|
1
|
Tai YC, Yin WR, Cheng KY, Chou XY, Lin YH, Hsu SH, Hou YT. Self-Healable Hydrogel for Regression of Liver Fibrosis. Biotechnol Bioeng 2025; 122:1496-1511. [PMID: 40051386 DOI: 10.1002/bit.28966] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2024] [Revised: 12/23/2024] [Accepted: 02/24/2025] [Indexed: 05/13/2025]
Abstract
Liver fibrosis is considered as a wound healing process in the presence of chronic hepatic injury. A hydrogel (CPDP) based on chitosan-phenol that undergoes fast gelling and owns self-healing and injectable properties was investigated for the effect on regression of liver fibrosis. For the purpose, we established both in vitro and in vivo liver fibrosis models and implanted CPDP hydrogel into the injured liver. The CPDP hydrogel not only provided a suitable microenvironment for hepatocyte spheroids, but also demonstrated a potential for the hepatocyte spheroid-embedded system to mimic the liver tissue in vitro. Furthermore, the urea synthesis of injured hepatocytes cultured on hepatocyte spheroid-embedded CPDP hydrogel was 1.12 times higher than that on hepatocyte spheroid-embedded collagen hydrogel after 7 days of culture, indicating that CPDP hydrogel effectively rescued hepatic function in the injured hepatocytes. Moreover, the hepatic injury was alleviated with improved hepatic function in the liver fibrosis model in vivo. A reduction of approximately 28% in serum AST/ALT ratios and a 70% decrease in the fibrotic area suggested the regression of liver fibrosis after 2 weeks of CPDP hydrogel administration. These findings suggest that CPDP hydrogel holds promise for applications in liver tissue engineering.
Collapse
Affiliation(s)
- Yu-Chai Tai
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
| | - Wei-Rong Yin
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
| | - Kai-Yi Cheng
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
| | - Xin-Yu Chou
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
| | - Yong-Heng Lin
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
| | - Shan-Hui Hsu
- Institute of Polymer Science and Engineering, National Taiwan University, Taipei, Taiwan
| | - Yung-Te Hou
- Department of Biomechatronics Engineering, National Taiwan University, Taipei, Taiwan
- Animal Resource Center, National Taiwan University, Taipei, Taiwan
| |
Collapse
|
|
2
|
Zhou Y, Zhong Y, Lauschke VM. Evaluating the synergistic use of advanced liver models and AI for the prediction of drug-induced liver injury. Expert Opin Drug Metab Toxicol 2025; 21:563-577. [PMID: 39893552 DOI: 10.1080/17425255.2025.2461484] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/28/2024] [Accepted: 01/29/2025] [Indexed: 02/04/2025]
Abstract
INTRODUCTION Drug-induced liver injury (DILI) is a leading cause of acute liver failure. Hepatotoxicity typically occurs only in a subset of individuals after prolonged exposure and constitutes a major risk factor for the termination of drug development projects. AREAS COVERED We provide an overview of available human liver models for DILI research and discuss how they have been used to aid in early risk assessments and to mitigate the risk of project closures due to DILI in clinical stages. We summarize the different data that can be provided by such models and illustrate how these diverse data types can be interfaced with machine learning strategies to improve predictions of liver safety liabilities. EXPERT OPINION Advanced human liver models closely mimic human liver phenotypes and functions for many weeks, allowing for the recapitulation of hepatotoxicity events in vitro. Integration of the biochemical, histological, and toxicogenomic output data from these models with physicochemical compound properties using different machine learning architectures holds promise to enhance preclinical DILI predictions. However, to realize this aim, it is important to benchmark the available liver models on test sets of DILI positive and negative compounds and to carefully annotate and share the resulting data.
Collapse
Affiliation(s)
- Yitian Zhou
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
| | - Yi Zhong
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
- Pharmaceutical Informatics Institute, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang, China
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
- Center for Molecular Medicine, Karolinska Institutet and University Hospital, Stockholm, Sweden
- Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany
- University of Tübingen, Tübingen, Germany
- Department of Pharmacy, The Second Xiangya Hospital, Central South University, Changsha, China
| |
Collapse
|
|
3
|
Arez F, Preiss L, Gal IR, Rebelo SP, Badolo L, Brito C, Spangenberg T, Alves PM. Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems. SLAS DISCOVERY : ADVANCING LIFE SCIENCES R & D 2025; 31:100210. [PMID: 39805483 DOI: 10.1016/j.slasd.2025.100210] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/19/2024] [Revised: 11/08/2024] [Accepted: 01/06/2025] [Indexed: 01/16/2025]
Abstract
Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability in vitro in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications. Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.
Collapse
Affiliation(s)
- Francisca Arez
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12 2781-901 Oeiras, Portugal; ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Av. Republica, Oeiras 2780-157, Portugal
| | - Lena Preiss
- Discovery and Development Technologies, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Isabella Ramella Gal
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12 2781-901 Oeiras, Portugal; ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Av. Republica, Oeiras 2780-157, Portugal
| | - Sofia P Rebelo
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12 2781-901 Oeiras, Portugal
| | - Lassina Badolo
- Discovery and Development Technologies, the healthcare business of Merck KGaA, Darmstadt, Germany
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12 2781-901 Oeiras, Portugal; ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Av. Republica, Oeiras 2780-157, Portugal
| | - Thomas Spangenberg
- Global Health R&D of Merck Healthcare, Ares Trading S.A., (a subsidiary of Merck KGaA, Darmstadt, Germany), Route de Crassier 1, Eysins 1262, Switzerland
| | - Paula M Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12 2781-901 Oeiras, Portugal; ITQB NOVA, Instituto de Tecnologia Química e Biológica António Xavier, Av. Republica, Oeiras 2780-157, Portugal.
| |
Collapse
|
|
4
|
Uehara MK, Bual R, Shafiq M, Yoshida K, Ijima H. Proposal for a non-adhesive single-cell culture technology for primary hepatocytes. Cytotechnology 2025; 77:30. [PMID: 39744312 PMCID: PMC11685352 DOI: 10.1007/s10616-024-00696-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2024] [Accepted: 12/24/2024] [Indexed: 03/08/2025] Open
Abstract
Primary hepatocytes (PHs) are indispensable for studying liver function, drug screening, and regenerative medicine. However, freshly isolated PHs only survive for a few hours in non-adherent suspension culture. This study proposes treatment with PEG-GRGDS, a polymer-peptide conjugate comprising polyethylene glycol (PEG) and the pentapeptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS), to sustain the viability of dispersed single PHs under non-adherent conditions. As a proof of concept, PHs treated with the PEG-GRGDS molecule were cultured in a microarray with single-cell-sized microwells. After 24 h of culture, enhanced cell survival was confirmed via esterase activity alongside activity for Cytochrome P450 1A1 (CYP1A1). Some liver-specific functionalities, including albumin secretion, were observed in the treated PHs. Additionally, it was observed that the length of the PEG-chain in the conjugates influenced the maintenance of single-cell dispersion and the levels of polymerized actin in the cells. These findings suggest that treatment with a polymer-peptide like PEG-GRGDS might provide a promising platform for the short-term culture of non-adherent single PHs. Supplementary Information The online version contains supplementary material available at 10.1007/s10616-024-00696-1.
Collapse
Affiliation(s)
- Mario K. Uehara
- Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395 Japan
| | - Ronald Bual
- Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395 Japan
- Department of Chemical Engineering & Technology, College of Engineering, Mindanao State University-Iligan Institute of Technology, A. Bonifacio Avenue, Tibanga, 9200 Iligan City, Philippines
| | - Muhammad Shafiq
- Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395 Japan
- Innovation Center of NanoMedicine (iCONM), Kawasaki Institute of Industrial Promotion, Kawasaki-Ku, Kawasaki, 210-0821 Japan
| | - Kozue Yoshida
- Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395 Japan
| | - Hiroyuki Ijima
- Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka, 819-0395 Japan
| |
Collapse
|
|
5
|
Wani SI, Mir TA, Nakamura M, Tsuchiya T, Alzhrani A, Iwanaga S, Arai K, Alshehri EA, Shamma T, Obeid DA, Chinnappan R, Assiri AM, Yaqinuddin A, Vashist YK, Broering DC. A review of current state-of-the-art materiobiology and technological approaches for liver tissue engineering. BIOPRINTING 2024; 42:e00355. [DOI: 10.1016/j.bprint.2024.e00355] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
|
|
6
|
Zhu X, Trehan R, Xie C. Primary liver cancer organoids and their application to research and therapy. JOURNAL OF THE NATIONAL CANCER CENTER 2024; 4:195-202. [PMID: 39281720 PMCID: PMC11401492 DOI: 10.1016/j.jncc.2024.06.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2024] [Revised: 05/02/2024] [Accepted: 06/13/2024] [Indexed: 09/18/2024] Open
Abstract
Primary liver cancer is a leading cause of death worldwide. To create advanced treatments for primary liver cancer, studies have utilized models such as 2D cell culture and in vivo animal models. Recent developments in cancer organoids have created the possibility for 3D in vitro cultures that recapitulates the cancer cell structure and operation as well as the tumor microenvironment (TME). However, before organoids can be directly translated to clinical use, tissue processing and culture medium must be standardized with unified protocols to decrease variability in results. Herein, we present the wide variety of published methodologies used to derive liver cancer organoids from patient tumor tissues. Additionally, we summarize validation methodologies for organoids in terms of marker expression levels with immunohistochemistry as well as the presence of mutations and variants through RNA-sequencing. Primary liver cancer organoids have exciting applications allowing for faster drug testing at a larger scale. Primary liver cancer organoids also assisit in uncovering new mechanisms. Through the coculture of different immune cells and cancer organoids, organoids are now better able to recapitulate the liver cancer TME. In addition, it further aids in the investigation of drug development and drug resistance. Lastly, we posit that the usage of liver cancer organoids in animal models provides researchers a methodology to overcome the current limitations of culture systems.
