom; and
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Abstract
In vitro systems are required to evaluate potential liver fibrogenic effects of drugs and compounds during drug development and toxicity screening, respectively. Upon liver injury or toxicity, hepatic stellate cells are activated, thereby acquiring a myofibroblastic phenotype and participating in extracellular matrix deposition and liver fibrosis. The most widely used in vitro models to investigate liver fibrogenesis are primary cultures of hepatic stellate cells, which can be isolated from healthy human livers. Currently, there are no effective methods to maintain hepatic stellate cells in vitro in a quiescent phenotype. Therefore, when cells are plated, they spontaneously become activated in few days. Most in vitro studies in this area have been performed with monocultures of hepatic stellate cells in order to assess the direct effects of a given factor on hepatic stellate cell activation or the induction of inflammatory and fibrogenic responses. In this chapter, focus is put on basic protocols to isolate hepatic stellate cells from human tissue and to maintain them in culture as well as on common in vitro assays to evaluate their response to profibrogenic factors.
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Affiliation(s)
- Luis Perea
- Laboratory of Liver Fibrosis, Institut d'Investigacions Biomèdiques, August Pi i Sunyer (IDIBAPS), Rossello 149-153, Barcelona, 08036, Spain
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Giraudi PJ, Becerra VJB, Marin V, Chavez-Tapia NC, Tiribelli C, Rosso N. The importance of the interaction between hepatocyte and hepatic stellate cells in fibrogenesis induced by fatty accumulation. Exp Mol Pathol 2014; 98:85-92. [PMID: 25533546 DOI: 10.1016/j.yexmp.2014.12.006] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/27/2014] [Accepted: 12/18/2014] [Indexed: 12/25/2022]
Abstract
BACKGROUND & AIMS Non-alcoholic fatty liver disease is characterized by an initial accumulation of triglycerides that can progress to non-alcoholic steatohepatitis, which can ultimately evolve to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells play a key role in liver fibrogenesis by an increased activation and an altered profile of genes involved in the turnover of extracellular matrix components. To reproduce in-vitro the functional cell connections observed in vivo it is essential to consider cell-to-cell proximity and interaction. The aim of this study was to determine the response to free fatty acids in a simultaneous co-culture model of hepatocytes and hepatic stellate cells. METHODS Simultaneous co-culture model and monoculture of each cell type (control) were exposed to FFA for 24 up to 144 h. Quantification of steatosis; stellate cell activation; assessment of fibrogenic response; expression and activity of metalloproteinases as well as collagen biosynthesis were evaluated. RESULTS Free fatty acids induced comparable steatosis in simultaneous co-culture and monoculture. However, the activation of the stellate cells assessed by alpha-smooth muscle actin expression is greater when cells were in close contact. Furthermore, a time-dependent increment of tissue inhibitor metalloproteinase-2 protein was observed, which was inversely correlated with protein expression and activity of matrix-metalloproteinases, suggesting enhanced collagen biosynthesis. This behavior was absent in cell monoculture. CONCLUSIONS These data indicate that cell-to-cell proximity between hepatocytes and stellate cells is necessary for the initiation of the fibrotic process.
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Affiliation(s)
- Pablo J Giraudi
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy
| | - Varenka J Barbero Becerra
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy; Liver Research Unit, Médica Sur Clinic & Foundation, Puente de Piedra 150, Col. Toriello Guerra, Tlalpan, C.P. 14050 Mexico City, Mexico
| | - Veronica Marin
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy
| | - Norberto C Chavez-Tapia
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy; Liver Research Unit, Médica Sur Clinic & Foundation, Puente de Piedra 150, Col. Toriello Guerra, Tlalpan, C.P. 14050 Mexico City, Mexico
| | - Claudio Tiribelli
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy; Department of Medical Sciences, University of Trieste, 34100 Trieste, Italy
| | - Natalia Rosso
- Fondazione Italiana Fegato, Centro Studi Fegato, Area Science Park Basovizza Bldg. Q SS14 Km 163.5, 34149 Trieste, Italy.