Collapse
Affiliation(s)
- Xiaobin Zhu
- Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA
| | - Rajiv Trehan
- Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA
| | - Changqing Xie
- Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, USA
| |
Collapse
|
|
7
|
Jamshed L, Jamshed S, Frank RA, Hewitt LM, Thomas PJ, Holloway AC. Assessing Receptor Activation in 2D and 3D Cultured Hepatocytes: Responses to a Single Compound and a Complex Mixture. TOXICS 2024; 12:631. [PMID: 39330559 PMCID: PMC11436198 DOI: 10.3390/toxics12090631] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/29/2024] [Revised: 08/07/2024] [Accepted: 08/22/2024] [Indexed: 09/28/2024]
Abstract
Responding to global standards and legislative updates in Canada, including Bill S-5 (2023), toxicity testing is shifting towards more ethical, in vitro methods. Traditional two-dimensional (2D) monolayer cell cultures, limited in replicating the complex in vivo environment, have prompted the development of more relevant three-dimensional (3D) spheroidal hepatocyte cultures. This study introduces the first 3D spheroid model for McA-RH7777 cells, assessing xenobiotic receptor activation, cellular signaling, and toxicity against dexamethasone and naphthenic acid (NA)-fraction components; NAFCs. Our findings reveal that 3D McA-RH7777 spheroids demonstrate enhanced sensitivity and more uniform dose-response patterns in gene expression related to xenobiotic metabolism (AhR and PPAR) for both single compounds and complex mixtures. Specifically, 3D cultures showed significant gene expression changes upon dexamethasone exposure and exhibited varying degrees of sensitivity and resistance to the apoptotic effects induced by NAFCs, in comparison to 2D cultures. The optimization of 3D culture conditions enhances the model's physiological relevance and enables the identification of genomic signatures under varied exposures. This study highlights the potential of 3D spheroid cultures in providing a more accurate representation of the liver's microenvironment and advancing our understanding of cellular mechanisms in toxicity testing.
Collapse
Affiliation(s)
- Laiba Jamshed
- Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON L8S 4L8, Canada; (L.J.); (S.J.)
| | - Shanza Jamshed
- Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON L8S 4L8, Canada; (L.J.); (S.J.)
| | - Richard A. Frank
- Water Science and Technology Directorate, Environment and Climate Change Canada, Burlington, ON L7S 1A1, Canada; (R.A.F.); (L.M.H.)
| | - L. Mark Hewitt
- Water Science and Technology Directorate, Environment and Climate Change Canada, Burlington, ON L7S 1A1, Canada; (R.A.F.); (L.M.H.)
| | - Philippe J. Thomas
- Environment and Climate Change Canada, National Wildlife Research Centre, Ottawa, ON K1S 5B6, Canada;
| | - Alison C. Holloway
- Department of Obstetrics and Gynecology, McMaster University, Hamilton, ON L8S 4L8, Canada; (L.J.); (S.J.)
| |
Collapse
|
|
8
|
Liu Y, Zhou Y, Ahodantin J, Jin Y, Zhu J, Sun Z, Wu X, Su L, Yang Y. Generation and characterization of mature hepatocyte organoids for liver metabolic studies. J Cell Sci 2024; 137:jcs261961. [PMID: 38700490 PMCID: PMC11166457 DOI: 10.1242/jcs.261961] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 04/12/2024] [Indexed: 05/05/2024] Open
Abstract
Hepatocyte organoids (HOs) generated in vitro are powerful tools for liver regeneration. However, previously reported HOs have mostly been fetal in nature with low expression levels of metabolic genes characteristic of adult liver functions, hampering their application in studies of metabolic regulation and therapeutic testing for liver disorders. Here, we report development of novel culture conditions that combine optimized levels of triiodothyronine (T3) with the removal of growth factors to enable successful generation of mature hepatocyte organoids (MHOs) of both mouse and human origin with metabolic functions characteristic of adult livers. We show that the MHOs can be used to study various metabolic functions including bile and urea production, zonal metabolic gene expression, and metabolic alterations in both alcoholic liver disease and non-alcoholic fatty liver disease, as well as hepatocyte proliferation, injury and cell fate changes. Notably, MHOs derived from human fetal hepatocytes also show improved hepatitis B virus infection. Therefore, these MHOs provide a powerful in vitro model for studies of human liver physiology and diseases. The human MHOs are potentially also a robust research tool for therapeutic development.
Collapse
Affiliation(s)
- Yuchen Liu
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Yaxing Zhou
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - James Ahodantin
- Division of Virology, Pathogenesis, and Cancer, Institute of Human Virology, Departments of Pharmacology and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Yu Jin
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Juanjuan Zhu
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
| | - Zhonghe Sun
- Cancer Research Technology Program, Frederick National Laboratory for Cancer, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA
| | - Xiaolin Wu
- Cancer Research Technology Program, Frederick National Laboratory for Cancer, Leidos Biomedical Research, Inc., Frederick, MD 21702, USA
| | - Lishan Su
- Division of Virology, Pathogenesis, and Cancer, Institute of Human Virology, Departments of Pharmacology and Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA
| | - Yingzi Yang
- Department of Developmental Biology, Harvard School of Dental Medicine, 188 Longwood Ave, Boston, MA 02115, USA
- Harvard Stem Cell Institute, Program in Gastrointestinal Malignancies, Dana-Farber/Harvard Cancer Center, 188 Longwood Ave, Boston, MA 02115, USA
- Program in Gastrointestinal Malignancies, Dana-Farber/Harvard Cancer Center, 188 Longwood Ave, Boston, MA 02115, USA
| |
Collapse
|
|
9
|
Sharma K, Dey S, Karmakar R, Rengan AK. A comprehensive review of 3D cancer models for drug screening and translational research. CANCER INNOVATION 2024; 3:e102. [PMID: 38948533 PMCID: PMC11212324 DOI: 10.1002/cai2.102] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/23/2023] [Revised: 09/06/2023] [Accepted: 09/11/2023] [Indexed: 07/02/2024]
Abstract
The 3D cancer models fill the discovery gap of 2D cancer models and play an important role in cancer research. In addition to cancer cells, a range of other factors include the stroma, density and composition of extracellular matrix, cancer-associated immune cells (e.g., cancer-associated fibroblasts cancer cell-stroma interactions and subsequent interactions, and a number of other factors (e.g., tumor vasculature and tumor-like microenvironment in vivo) has been widely ignored in the 2D concept of culture. Despite this knowledge, the continued use of monolayer cell culture methods has led to the failure of a series of clinical trials. This review discusses the immense importance of tumor microenvironment (TME) recapitulation in cancer research, prioritizing the individual roles of TME elements in cancer histopathology. The TME provided by the 3D model fulfills the requirements of in vivo spatiotemporal arrangement, components, and is helpful in analyzing various different aspects of drug sensitivity in preclinical and clinical trials, some of which are discussed here. Furthermore, it discusses models for the co-assembly of different TME elements in vitro and focuses on their synergistic function and responsiveness as tumors. Furthermore, this review broadly describes of a handful of recently developed 3D models whose main focus is limited to drug development and their screening and/or the impact of this approach in preclinical and translational research.
Collapse
Affiliation(s)
- Karthikey Sharma
- Department of Biomedical EngineeringIndian Institute of Technology (IIT)HyderabadIndia
| | - Sreenath Dey
- Department of Biomedical EngineeringIndian Institute of Technology (IIT)HyderabadIndia
| | - Rounik Karmakar
- Department of Biomedical EngineeringIndian Institute of Technology (IIT)HyderabadIndia
| | - Aravind Kumar Rengan
- Department of Biomedical EngineeringIndian Institute of Technology (IIT)HyderabadIndia
| |
Collapse
|
|
10
|
Iqbal W, Wang Y, Sun P, Zhou X. Modeling Liver Development and Disease in a Dish. Int J Mol Sci 2023; 24:15921. [PMID: 37958904 PMCID: PMC10650907 DOI: 10.3390/ijms242115921] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Revised: 10/19/2023] [Accepted: 10/23/2023] [Indexed: 11/15/2023] Open
Abstract
Historically, biological research has relied primarily on animal models. While this led to the understanding of numerous human biological processes, inherent species-specific differences make it difficult to answer certain liver-related developmental and disease-specific questions. The advent of 3D organoid models that are either derived from pluripotent stem cells or generated from healthy or diseased tissue-derived stem cells have made it possible to recapitulate the biological aspects of human organs. Organoid technology has been instrumental in understanding the disease mechanism and complements animal models. This review underscores the advances in organoid technology and specifically how liver organoids are used to better understand human-specific biological processes in development and disease. We also discuss advances made in the application of organoid models in drug screening and personalized medicine.
Collapse
Affiliation(s)
- Waqas Iqbal
- Stem Cell Research Center, Shantou University Medical College, Shantou 515041, China; (W.I.); (Y.W.); (P.S.)
- Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
| | - Yaru Wang
- Stem Cell Research Center, Shantou University Medical College, Shantou 515041, China; (W.I.); (Y.W.); (P.S.)
- Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
| | - Pingnan Sun
- Stem Cell Research Center, Shantou University Medical College, Shantou 515041, China; (W.I.); (Y.W.); (P.S.)
- Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
| | - Xiaoling Zhou
- Stem Cell Research Center, Shantou University Medical College, Shantou 515041, China; (W.I.); (Y.W.); (P.S.)
- Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, China
- Guangdong Provincial Key Laboratory of Infectious Diseases and Molecular Immunopathology, Shantou University Medical College, Shantou 515041, China
| |
Collapse
|
|
11
|
KHAWAR MUHAMMADBABAR, WANG YAJUN, MAJEED ANEEQA, AFZAL ALI, HANEEF KABEER, SUN HAIBO. Mini-organs with big impact: Organoids in liver cancer studies. Oncol Res 2023; 31:677-688. [PMID: 37547759 PMCID: PMC10398413 DOI: 10.32604/or.2023.029718] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2023] [Accepted: 06/14/2023] [Indexed: 08/08/2023] Open
Abstract
Hepatocellular carcinoma, the most common primary liver cancer and a leading cause of death, is a difficult disease to treat due to its heterogeneous nature. Traditional models, such as 2D culture and patient-derived xenografts, have not proven effective. However, the development of 3D culture techniques, such as organoids, which can mimic the tumor microenvironment (TME) and preserve heterogeneity and pathophysiological properties of tumor cells, offers new opportunities for treatment and research. Organoids also have the potential for biomarker detection and personalized medication, as well as genome editing using CRISPR/Cas9 to study the behavior of certain genes and therapeutic interventions. This review explores to-the-date development of organoids with a focus on TME modeling in 3D organoid cultures. Further, it discusses gene editing using CRISPR/Cas9 in organoids, the challenges faced, and the prospects in the field of organoids.
Collapse
Affiliation(s)
- MUHAMMAD BABAR KHAWAR
- Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Experimental and Translational Non-Coding RNA Research, Yangzhou, China
- Applied Molecular Biology and Biomedicine Lab, Department of Zoology, University of Narowal, Narowal, Pakistan
| | - YAJUN WANG
- Department of Oncology, Haian Hospital of Traditional Chinese Medicine, Haian, China
| | - ANEEQA MAJEED
- Applied Molecular Biology and Biomedicine Lab, Department of Zoology, University of Narowal, Narowal, Pakistan
| | - ALI AFZAL
- Molecular Medicine and Cancer Therapeutics Lab, Department of Zoology, Faculty of Sciences Technology, University of Central Punjab, Lahore, Pakistan
| | - KABEER HANEEF
- Chinese Institute for Brain Research (CIBR), Beijing, China
| | - HAIBO SUN
- Institute of Translational Medicine, Medical College, Yangzhou University, Yangzhou, China
- Jiangsu Key Laboratory of Experimental and Translational Non-Coding RNA Research, Yangzhou, China
| |
Collapse
|
|
12
|
Alves RF, Lopes C, Rocha E, Madureira TV. A Step Forward in the Characterization of Primary Brown Trout Hepatocytic Spheroids as Experimental Models. Animals (Basel) 2023; 13:2277. [PMID: 37508054 PMCID: PMC10376616 DOI: 10.3390/ani13142277] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Revised: 07/03/2023] [Accepted: 07/08/2023] [Indexed: 07/30/2023] Open
Abstract
Mammal hepatocyte spheroids have been investigated as alternative experimental models in several contexts, since three-dimensional (3D) systems have shown the potential to mimic in vivo scenarios. The description of fish hepatocyte 3D models is still minimal. This study intends to further characterize brown trout primary hepatocyte spheroids at distinct time points up to 25 days in culture. Viability, biometry, histomorphology, and basal expression of a selection of genes (metabolism and detoxification, efflux transport, and estrogenic signalling) were considered. The gene expression of whole liver samples from the same fish donor were evaluated concurrently. After 12 days in culture, the hepatocyte spheroids exhibited biometric and morphological stability. From the 12th to the 20th day in culture, the basal expression levels for most of the selected genes did not vary. The targeted mRNA levels were higher in brown trout liver samples compared to hepatocyte spheroids. Despite that, data supported that this model resembles some in vivo features. As an experimental alternative model, it showed potential to be used in a stable time window that can be exploited for exposure tests to different xenobiotics, namely, estrogenic compounds.
Collapse
Affiliation(s)
- Rodrigo F Alves
- Team of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
- Laboratory of Histology and Embryology, Department of Microscopy, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
| | - Célia Lopes
- Team of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
- Laboratory of Histology and Embryology, Department of Microscopy, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
| | - Eduardo Rocha
- Team of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
- Laboratory of Histology and Embryology, Department of Microscopy, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
| | - Tânia V Madureira
- Team of Animal Morphology and Toxicology, Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
- Laboratory of Histology and Embryology, Department of Microscopy, ICBAS-School of Medicine and Biomedical Sciences, University of Porto, Rua Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
| |
Collapse
|
|
13
|
Liver Organoids, Novel and Promising Modalities for Exploring and Repairing Liver Injury. Stem Cell Rev Rep 2023; 19:345-357. [PMID: 36199007 PMCID: PMC9534590 DOI: 10.1007/s12015-022-10456-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 09/24/2022] [Indexed: 02/07/2023]
Abstract
The past decades have witnessed great advances in organoid technology. Liver is the biggest solid organ, performing multifaceted physiological functions. Nowadays, liver organoids have been applied in many fields including pharmaceutical research, precision medicine and disease models. Compared to traditional 2-dimensional cell line cultures and animal models, liver organoids showed the unique advantages. More importantly, liver organoids can well model the features of the liver and tend to be novel and promising modalities for exploring liver injury, thus finding potential treatment targets and repairing liver injury. In this review, we reviewed the history of the development of liver organoids and summarized the application of liver organoids and recent studies using organoids to explore and further repair the liver injury. These novel modalities could provide new insights about the process of liver injury.
Collapse
|
|
14
|
Jalan-Sakrikar N, Brevini T, Huebert RC, Sampaziotis F. Organoids and regenerative hepatology. Hepatology 2023; 77:305-322. [PMID: 35596930 PMCID: PMC9676408 DOI: 10.1002/hep.32583] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/15/2022] [Revised: 05/13/2022] [Accepted: 05/14/2022] [Indexed: 02/03/2023]
Abstract
The burden of liver diseases is increasing worldwide, with liver transplantation remaining the only treatment option for end-stage liver disease. Regenerative medicine holds great potential as a therapeutic alternative, aiming to repair or replace damaged liver tissue with healthy functional cells. The properties of the cells used are critical for the efficacy of this approach. The advent of liver organoids has not only offered new insights into human physiology and pathophysiology, but also provided an optimal source of cells for regenerative medicine and translational applications. Here, we discuss various historical aspects of 3D organoid culture, how it has been applied to the hepatobiliary system, and how organoid technology intersects with the emerging global field of liver regenerative medicine. We outline the hepatocyte, cholangiocyte, and nonparenchymal organoids systems available and discuss their advantages and limitations for regenerative medicine as well as future directions.
Collapse
Affiliation(s)
- Nidhi Jalan-Sakrikar
- Division of Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, Minnesota, USA
- Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota, USA
| | - Teresa Brevini
- Wellcome-MRC Cambridge Stem Cell Institute, Cambridge, UK
| | - Robert C. Huebert
- Division of Gastroenterology and Hepatology, Mayo Clinic and Foundation, Rochester, Minnesota, USA
- Gastroenterology Research Unit, Mayo Clinic and Foundation, Rochester, Minnesota, USA
| | - Fotios Sampaziotis
- Wellcome-MRC Cambridge Stem Cell Institute, Cambridge, UK
- Department of Medicine, University of Cambridge, Cambridge, UK
- Cambridge Liver Unit, Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK
| |
Collapse
|
|
15
|
Bioengineering Liver Organoids for Diseases Modelling and Transplantation. BIOENGINEERING (BASEL, SWITZERLAND) 2022; 9:bioengineering9120796. [PMID: 36551002 PMCID: PMC9774794 DOI: 10.3390/bioengineering9120796] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Revised: 12/05/2022] [Accepted: 12/08/2022] [Indexed: 12/15/2022]
Abstract
Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.
Collapse
|
|
16
|
Tsujimura M, Kusamori K, Takamura K, Ito T, Kaya T, Shimizu K, Konishi S, Nishikawa M. Quality evaluation of cell spheroids for transplantation by monitoring oxygen consumption using an on-chip electrochemical device. BIOTECHNOLOGY REPORTS (AMSTERDAM, NETHERLANDS) 2022; 36:e00766. [PMID: 36245695 PMCID: PMC9562952 DOI: 10.1016/j.btre.2022.e00766] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 09/19/2022] [Accepted: 10/01/2022] [Indexed: 11/18/2022]
Abstract
Three-dimensional cell spheroids are superior cell-administration form for cell-based therapy which generally exhibit superior functionality and long-term survival after transplantation. Here, we nondestructively measured the oxygen consumption rate of cell spheroids using an on-chip electrochemical device (OECD) and examined whether this rate can be used as a marker to estimate the quality of cell spheroids. Cell spheroids containing NanoLuc luciferase-expressing mouse mesenchymal stem cell line C3H10T1/2 (C3H10T1/2/Nluc) were prepared. Spheroids of high or low quality were prepared by altering the medium change frequency. After transplantation into mice, the high-quality C3H10T1/2/Nluc spheroids exhibited a higher survival rate than the low-quality ones. The oxygen consumption rate of the high-quality C3H10T1/2/Nluc spheroids was maintained at high levels, whereas that of the low-quality spheroids decreased with time. These results indicate that OECD-based measurement of the oxygen consumption rate can be used to estimate the quality of cell spheroids without destructive analysis of the spheroids.
Collapse
Affiliation(s)
- Mari Tsujimura
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
| | - Kosuke Kusamori
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
- Corresponding author.
| | - Kodai Takamura
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
| | - Temmei Ito
- KONICA MINOLTA, INC., No.1 Sakura-machi, Hino-shi, Tokyo, 191-8511, Japan
| | - Takatoshi Kaya
- KONICA MINOLTA, INC., No.1 Sakura-machi, Hino-shi, Tokyo, 191-8511, Japan
| | - Kazunori Shimizu
- Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Aichi, 464-8603, Japan
| | - Satoshi Konishi
- Department of Mechanical Engineering, Graduate School of Science and Engineering, Ritsumeikan University, 1-1-1 Noji-higashi, Kusatsu, Shiga, 525-8577, Japan
| | - Makiya Nishikawa
- Laboratory of Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba, 278-8510, Japan
| |
Collapse
|
|
17
|
Wolf KJ, Weiss JD, Uzel SGM, Skylar-Scott MA, Lewis JA. Biomanufacturing human tissues via organ building blocks. Cell Stem Cell 2022; 29:667-677. [PMID: 35523137 PMCID: PMC9617289 DOI: 10.1016/j.stem.2022.04.012] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
The construction of human organs on demand remains a tantalizing vision to solve the organ donor shortage. Yet, engineering tissues that recapitulate the cellular and architectural complexity of native organs is a grand challenge. The use of organ building blocks (OBBs) composed of multicellular spheroids, organoids, and assembloids offers an important pathway for creating organ-specific tissues with the desired cellular-to-tissue-level organization. Here, we review the differentiation, maturation, and 3D assembly of OBBs into functional human tissues and, ultimately, organs for therapeutic repair and replacement. We also highlight future challenges and areas of opportunity for this nascent field.