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Fisher CP, Kierzek AM, Plant NJ, Moore JB. Systems biology approaches for studying the pathogenesis of non-alcoholic fatty liver disease. World J Gastroenterol 2014; 20:15070-15078. [PMID: 25386055 PMCID: PMC4223240 DOI: 10.3748/wjg.v20.i41.15070] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/05/2013] [Accepted: 03/13/2014] [Indexed: 02/06/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD) is a progressive disease of increasing public health concern. In western populations the disease has an estimated prevalence of 20%-40%, rising to 70%-90% in obese and type II diabetic individuals. Simplistically, NAFLD is the macroscopic accumulation of lipid in the liver, and is viewed as the hepatic manifestation of the metabolic syndrome. However, the molecular mechanisms mediating both the initial development of steatosis and its progression through non-alcoholic steatohepatitis to debilitating and potentially fatal fibrosis and cirrhosis are only partially understood. Despite increased research in this field, the development of non-invasive clinical diagnostic tools and the discovery of novel therapeutic targets has been frustratingly slow. We note that, to date, NAFLD research has been dominated by in vivo experiments in animal models and human clinical studies. Systems biology tools and novel computational simulation techniques allow the study of large-scale metabolic networks and the impact of their dysregulation on health. Here we review current systems biology tools and discuss the benefits to their application to the study of NAFLD. We propose that a systems approach utilising novel in silico modelling and simulation techniques is key to a more comprehensive, better targeted NAFLD research strategy. Such an approach will accelerate the progress of research and vital translation into clinic.
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Gómez-Lechón MJ, Tolosa L, Conde I, Donato MT. Competency of different cell models to predict human hepatotoxic drugs. Expert Opin Drug Metab Toxicol 2014; 10:1553-68. [PMID: 25297626 DOI: 10.1517/17425255.2014.967680] [Citation(s) in RCA: 143] [Impact Index Per Article: 13.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
INTRODUCTION The liver is the most important target for drug-induced toxicity. This vulnerability results from functional liver features and its role in the metabolic elimination of most drugs. Drug-induced liver injury is a significant leading cause of acute, chronic liver disease and an important safety issue when developing new drugs. AREAS COVERED This review describes the advantages and limitations of hepatic cell-based models for early safety risk assessment during drug development. These models include hepatocytes cultured as monolayer, collagen-sandwich; emerging complex 3D configuration; liver-derived cell lines; stem cell-derived hepatocytes. EXPERT OPINION In vitro toxicity assays performed in hepatocytes or hepatoma cell lines can potentially provide rapid and cost-effective early feedback to identify toxic candidates for compound prioritization. However, their capacity to predict hepatotoxicity depends critically on cells' functional performance. In an attempt to improve and prolong functional properties of cultured cells, different strategies to recreate the in vivo hepatocyte environment have been explored. 3D cultures, co-cultures of hepatocytes with other cell types and microfluidic devices seem highly promising for toxicological studies. Moreover, hepatocytes derived from human pluripotent stem cells are emerging cell-based systems that may provide a stable source of hepatocytes to reliably screen metabolism and toxicity of candidate compounds.