Collapse
Affiliation(s)
- Kayla J Wolf
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA
| | - Jonathan D Weiss
- Department of Bioengineering, Stanford University, 240 Pasteur Drive, Stanford, CA 94304, USA
| | - Sebastien G M Uzel
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA
| | - Mark A Skylar-Scott
- Department of Bioengineering, Stanford University, 240 Pasteur Drive, Stanford, CA 94304, USA; BASE Initiative, Betty Irene Moore Children's Heart Center, Lucile Packard Children's Hospital, Stanford University School of Medicine, Stanford, CA 94304, USA.
| | - Jennifer A Lewis
- Wyss Institute for Biologically Inspired Engineering & John A. Paulson School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02138, USA.
| |
Collapse
|
|
18
|
Hasan MF, Trushina E. Advances in Recapitulating Alzheimer's Disease Phenotypes Using Human Induced Pluripotent Stem Cell-Based In Vitro Models. Brain Sci 2022; 12:552. [PMID: 35624938 PMCID: PMC9138647 DOI: 10.3390/brainsci12050552] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Revised: 04/24/2022] [Accepted: 04/24/2022] [Indexed: 12/12/2022] Open
Abstract
Alzheimer's disease (AD) is an incurable neurodegenerative disorder and the leading cause of death among older individuals. Available treatment strategies only temporarily mitigate symptoms without modifying disease progression. Recent studies revealed the multifaceted neurobiology of AD and shifted the target of drug development. Established animal models of AD are mostly tailored to yield a subset of disease phenotypes, which do not recapitulate the complexity of sporadic late-onset AD, the most common form of the disease. The use of human induced pluripotent stem cells (HiPSCs) offers unique opportunities to fill these gaps. Emerging technology allows the development of disease models that recapitulate a brain-like microenvironment using patient-derived cells. These models retain the individual's unraveled genetic background, yielding clinically relevant disease phenotypes and enabling cost-effective, high-throughput studies for drug discovery. Here, we review the development of various HiPSC-based models to study AD mechanisms and their application in drug discovery.
Collapse
Affiliation(s)
- Md Fayad Hasan
- Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA;
| | - Eugenia Trushina
- Department of Neurology, Mayo Clinic, Rochester, MN 55905, USA;
- Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, MN 55905, USA
| |
Collapse
|
|
19
|
Sun S, Wang J, Yao J, Guo H, Dai J. Transcriptome analysis of 3D primary mouse liver spheroids shows that long-term exposure to hexafluoropropylene oxide trimer acid disrupts hepatic bile acid metabolism. THE SCIENCE OF THE TOTAL ENVIRONMENT 2022; 812:151509. [PMID: 34762948 DOI: 10.1016/j.scitotenv.2021.151509] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 09/02/2021] [Revised: 10/27/2021] [Accepted: 11/03/2021] [Indexed: 06/13/2023]
Abstract
Hexafluoropropylene oxide trimer acid (HFPO-TA), an alternative to perfluorooctanoic acid (PFOA), has been detected in various environmental and human matrices. However, information regarding its toxicity remains limited. Here, we established a three-dimensional (3D) primary mouse liver spheroid model to compare the hepatotoxicity of HFPO-TA and PFOA. The 3D spheroids were repeatedly exposed to 25-, 50-, or 100-μM HFPO-TA and PFOA for 28 d. Compared with the PFOA groups, the HFPO-TA groups showed higher bioaccumulation potential, higher lactate dehydrogenase (LDH) leakage, and lower adenosine triphosphate (ATP), albumin, and urea secretion. Transcriptome analysis identified 1603 and 772 differentially expressed genes in the 100-μM HFPO-TA- and PFOA-treated groups, respectively. Bioinformatics analysis indicated that cholesterol metabolism, bile acid metabolism, and inflammatory response were significantly altered. Exposure to 100-μM HFPO-TA increased triglyceride content but decreased total cholesterol content, while no changes were observed in the 100-μM PFOA-treated group. Total bile acids in the re-polarized 3D spheroids increased significantly after 100-μM HFPO-TA and PFOA treatment, which did not affect bile acid synthesis but inhibited the expression levels of Bsep and Mrp2 related to bile acid transport. Thus, HFPO-TA exhibited more serious hepatotoxicity than PFOA in 3D primary liver spheroids and may not be a safe alternative.
Collapse
Affiliation(s)
- Sujie Sun
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jianshe Wang
- School of Pharmacy, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Yantai University, Yantai 264005, Shandong Province, China
| | - Jingzhi Yao
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Hua Guo
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China
| | - Jiayin Dai
- State Environmental Protection Key Laboratory of Environmental Health Impact Assessment of Emerging Contaminants, School of Environmental Sciences and Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang District, Shanghai 200240, China; Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.
| |
Collapse
|
|
20
|
Youhanna S, Kemas AM, Preiss L, Zhou Y, Shen JX, Cakal SD, Paqualini FS, Goparaju SK, Shafagh RZ, Lind JU, Sellgren CM, Lauschke VM. Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development-Current State-of-the-Art and Future Perspectives. Pharmacol Rev 2022; 74:141-206. [PMID: 35017176 DOI: 10.1124/pharmrev.120.000238] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Accepted: 10/12/2021] [Indexed: 12/11/2022] Open
Abstract
The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.
Collapse
Affiliation(s)
- Sonia Youhanna
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Aurino M Kemas
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Lena Preiss
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Yitian Zhou
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Joanne X Shen
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Selgin D Cakal
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Francesco S Paqualini
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Sravan K Goparaju
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Reza Zandi Shafagh
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Johan Ulrik Lind
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Carl M Sellgren
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| |
Collapse
|
|
21
|
Liao W, Yang W, Xu J, Yan Z, Pan M, Xu X, Zhou S, Zhu Y, Lan J, Zeng M, Han X, Li S, Li Y, Liang K, Gao Y, Peng Q. Therapeutic Potential of CUDC-907 (Fimepinostat) for Hepatocarcinoma Treatment Revealed by Tumor Spheroids-Based Drug Screening. Front Pharmacol 2021; 12:658197. [PMID: 34776939 PMCID: PMC8585736 DOI: 10.3389/fphar.2021.658197] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 09/22/2021] [Indexed: 12/17/2022] Open
Abstract
Background: Cancer is the second leading cause of death globally. However, most of the new anti-cancer agents screened by traditional drug screening methods fail in the clinic because of lack of efficacy. Choosing an appropriate in vitro tumor model is crucial for preclinical drug screening. In this study, we screened anti-hepatocarcinoma (HCC) drugs using a novel spheroid cell culture device. Methods: Four HCC cell lines were three-dimensionally (3D) cultured to screen 19 small molecular agents. 3D-cultured primary HCC cells and a tumor-bearing mouse model were used to verify the candidate anti-hepatocarcinoma agent. Cell function experiments and western blotting were conducted to explore the anti-hepatocarcinoma mechanism of the candidate agent. Results: We found that CUDC-907 can serve as a potent anti-hepatocarcinoma agent. The study data show that CUDC-907 (fimepinostat), a novel dual acting inhibitor of phosphoinositide 3-kinase (PI3K) and histone deacetylase (HDAC), has potent inhibitory effects on HCC cell lines and primary HCC cells in vitro, Animal studies have shown that CUDC-907 can also suppress HCC cells in vivo. Furthermore, we found that CUDC-907 inhibits the PI3K/AKT/mTOR pathway and downregulates the expression of c-Myc, leading to the suppression of HCC cells. Conclusion: Our results suggest that CUDC-907 can be a candidate anti-HCC drug, and the 3D in vitro drug screening method based on our novel spheroid culture device is promising for future drug screening efforts.
Collapse
Affiliation(s)
- Wei Liao
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Wanren Yang
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Jiecheng Xu
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Zhengming Yan
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Mingxin Pan
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Xiaoping Xu
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Shuqin Zhou
- Department of Anesthesiology, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Yu Zhu
- Accurate International Biotechnology Co., Guangzhou, China
| | - Jianqiang Lan
- Accurate International Biotechnology Co., Guangzhou, China
| | - Min Zeng
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Xu Han
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Shao Li
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Yang Li
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Kangyan Liang
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Yi Gao
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China.,State Key Laboratory of Organ Failure Research, Southern Medical University, Guangzhou, China
| | - Qing Peng
- General Surgery Center, Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| |
Collapse
|
|
22
|
Habanjar O, Diab-Assaf M, Caldefie-Chezet F, Delort L. 3D Cell Culture Systems: Tumor Application, Advantages, and Disadvantages. Int J Mol Sci 2021; 22:12200. [PMID: 34830082 PMCID: PMC8618305 DOI: 10.3390/ijms222212200] [Citation(s) in RCA: 243] [Impact Index Per Article: 60.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2021] [Revised: 11/05/2021] [Accepted: 11/07/2021] [Indexed: 01/09/2023] Open
Abstract
The traditional two-dimensional (2D) in vitro cell culture system (on a flat support) has long been used in cancer research. However, this system cannot be fully translated into clinical trials to ideally represent physiological conditions. This culture cannot mimic the natural tumor microenvironment due to the lack of cellular communication (cell-cell) and interaction (cell-cell and cell-matrix). To overcome these limitations, three-dimensional (3D) culture systems are increasingly developed in research and have become essential for tumor research, tissue engineering, and basic biology research. 3D culture has received much attention in the field of biomedicine due to its ability to mimic tissue structure and function. The 3D matrix presents a highly dynamic framework where its components are deposited, degraded, or modified to delineate functions and provide a platform where cells attach to perform their specific functions, including adhesion, proliferation, communication, and apoptosis. So far, various types of models belong to this culture: either the culture based on natural or synthetic adherent matrices used to design 3D scaffolds as biomaterials to form a 3D matrix or based on non-adherent and/or matrix-free matrices to form the spheroids. In this review, we first summarize a comparison between 2D and 3D cultures. Then, we focus on the different components of the natural extracellular matrix that can be used as supports in 3D culture. Then we detail different types of natural supports such as matrigel, hydrogels, hard supports, and different synthetic strategies of 3D matrices such as lyophilization, electrospiding, stereolithography, microfluid by citing the advantages and disadvantages of each of them. Finally, we summarize the different methods of generating normal and tumor spheroids, citing their respective advantages and disadvantages in order to obtain an ideal 3D model (matrix) that retains the following characteristics: better biocompatibility, good mechanical properties corresponding to the tumor tissue, degradability, controllable microstructure and chemical components like the tumor tissue, favorable nutrient exchange and easy separation of the cells from the matrix.