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Affiliation(s)
- M José Gómez-Lechón
- Unidad de Hepatología Experimental Instituto de Investigación Sanitaria La Fe (IIS LA Fe) , Torre A Avda. Fernando Abril Martorell 106, 46026 Valencia , Spain +34 961246619 ;
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Rosso N, Chavez-Tapia NC, Tiribelli C, Bellentani S. Translational approaches: From fatty liver to non-alcoholic steatohepatitis. World J Gastroenterol 2014; 20:9038-9049. [PMID: 25083077 PMCID: PMC4112858 DOI: 10.3748/wjg.v20.i27.9038] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/21/2013] [Revised: 12/04/2013] [Accepted: 04/23/2014] [Indexed: 02/06/2023] Open
Abstract
Over the past few decades, non-alcoholic fatty liver disease (NAFLD) has become one, if not the most common, cause of chronic liver disease affecting both adults and children. The increasing number of cases at an early age is the most worrying aspect of this pathology, since it provides more time for its evolution. The spectrum of this disease ranges from liver steatosis to steatohepatitis, fibrosis and in some cases, hepatocellular carcinoma. NAFLD may not always be considered a benign disease and hepatologists must be cautious in the presence of fatty liver. This should prompt the use of the available experimental models to understand better the pathogenesis and to develop a rational treatment of a disease that is dangerously increasing. In spite of the growing efforts, the pathogenesis of NAFLD is still poorly understood. In the present article we review the most relevant hypotheses and evidence that account for the progression of NAFLD to non-alcoholic steatohepatitis (NASH) and fibrosis. The available in vitro and in vivo experimental models of NASH are discussed and revised in terms of their validity in translational studies. These studies must be aimed at the discovery of the still unknown triggers or mediators that induce the progression of hepatic inflammation, apoptosis and fibrosis.
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44
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An in vitro liver model consisting of endothelial vascular networks surrounded by human hepatoma cell lines allows for improved hepatitis B virus replication. J Biosci Bioeng 2014; 118:107-11. [DOI: 10.1016/j.jbiosc.2013.12.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2013] [Revised: 12/13/2013] [Accepted: 12/16/2013] [Indexed: 12/12/2022]
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46
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Lee SA, No DY, Kang E, Ju J, Kim DS, Lee SH. Spheroid-based three-dimensional liver-on-a-chip to investigate hepatocyte-hepatic stellate cell interactions and flow effects. LAB ON A CHIP 2013; 13:3529-37. [PMID: 23657720 DOI: 10.1039/c3lc50197c] [Citation(s) in RCA: 206] [Impact Index Per Article: 17.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/20/2023]
Abstract
We have developed a three-dimensional (3D) liver-on-a-chip to investigate the interaction of hepatocytes and hepatic stellate cells (HSCs) in which primary 3D hepatocyte spheroids and HSCs are co-cultured without direct cell-cell contact. Here, we show that the 3D liver chip offers substantial advantages for the formation and harvesting of spheroids. The most important feature of this liver chip is that it enables continuous flow of medium to the cells through osmotic pumping, and thus requires only minimal handling and no external power source. We also demonstrate that flow assists the formation and long-term maintenance of spheroids. Additionally, we quantitatively and qualitatively investigated the paracrine effects of HSCs, demonstrating that HSCs assist in the maintenance of hepatocyte spheroids and play an important role in the formation of tight cell-cell contacts, thereby improving liver-specific function. Spheroids derived from co-cultures exhibited improved albumin and urea secretion rates compared to mono-cultured spheroids after 9 days. Immunostaining for cytochrome P450 revealed that the enzymatic activity of spheroids co-cultured for 8 days was greater than that of mono-cultured spheroids. These results indicate that this system has the potential for further development as a unique model for studying cellular interactions or as a tool that can be incorporated into other models aimed at creating hepatic structure and prolonging hepatocyte function in culture.
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Affiliation(s)
- Seung-A Lee
- Department of Biomedical Engineering, Korea University, Seoul, Republic of Korea
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Samluk A, Zakrzewska KE, Pluta KD. Generation of fluorescently labeled cell lines, C3A hepatoma cells, and human adult skin fibroblasts to study coculture models. Artif Organs 2013; 37:E123-30. [PMID: 23581829 DOI: 10.1111/aor.12064] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Hepatic/nonhepatic cell cocultures are widely used in studies on the role of homo- and heterotypic interactions in liver physiology and pathophysiology. In this article, for the first time, establishment of the coculture model employing hepatoma C3A cells and human skin fibroblasts, stably expressing fluorescent markers, is described. Suitability of the model in studying coculture conditions using fluorescence microscopy and flow cytometry was examined. C3A cells spontaneously formed island-like growth patterns surrounded by fibroblasts. The "islands" size and resulting intensity of the homo- and heterotypic interactions can easily be tuned by applying various plated cells ratios. We examined the capability of the hepatoma cells to produce albumin in hepatic/nonhepatic cell cocultures. The enzyme-linked immunosorbent assay (ELISA) tests showed that greater number of fibroblasts in coculture, resulting in smaller sizes of hepatoma "islands," and thus, a larger heterotypic interface, promoted higher albumin synthesis. The use of fluorescently labeled cells in flow cytometry measurements enabled us to separately gate two cell populations and to evaluate protein expression only in/on cells of interest. Flow cytometry confirmed ELISA results indicating the highest albumin production in hepatoma cells cocultured with the greatest number of fibroblasts and the inhibited protein synthesis in coculture with osteosarcoma cells.