Collapse
Affiliation(s)
- Ola Habanjar
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
| | - Mona Diab-Assaf
- Equipe Tumorigénèse Pharmacologie Moléculaire et Anticancéreuse, Faculté des Sciences II, Université Libanaise Fanar, Beyrouth 1500, Liban;
| | - Florence Caldefie-Chezet
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
| | - Laetitia Delort
- Université Clermont-Auvergne, INRAE, UNH, Unité de Nutrition Humaine, CRNH-Auvergne, 63000 Clermont-Ferrand, France; (O.H.); (F.C.-C.)
| |
Collapse
|
|
23
|
Peng WC, Kraaier LJ, Kluiver TA. Hepatocyte organoids and cell transplantation: What the future holds. Exp Mol Med 2021; 53:1512-1528. [PMID: 34663941 PMCID: PMC8568948 DOI: 10.1038/s12276-021-00579-x] [Citation(s) in RCA: 20] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2020] [Revised: 01/11/2021] [Accepted: 01/14/2021] [Indexed: 12/29/2022] Open
Abstract
Historically, primary hepatocytes have been difficult to expand or maintain in vitro. In this review, we will focus on recent advances in establishing hepatocyte organoids and their potential applications in regenerative medicine. First, we provide a background on the renewal of hepatocytes in the homeostatic as well as the injured liver. Next, we describe strategies for establishing primary hepatocyte organoids derived from either adult or fetal liver based on insights from signaling pathways regulating hepatocyte renewal in vivo. The characteristics of these organoids will be described herein. Notably, hepatocyte organoids can adopt either a proliferative or a metabolic state, depending on the culture conditions. Furthermore, the metabolic gene expression profile can be modulated based on the principles that govern liver zonation. Finally, we discuss the suitability of cell replacement therapy to treat different types of liver diseases and the current state of cell transplantation of in vitro-expanded hepatocytes in mouse models. In addition, we provide insights into how the regenerative microenvironment in the injured host liver may facilitate donor hepatocyte repopulation. In summary, transplantation of in vitro-expanded hepatocytes holds great potential for large-scale clinical application to treat liver diseases.
Collapse
Affiliation(s)
- Weng Chuan Peng
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands.
| | - Lianne J Kraaier
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands
| | - Thomas A Kluiver
- Princess Máxima Center for Pediatric Oncology, Heidelberglaan 25, 3584 CS, Utrecht, The Netherlands
| |
Collapse
|
|
24
|
Shibuya K, Watanabe M, Goto R, Zaitsu M, Ganchiku Y, Taketomi A. The Efficacy of the Hepatocyte Spheroids for Hepatocyte Transplantation. Cell Transplant 2021; 30:9636897211000014. [PMID: 33900126 PMCID: PMC8085376 DOI: 10.1177/09636897211000014] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
The safety and short-term efficacy of hepatocyte transplantation (HCTx) have been widely proven. However, issues such as reduced viability and/or function of hepatocytes, insufficient engraftment, and lack of a long-term effect have to be overcome for widespread application of HCTx. In this study, we evaluated hepatocyte spheroids (HSs), formed by self-aggregation of hepatocytes, as an alternative to hepatocytes in single-cell suspension. Hepatocytes were isolated from C57BL/6 J mice liver using a three-step collagenase perfusion technique and HSs were formed by the hanging drop method. After the spheroids formation, the HSs showed significantly higher mRNA expression of albumin, ornithine transcarbamylase, glucose-6-phosphate, alpha-1-antitrypsin, low density lipoprotein receptor, coagulation factors, and apolipoprotein E (ApoE) than 2 dimensional (2D)-cultured hepatocytes (p < 0.05). Albumin production by HSs was significantly higher than that by 2D-cultured hepatocytes (9.5 ± 2.5 vs 3.5 ± 1.8 μg/dL, p < 0.05). The HSs, but not single hepatocytes, maintained viability and albumin mRNA expression in suspension (92.0 ± 2.8% and 1.03 ± 0.09 at 6 h). HSs (3.6 × 106 cells) or isolated hepatocytes (fSH, 3.6 × 106 cells) were transplanted into the liver of ApoE knockout (KO-/-) mice via the portal vein. Following transplantation, serum ApoE concentration (ng/mL) of HS-transplanted mice (1w: 63.1 ± 56.7, 4w: 17.0 ± 10.9) was higher than that of fSH-transplanted mice (1 w: 33.4 ± 13.0, 4w: 13.7 ± 9.6). In both groups, the mRNA levels of pro-inflammatory cytokines (IL-6, IL-1β, TNF-α, MCP-1, and MIP-1β) were upregulated in the liver following transplantation; however, no significant differences were observed. Pathologically, transplanted HSs were observed as flat cell clusters in contact with the portal vein wall on day 7. Additionally, ApoE positive cells were observed in the liver parenchyma distant from the portal vein on day 28. Our results indicate that HS is a promising alternative to single hepatocytes and can be applied for HCTx.
Collapse
Affiliation(s)
- Kazuaki Shibuya
- Department of Gastroenterological surgery I, 12810Hokkaido university graduate school, kita-ku, Sapporo, Japan
| | - Masaaki Watanabe
- Transplant surgery, 163693Hokkaido University Hospital, kita-ku, Sapporo, Japan
| | - Ryoichi Goto
- Department of Gastroenterological surgery I, 12810Hokkaido university graduate school, kita-ku, Sapporo, Japan
| | - Masaaki Zaitsu
- Department of Gastroenterological surgery I, 12810Hokkaido university graduate school, kita-ku, Sapporo, Japan
| | - Yoshikazu Ganchiku
- Department of Gastroenterological surgery I, 12810Hokkaido university graduate school, kita-ku, Sapporo, Japan
| | - Akinobu Taketomi
- Department of Gastroenterological surgery I, 12810Hokkaido university graduate school, kita-ku, Sapporo, Japan
| |
Collapse
|
|
25
|
Ingelman-Sundberg M, Lauschke VM. 3D human liver spheroids for translational pharmacology and toxicology. Basic Clin Pharmacol Toxicol 2021; 130 Suppl 1:5-15. [PMID: 33872466 DOI: 10.1111/bcpt.13587] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Revised: 04/12/2021] [Accepted: 04/13/2021] [Indexed: 12/14/2022]
Abstract
Drug development is a failure-prone endeavour, and more than 85% of drugs fail during clinical development, showcasing that current preclinical systems for compound selection are clearly inadequate. Liver toxicity remains a major reason for safety failures. Furthermore, all efforts to develop pharmacological therapies for a variety of chronic liver diseases, such as non-alcoholic steatohepatitis (NASH) and fibrosis, remain unsuccessful. Considering the time and expense of clinical trials, as well as the substantial burden on patients, new strategies are thus of paramount importance to increase clinical success rates. To this end, human liver spheroids are becoming increasingly utilized as they allow to preserve patient-specific phenotypes and functions for multiple weeks in culture. We here review the recent application of such systems for i) predictive and mechanistic analyses of drug hepatotoxicity, ii) the evaluation of hepatic disposition and metabolite formation of low clearance drugs and iii) the development of drugs for metabolic and infectious liver diseases, including NASH, fibrosis, malaria and viral hepatitis. We envision that with increasing dissemination, liver spheroids might become the new gold standard for such applications in translational pharmacology and toxicology.
Collapse
Affiliation(s)
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| |
Collapse
|
|
26
|
Advanced Multi-Dimensional Cellular Models as Emerging Reality to Reproduce In Vitro the Human Body Complexity. Int J Mol Sci 2021; 22:ijms22031195. [PMID: 33530487 PMCID: PMC7865724 DOI: 10.3390/ijms22031195] [Citation(s) in RCA: 34] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 01/19/2021] [Accepted: 01/22/2021] [Indexed: 02/07/2023] Open
Abstract
A hot topic in biomedical science is the implementation of more predictive in vitro models of human tissues to significantly improve the knowledge of physiological or pathological process, drugs discovery and screening. Bidimensional (2D) culture systems still represent good high-throughput options for basic research. Unfortunately, these systems are not able to recapitulate the in vivo three-dimensional (3D) environment of native tissues, resulting in a poor in vitro–in vivo translation. In addition, intra-species differences limited the use of animal data for predicting human responses, increasing in vivo preclinical failures and ethical concerns. Dealing with these challenges, in vitro 3D technological approaches were recently bioengineered as promising platforms able to closely capture the complexity of in vivo normal/pathological tissues. Potentially, such systems could resemble tissue-specific extracellular matrix (ECM), cell–cell and cell–ECM interactions and specific cell biological responses to mechanical and physical/chemical properties of the matrix. In this context, this review presents the state of the art of the most advanced progresses of the last years. A special attention to the emerging technologies for the development of human 3D disease-relevant and physiological models, varying from cell self-assembly (i.e., multicellular spheroids and organoids) to the use of biomaterials and microfluidic devices has been given.
Collapse
|
|
27
|
Kang SM, Kim D, Lee JH, Takayama S, Park JY. Engineered Microsystems for Spheroid and Organoid Studies. Adv Healthc Mater 2021; 10:e2001284. [PMID: 33185040 PMCID: PMC7855453 DOI: 10.1002/adhm.202001284] [Citation(s) in RCA: 57] [Impact Index Per Article: 14.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2020] [Revised: 10/01/2020] [Indexed: 01/09/2023]
Abstract
3D in vitro model systems such as spheroids and organoids provide an opportunity to extend the physiological understanding using recapitulated tissues that mimic physiological characteristics of in vivo microenvironments. Unlike 2D systems, 3D in vitro systems can bridge the gap between inadequate 2D cultures and the in vivo environments, providing novel insights on complex physiological mechanisms at various scales of organization, ranging from the cellular, tissue-, to organ-levels. To satisfy the ever-increasing need for highly complex and sophisticated systems, many 3D in vitro models with advanced microengineering techniques have been developed to answer diverse physiological questions. This review summarizes recent advances in engineered microsystems for the development of 3D in vitro model systems. The relationship between the underlying physics behind the microengineering techniques, and their ability to recapitulate distinct 3D cellular structures and functions of diverse types of tissues and organs are highlighted and discussed in detail. A number of 3D in vitro models and their engineering principles are also introduced. Finally, current limitations are summarized, and perspectives for future directions in guiding the development of 3D in vitro model systems using microengineering techniques are provided.
Collapse
Affiliation(s)
- Sung-Min Kang
- Department of Green Chemical Engineering, Sangmyung University, Cheonan, Chungnam, 31066, Republic of Korea
| | - Daehan Kim
- Department of Mechanical Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| | - Ji-Hoon Lee
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, 30332, USA
- The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Shuichi Takayama
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory School of Medicine, Atlanta, GA, 30332, USA
- The Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA, 30332, USA
| | - Joong Yull Park
- Department of Mechanical Engineering, Chung-Ang University, Seoul, 06974, Republic of Korea
| |
Collapse
|
|
28
|
Lee SW, Jung DJ, Jeong GS. Gaining New Biological and Therapeutic Applications into the Liver with 3D In Vitro Liver Models. Tissue Eng Regen Med 2020; 17:731-745. [PMID: 32207030 PMCID: PMC7710770 DOI: 10.1007/s13770-020-00245-9] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2019] [Revised: 02/17/2020] [Accepted: 02/18/2020] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Three-dimensional (3D) cell cultures with architectural and biomechanical properties similar to those of natural tissue have been the focus for generating liver tissue. Microarchitectural organization is believed to be crucial to hepatic function, and 3D cell culture technologies have enabled the construction of tissue-like microenvironments, thereby leading to remarkable progress in vitro models of human tissue and organs. Recently, to recapitulate the 3D architecture of tissues, spheroids and organoids have become widely accepted as new practical tools for 3D organ modeling. Moreover, the combination of bioengineering approach offers the promise to more accurately model the tissue microenvironment of human organs. Indeed, the employment of sophisticated bioengineered liver models show long-term viability and functional enhancements in biochemical parameters and disease-orient outcome. RESULTS Various 3D in vitro liver models have been proposed as a new generation of liver medicine. Likewise, new biomedical engineering approaches and platforms are available to more accurately replicate the in vivo 3D microarchitectures and functions of living organs. This review aims to highlight the recent 3D in vitro liver model systems, including micropatterning, spheroids, and organoids that are either scaffold-based or scaffold-free systems. Finally, we discuss a number of challenges that will need to be addressed moving forward in the field of liver tissue engineering for biomedical applications. CONCLUSION The ongoing development of biomedical engineering holds great promise for generating a 3D biomimetic liver model that recapitulates the physiological and pathological properties of the liver and has biomedical applications.
Collapse
Affiliation(s)
- Sang Woo Lee
- Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, 88 Olympic-Ro 43 Gil, Songpa-Gu, Seoul, 05505, Republic of Korea
| | - Da Jung Jung
- Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, 88 Olympic-Ro 43 Gil, Songpa-Gu, Seoul, 05505, Republic of Korea
| | - Gi Seok Jeong
- Biomedical Engineering Research Center, Asan Institute for Life Sciences, Asan Medical Center, 88 Olympic-Ro 43 Gil, Songpa-Gu, Seoul, 05505, Republic of Korea.
- Department of Convergence Medicine, University of Ulsan College of Medicine, 88 Olympic-Ro 43 Gil, Songpa-Gu, Seoul, 05505, Republic of Korea.
| |
Collapse
|
|
29
|
Nudischer R, Renggli K, Hierlemann A, Roth AB, Bertinetti-Lapatki C. Characterization of a long-term mouse primary liver 3D tissue model recapitulating innate-immune responses and drug-induced liver toxicity. PLoS One 2020; 15:e0235745. [PMID: 32645073 PMCID: PMC7347206 DOI: 10.1371/journal.pone.0235745] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/07/2019] [Accepted: 06/22/2020] [Indexed: 02/07/2023] Open
Abstract
Three-dimensional liver in vitro systems have recently attracted a lot of attention in drug development. These systems help to gain unprecedented insights into drug-induced liver injury (DILI), as they more closely reproduce liver biology, and as drug effects can be studied in isolated and controllable microenvironments. Many groups established human-based in vitro models but so far neglected the animal equivalent, although the availability of both models would be desirable. Animal in vitro models enable back- and forward translation of in vitro and in vivo findings, bridge the gap between rodent in vivo and human in vitro scenarios, and ultimately support the interpretation of data generated with preclinical species and humans. Since mice are often used in drug development and physiologically relevant in vitro systems are lacking, we established, for the first time, a mouse liver model that encompasses primary parenchymal and non-parenchymal cells with preserved viability and functionality over three weeks. Using our three-dimensional liver spheroids, we were able to predict the toxicity of known DILI compounds, demonstrated the interaction cascades between the different cell types and showed evidence of drug-induced steatosis and cholestasis. In summary, our mouse liver spheroids represent a valuable in vitro model that can be applied to study DILI findings, reported from mouse studies, and offers the potential to detect immune-mediated drug-induced liver toxicity.
Collapse
Affiliation(s)
- Ramona Nudischer
- Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland
- * E-mail:
| | - Kasper Renggli
- Bioengineering Laboratory, Department of Biosystems Science and Engineering, ETH Zürich, Zurich, Switzerland
| | - Andreas Hierlemann
- Bioengineering Laboratory, Department of Biosystems Science and Engineering, ETH Zürich, Zurich, Switzerland
| | - Adrian B. Roth
- Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland
| | - Cristina Bertinetti-Lapatki
- Roche Pharmaceutical Research and Early Development, Pharmaceutical Sciences, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland
| |
Collapse
|
|
30
|
Ehrlich A, Duche D, Ouedraogo G, Nahmias Y. Challenges and Opportunities in the Design of Liver-on-Chip Microdevices. Annu Rev Biomed Eng 2020; 21:219-239. [PMID: 31167098 DOI: 10.1146/annurev-bioeng-060418-052305] [Citation(s) in RCA: 78] [Impact Index Per Article: 15.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
The liver is the central hub of xenobiotic metabolism and consequently the organ most prone to cosmetic- and drug-induced toxicity. Failure to detect liver toxicity or to assess compound clearance during product development is a major cause of postmarketing product withdrawal, with disastrous clinical and financial consequences. While small animals are still the preferred model in drug development, the recent ban on animal use in the European Union created a pressing need to develop precise and efficient tools to detect human liver toxicity during cosmetic development. This article includes a brief review of liver development, organization, and function and focuses on the state of the art of long-term cell culture, including hepatocyte cell sources, heterotypic cell-cell interactions, oxygen demands, and culture medium formulation. Finally, the article reviews emerging liver-on-chip devices and discusses the advantages and pitfalls of individual designs. The goal of this review is to provide a framework to design liver-on-chip devices and criteria with which to evaluate this emerging technology.
Collapse
Affiliation(s)
- Avner Ehrlich
- Grass Center for Bioengineering, Benin School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem 91904, Israel
| | - Daniel Duche
- L'Oréal Research and Innovation, Aulnay-sous-Bois 93600, France
| | | | - Yaakov Nahmias
- Grass Center for Bioengineering, Benin School of Computer Science and Engineering, Hebrew University of Jerusalem, Jerusalem 91904, Israel.,Department of Cell and Developmental Biology, Silberman Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel.,Tissue Dynamics Ltd., Jerusalem 91904, Israel
| |
Collapse
|
|
31
|
Teng CL, Chen JY, Chang TL, Hsiao SK, Hsieh YK, Villalobos Gorday K, Cheng YL, Wang J. Design of photocurable, biodegradable scaffolds for liver lobule regeneration via digital light process-additive manufacturing. Biofabrication 2020; 12:035024. [DOI: 10.1088/1758-5090/ab69da] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
|
|
32
|
Shimura R, Suematsu Y, Horiuchi H, Takeoka S, Oshima A, Washio M. Fabrication of thermo-responsive cell-culture membranes with Poly(N-isopropylacrylamide) by electron-beam graft polymerization. Radiat Phys Chem Oxf Engl 1993 2020. [DOI: 10.1016/j.radphyschem.2020.108741] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/25/2022]
|
|
33
|
Zhang X, Jiang T, Chen D, Wang Q, Zhang LW. Three-dimensional liver models: state of the art and their application for hepatotoxicity evaluation. Crit Rev Toxicol 2020; 50:279-309. [DOI: 10.1080/10408444.2020.1756219] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Affiliation(s)
- Xihui Zhang
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, P. R. China
| | - Tianyan Jiang
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, P. R. China
| | - Dandan Chen
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, P. R. China
| | - Qi Wang
- Institute for Control of Chinese Traditional Medicine and Ethnic Medicine, National Institutes for Food and Drug Control (NIFDC), China Food and Drug Administration (CFDA), Beijing, P. R. China
| | - Leshuai W. Zhang
- State Key Laboratory of Radiation Medicine and Protection, School of Radiation Medicine and Protection, School for Radiological and Interdisciplinary Sciences (RAD-X), Collaborative Innovation Center of Radiation Medicine of Jiangsu Higher Education Institutions, Soochow University, Suzhou, P. R. China
| |
Collapse
|
|
34
|
Ruoß M, Vosough M, Königsrainer A, Nadalin S, Wagner S, Sajadian S, Huber D, Heydari Z, Ehnert S, Hengstler JG, Nussler AK. Towards improved hepatocyte cultures: Progress and limitations. Food Chem Toxicol 2020; 138:111188. [PMID: 32045649 DOI: 10.1016/j.fct.2020.111188] [Citation(s) in RCA: 33] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2019] [Revised: 01/31/2020] [Accepted: 02/07/2020] [Indexed: 12/14/2022]
Abstract
Hepatotoxicity is among the most frequent reasons for drug withdrawal from the market. Therefore, there is an urgent need for reliable predictive in vitro tests, which unfailingly identify hepatotoxic drug candidates, reduce drug development time, expenses and the number of test animals. Currently, human hepatocytes represent the gold standard. However, the use of hepatocytes is challenging since the cells are not constantly available and lose their metabolic activity in culture. To solve these problems many different approaches have been developed in the past decades. The aim of this review is to present these approaches and to discuss the possibilities and limitations as well as future opportunities and directions.