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Affiliation(s)
- Anna Samluk
- Department of Hybrid Microbiosystem Engineering, Nalecz Institute of Biocybernetics and Biomedical Engineering, PAS, Warsaw, Poland
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Soldatow VY, Lecluyse EL, Griffith LG, Rusyn I. In vitro models for liver toxicity testing. Toxicol Res (Camb) 2012; 2:23-39. [PMID: 23495363 DOI: 10.1039/c2tx20051a] [Citation(s) in RCA: 320] [Impact Index Per Article: 24.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022] Open
Abstract
Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system.
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Affiliation(s)
- Valerie Y Soldatow
- Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599, USA
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Dash A, Blackman BR, Wamhoff BR. Organotypic systems in drug metabolism and toxicity: challenges and opportunities. Expert Opin Drug Metab Toxicol 2012; 8:999-1014. [DOI: 10.1517/17425255.2012.693161] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
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Barakat O, Abbasi S, Rodriguez G, Rios J, Wood RP, Ozaki C, Holley LS, Gauthier PK. Use of decellularized porcine liver for engineering humanized liver organ. J Surg Res 2011; 173:e11-25. [PMID: 22099595 DOI: 10.1016/j.jss.2011.09.033] [Citation(s) in RCA: 168] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2011] [Revised: 09/13/2011] [Accepted: 09/15/2011] [Indexed: 12/11/2022]
Abstract
BACKGROUND New bioartificial liver devices are needed to supplement the limited supply of organ donors available for patients with end-stage liver disease. Here, we report the results of a pilot study aimed at developing a humanized porcine liver by transplanting second trimester human fetal hepatocytes (Hfh) co-cultured with fetal stellate cells (Hfsc) into the decellularized matrix of a porcine liver. MATERIAL AND METHODS Ischemic livers were removed from 19 Yorkshire swine. Liver decellularization was achieved by an anionic detergent (SDS). The decellularized matrix of three separate porcine liver matrices was seeded with 3.5 × 10(8) and 1 × 10(9) of Hfsc and Hfh, respectively, and perfused for 3, 7, and 13 d. The metabolic and synthetic activities of the engrafted cells were assessed during and after perfusion. RESULTS Immunohistologic examination of the decellularized matrix showed removal of nuclear materials with intact architecture and preserved extracellular matrix (ECM) proteins. During perfusion of the recellularized matrices, measurement of metabolic parameters (i.e., oxygen concentration, glucose consumption, and lactate and urea production) indicated active metabolism. The average human albumin concentration was 29.48 ± 7.4 μg/mL. Immunohistochemical analysis revealed cell differentiation into mature hepatocytes. Moreover, 40% of the engrafted cells were actively proliferating, and less than 30% of cells were apoptotic. CONCLUSION We showed that our decellularization protocol successfully removed the cellular components of porcine livers while preserving the native architecture and most ECM protein. We also demonstrated the ability of the decellularized matrix to support and induce phenotypic maturation of engrafted Hfh in a continuously perfused system.
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Affiliation(s)
- Omar Barakat
- Department of Hepatobiliary and Transplantation Surgery, St. Luke's Episcopal Hospital, Houston, Texas 77030, USA.
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