Collapse
Affiliation(s)
- Marc Ruoß
- Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Massoud Vosough
- Department of Regenerative Medicine, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Alfred Königsrainer
- Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, Germany
| | - Silvio Nadalin
- Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, Germany
| | - Silvia Wagner
- Department of General, Visceral and Transplant Surgery, University Hospital Tübingen, Tübingen, Germany
| | - Sahar Sajadian
- Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Diana Huber
- Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Zahra Heydari
- Department of Regenerative Medicine, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
| | - Sabrina Ehnert
- Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany
| | - Jan G Hengstler
- Leibniz Research Centre for Working Environment and Human Factors (IfADo), Technical University of Dortmund, Dortmund, Germany
| | - Andreas K Nussler
- Department of Traumatology, Siegfried Weller Institute, Eberhard Karls University Tübingen, Tübingen, Germany.
| |
Collapse
|
|
35
|
Chao C, Ngo Le P, Engelward BP. SpheroidChip: Patterned Agarose Microwell Compartments Harboring HepG2 Spheroids are Compatible with Genotoxicity Testing. ACS Biomater Sci Eng 2020; 6:2427-2439. [PMID: 33145399 DOI: 10.1021/acsbiomaterials.9b01951] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Three-dimensional tissue culture models are emerging as effective alternatives to animal testing. They are especially beneficial for liver toxicity studies, enabling hepatocytes to display improved levels of liver-specific functions. One common model is hepatocyte spheroids, which are spontaneously formed cell aggregates. Techniques for spheroid formation include the use of ultralow attachment plates and the hanging drop method, both of which are technically challenging and relatively low throughput. Here, we describe a simple-to-use platform that improves spheroid production and is compatible with genotoxicity testing by the comet assay. To achieve this, we created a chip containing a microwell array where dozens of spheroids are contained within a single well of a 96-well plate. The microwells are made from agarose, a nontoxic material suitable for cell growth and spheroid formation. HepG2 cells loaded into customizable microwells formed spheroids through agarose-assisted aggregation within one to two days. In addition, the agarose matrix allows the same platform to be used in DNA damage analysis. Specifically, the comet assay enables quantification of DNA breaks based on the increased migration of damaged DNA through agarose during electrophoresis. Here, we developed a modified comet assay and show that intact HepG2 spheroids cultured in microwells can be electrophoresed to reveal the extent of DNA damage following exposure to inflammatory chemicals (H2O2 and SIN-1). With this SpheroidChip analysis method, we detected a dose-dependent increase in DNA damage and observed rapid repair of H2O2-induced DNA damage. In summary, we utilized an agarose microarray to condense what had required an entire 96-well plate into a single well, enabling analysis techniques that were cumbersome or impossible under conditions of a single spheroid per well of a 96-well plate.
Collapse
Affiliation(s)
- Christy Chao
- Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
| | - P Ngo Le
- Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
| | - Bevin P Engelward
- Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA
| |
Collapse
|
|
36
|
Li Y, Wu Q, Yang Z, He Y, Weng C, Gao M, Zhang B, Wang Y, Li L, Chen F, Bu H, Bao J. Heterotopic vascularization and functionalization of implantable bio engineered hepatic tissue alleviates liver injury in rats. Liver Int 2020; 40:712-726. [PMID: 31571356 DOI: 10.1111/liv.14267] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2019] [Revised: 09/20/2019] [Accepted: 09/24/2019] [Indexed: 02/05/2023]
Abstract
BACKGROUND The challenge of using bioengineered liver lies in sustaining the quantity of high-quality hepatocytes and the vasculature for blood perfusion. We characterized the heparinization of a porcine decellularized liver scaffold (DLS) as a carrier to support hepatocyte angiogenesis, thereby developing functional and vascularized hepatic tissue useful to treat liver injury. METHOD The porcine DLS was obtained by the removal of cellular components and then subjected to heparinization by the end-point attachment technique. The heparinized DLSs were recellularized with rat hepatocyte spheroids to construct engineered hepatic tissue. The hepatic tissue was heterotopically implanted in the omentum majus of a rat model with liver injury induced by carbon tetrachloride (CCl4 ). RESULTS Hepatocyte spheroids in the heparinized DLS remained viable for at least 10 weeks in vivo. The entire scaffold was populated with hepatocytes and arranged well. The volume of the heparinized DLS group was expanded over 400-fold. Liver-specific functions such as albumin synthesis, glycogen storage and cytochrome P 3A4 activity were highly expressed in the hepatic tissue. In addition, endothelial cells were recruited, as shown by CD31 staining, and new blood vessels formed, as visualized by fluorescein isothiocyanate-labelled dextran intravital confocal microscopy. The heparinized bioengineered hepatic tissue alleviated CCl4 -induced liver injury by regulating the deposition and degradation of the extracellular matrix. CONCLUSION Primary hepatocyte spheroids survived for an extended time on the heparinized DLS and expanded to generate vascularized and functional bioengineered hepatic tissue that can alleviate liver injury in rats.
Collapse
Affiliation(s)
- Yi Li
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China.,Precision Medicine Key Laboratory, West China Hospital, Sichuan University, Chengdu, China
| | - Qiong Wu
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Zhen Yang
- Core Facility of West China Hospital, Sichuan University, Chengdu, China
| | - Yuting He
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Chengxin Weng
- Department of Vascular Surgery, West China School of Medicine, Sichuan University, Chengdu, China
| | - Mengyu Gao
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Bingqi Zhang
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Yujia Wang
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Li Li
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Fei Chen
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| | - Hong Bu
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China.,Department of Pathology, West China Hospital, Sichuan University, Chengdu, China
| | - Ji Bao
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China
| |
Collapse
|
|
37
|
Salehi SS, Shamloo A, Hannani SK. Microfluidic technologies to engineer mesenchymal stem cell aggregates-applications and benefits. Biophys Rev 2020; 12:123-133. [PMID: 31953794 PMCID: PMC7040154 DOI: 10.1007/s12551-020-00613-8] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2019] [Accepted: 01/07/2020] [Indexed: 02/07/2023] Open
Abstract
Three-dimensional cell culture and the forming multicellular aggregates are superior over traditional monolayer approaches due to better mimicking of in vivo conditions and hence functions of a tissue. A considerable amount of attention has been devoted to devising efficient methods for the rapid formation of uniform-sized multicellular aggregates. Microfluidic technology describes a platform of techniques comprising microchannels to manipulate the small number of reagents with unique properties and capabilities suitable for biological studies. The focus of this review is to highlight recent studies of using microfluidics, especially droplet-based types for the formation, culture, and harvesting of mesenchymal stem cell aggregates and their subsequent application in stem cell biology, tissue engineering, and drug screening. Droplet-based microfluidics can be used to form microgels as carriers for delivering cells and to provide biological cues to the target tissue so as to be minimally invasive. Stem cell-laden microgels with a shape-forming property can be used as smart building blocks by injecting them into the injured tissue thereby constituting the cornerstone of tissue regeneration.
Collapse
Affiliation(s)
| | - Amir Shamloo
- School of Mechanical Engineering, Sharif University of Technology, Tehran, Iran.
| | | |
Collapse
|
|
38
|
Heydari Z, Najimi M, Mirzaei H, Shpichka A, Ruoss M, Farzaneh Z, Montazeri L, Piryaei A, Timashev P, Gramignoli R, Nussler A, Baharvand H, Vosough M. Tissue Engineering in Liver Regenerative Medicine: Insights into Novel Translational Technologies. Cells 2020; 9:304. [PMID: 32012725 PMCID: PMC7072533 DOI: 10.3390/cells9020304] [Citation(s) in RCA: 57] [Impact Index Per Article: 11.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2019] [Revised: 01/17/2020] [Accepted: 01/21/2020] [Indexed: 12/15/2022] Open
Abstract
Organ and tissue shortage are known as a crucially important public health problem as unfortunately a small percentage of patients receive transplants. In the context of emerging regenerative medicine, researchers are trying to regenerate and replace different organs and tissues such as the liver, heart, skin, and kidney. Liver tissue engineering (TE) enables us to reproduce and restore liver functions, fully or partially, which could be used in the treatment of acute or chronic liver disorders and/or generate an appropriate functional organ which can be transplanted or employed as an extracorporeal device. In this regard, a variety of techniques (e.g., fabrication technologies, cell-based technologies, microfluidic systems and, extracorporeal liver devices) could be applied in tissue engineering in liver regenerative medicine. Common TE techniques are based on allocating stem cell-derived hepatocyte-like cells or primary hepatocytes within a three-dimensional structure which leads to the improvement of their survival rate and functional phenotype. Taken together, new findings indicated that developing liver tissue engineering-based techniques could pave the way for better treatment of liver-related disorders. Herein, we summarized novel technologies used in liver regenerative medicine and their future applications in clinical settings.
Collapse
Affiliation(s)
- Zahra Heydari
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran; (Z.H.); (Z.F.)
- Department of Developmental Biology, University of Science and Culture, ACECR, Tehran 1665659911, Iran
| | - Mustapha Najimi
- Laboratory of Pediatric Hepatology and Cell Therapy, Institute of Experimental & Clinical Research, Université Catholique de Louvain, B-1200 Brussels, Belgium;
| | - Hamed Mirzaei
- Research Center for Biochemistry and Nutrition in Metabolic Diseases, Kashan University of Medical Sciences, Kashan 121135879, Iran;
| | - Anastasia Shpichka
- Institute for Regenerative Medicine, Sechenov University, 119146 Moscow, Russia; (A.S.); (P.T.)
| | - Marc Ruoss
- Siegfried Weller Institute for Trauma Research, University of Tübingen, 72076 Tübingen, Germany; (M.R.); (A.N.)
| | - Zahra Farzaneh
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran; (Z.H.); (Z.F.)
| | - Leila Montazeri
- Department of Cell Engineering, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran;
| | - Abbas Piryaei
- Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran 1985717443, Iran
| | - Peter Timashev
- Institute for Regenerative Medicine, Sechenov University, 119146 Moscow, Russia; (A.S.); (P.T.)
- Department of Polymers and Composites, N.N.Semenov Institute of Chemical Physics, 117977 Moscow, Russia
| | - Roberto Gramignoli
- Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, 171 77 Stockholm, Sweden;
| | - Andreas Nussler
- Siegfried Weller Institute for Trauma Research, University of Tübingen, 72076 Tübingen, Germany; (M.R.); (A.N.)
| | - Hossein Baharvand
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran; (Z.H.); (Z.F.)
- Department of Developmental Biology, University of Science and Culture, ACECR, Tehran 1665659911, Iran
| | - Massoud Vosough
- Department of Stem Cells and Developmental Biology, Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran; (Z.H.); (Z.F.)
- Department of Regenerative Medicine, Cell Science Research Centre, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran 1665659911, Iran
| |
Collapse
|
|
39
|
Inubushi Y, Tachibana A. Uniform spheroid formation on a laboratory-made, low cell attachment surface consisting of a chitin sheet. Biosci Biotechnol Biochem 2020; 84:997-1000. [PMID: 31928142 DOI: 10.1080/09168451.2020.1714423] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
We utilized the reaction of chitosan with acetic anhydride to form a chitin gel. This gel was then dried, which formed a chitin sheet. The procedure was extremely easy for a biologist unfamiliar with materials engineering. Spheroids derived from HEK293T cells were formed on the chitin sheet, because the spheroids slightly attached and slowly moved on the chitin sheet.
Collapse
Affiliation(s)
- Yusaku Inubushi
- Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan
| | - Akira Tachibana
- Department of Bioengineering, Graduate School of Engineering, Osaka City University, Osaka, Japan
| |
Collapse
|
|
40
|
Zhao L, Xiu J, Liu Y, Zhang T, Pan W, Zheng X, Zhang X. A 3D Printed Hanging Drop Dripper for Tumor Spheroids Analysis Without Recovery. Sci Rep 2019; 9:19717. [PMID: 31873199 PMCID: PMC6928160 DOI: 10.1038/s41598-019-56241-0] [Citation(s) in RCA: 61] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 12/09/2019] [Indexed: 12/15/2022] Open
Abstract
Compared with traditional monolayer cell culture, the three-dimensional tumor spheroid has emerged as an essential in vitro model for cancer research due to the recapitulation of the architecture and physiology of solid human tumors. Herein, by implementing the rapid prototyping of a benchtop 3D printer, we developed a new strategy to generate and analyze tumor spheroids on a commonly used multi-well plate. In this method, the printed artifact can be directly mounted on a 96/384-well plate, enables hanging drop-based spheroid formation, avoiding the tedious fabrication process from micromechanical systems. Besides long-term spheroid culture (20 days), this method supports subsequent analysis of tumor spheroid by seamlessly dripping from the printed array, thereby eliminating the need for spheroids retrieval for downstream characterization. We demonstrated several tumor spheroid-based assays, including tumoroid drug testing, metastasis on or inside extracellular matrix gel, and tumor transendothelial (TEM) assay. Based on quantitative phenotypical and molecular analysis without any precarious retrieval and transfer, we found that the malignant breast cancer (MDA-MB-231) cell aggregate presents a more metastatic morphological phenotype than the non-malignant breast cancer (MCF-7) and colonial cancer (HCT-116) cell spheroid, and shows an up-regulation of epithelial-mesenchymal transition (EMT) relevant genes (fold change > 2). Finally, we validated this tumor malignancy by the TEM assay, which could be easily performed using our approach. This methodology could provide a useful workflow for expediting tumoroid modeled in vitro assay, allowing the “Lab-on-a-Cloud” scenario for routine study.
Collapse
Affiliation(s)
- Liang Zhao
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China. .,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China. .,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China. .,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China.
| | - Jidong Xiu
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China.,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China.,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China.,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China
| | - Yang Liu
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China.,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China.,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China.,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China
| | - Tianye Zhang
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China.,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China.,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China.,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China
| | - Wenjie Pan
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China.,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China.,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China.,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China
| | - Xiaonan Zheng
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China.,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China.,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China.,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China
| | - Xueji Zhang
- Institute of Precision Medicine and Health, University of Science and Technology Beijing, Beijing, 100083, China. .,Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, 100083, China. .,Beijing Key Laboratory for Bioengineering and Sensing Technology University of Science and Technology Beijing, Beijing, 100083, China. .,School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, China.
| |
Collapse
|
|
41
|
Liao W, Wang J, Xu J, You F, Pan M, Xu X, Weng J, Han X, Li S, Li Y, Liang K, Peng Q, Gao Y. High-throughput three-dimensional spheroid tumor model using a novel stamp-like tool. J Tissue Eng 2019; 10:2041731419889184. [PMID: 31827757 PMCID: PMC6886283 DOI: 10.1177/2041731419889184] [Citation(s) in RCA: 38] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2019] [Accepted: 10/29/2019] [Indexed: 12/28/2022] Open
Abstract
Spheroid culture is a widely used three-dimensional culture technology that simulates the three-dimensional structure of tumors in vivo and has been considered a good model for tumor research. However, current commercialized spheroid culture tools have the shortcomings of high cost or relatively poor spheroid-forming results for some special cells. To solve such problems, we designed a 3D printed, reusable, stamp-like resin mold that could shape microstructures for spheroid culture of tumor cells on the surface of agarose substrate in a 96-well plate. We applied this homemade three-dimensional culture tool in spheroid formation for hepatocellular carcinoma cells. The experimental data show that the effect of spheroid culture on four hepatocellular carcinoma cell lines in our homemade spheroid culture plate is better than that of the commercialized ultralow attachment spheroid culture plate, and compared to two-dimensional culture, three-dimensional culture improves cell functions. In addition, the drug-sensitive test based on patient-derived hepatocellular carcinoma cells showed a different pattern between spheroid and two-dimensional cultures. In conclusion, our spheroid culture tool is characterized by its low cost, reusability, low cell consumption, convenience in medium exchange, and good effect of spheroid formation, suggesting that this technique could be widely used in individual treatment and high-throughput drug screening.
Collapse
Affiliation(s)
- Wei Liao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Jieqing Wang
- Department of Pancreatobiliary Surgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China
| | - Jiecheng Xu
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Fuyu You
- Department of Hepatobiliary Surgery, Guangzhou Women and Children's Medical Center, Guangzhou, China
| | - Mingxin Pan
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Xiaoping Xu
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Jun Weng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Xu Han
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Shao Li
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Yang Li
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Kangyan Liang
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Qing Peng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| | - Yi Gao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Guangzhou Clinical Research and Transformation Center for Artificial Liver, Institute of Regenerative Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou, China
| |
Collapse
|
|
42
|
Deferm N, De Vocht T, Qi B, Van Brantegem P, Gijbels E, Vinken M, de Witte P, Bouillon T, Annaert P. Current insights in the complexities underlying drug-induced cholestasis. Crit Rev Toxicol 2019; 49:520-548. [PMID: 31589080 DOI: 10.1080/10408444.2019.1635081] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Drug-induced cholestasis (DIC) poses a major challenge to the pharmaceutical industry and regulatory agencies. It causes both drug attrition and post-approval withdrawal of drugs. DIC represents itself as an impaired secretion and flow of bile, leading to the pathological hepatic and/or systemic accumulation of bile acids (BAs) and their conjugate bile salts. Due to the high number of mechanisms underlying DIC, predicting a compound's cholestatic potential during early stages of drug development remains elusive. A profound understanding of the different molecular mechanisms of DIC is, therefore, of utmost importance. Although many knowledge gaps and caveats still exist, it is generally accepted that alterations of certain hepatobiliary membrane transporters and changes in hepatocellular morphology may cause DIC. Consequently, liver models, which represent most of these mechanisms, are valuable tools to predict human DIC. Some of these models, such as membrane-based in vitro models, are exceptionally well-suited to investigate specific mechanisms (i.e. transporter inhibition) of DIC, while others, such as liver slices, encompass all relevant biological processes and, therefore, offer a better representation of the in vivo situation. In the current review, we highlight the principal molecular mechanisms associated with DIC and offer an overview and critical appraisal of the different liver models that are currently being used to predict the cholestatic potential of drugs.
Collapse
Affiliation(s)
- Neel Deferm
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Tom De Vocht
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Bing Qi
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Pieter Van Brantegem
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Eva Gijbels
- Entity of In Vitro Toxicology and Dermato-Cosmetology, Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mathieu Vinken
- Entity of In Vitro Toxicology and Dermato-Cosmetology, Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Peter de Witte
- Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Thomas Bouillon
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Pieter Annaert
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| |
Collapse
|
|
43
|
|
|
44
|
Zhou Y, Shen JX, Lauschke VM. Comprehensive Evaluation of Organotypic and Microphysiological Liver Models for Prediction of Drug-Induced Liver Injury. Front Pharmacol 2019; 10:1093. [PMID: 31616302 PMCID: PMC6769037 DOI: 10.3389/fphar.2019.01093] [Citation(s) in RCA: 63] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Accepted: 08/26/2019] [Indexed: 12/21/2022] Open
Abstract
Drug-induced liver injury (DILI) is a major concern for the pharmaceutical industry and constitutes one of the most important reasons for the termination of promising drug development projects. Reliable prediction of DILI liability in preclinical stages is difficult, as current experimental model systems do not accurately reflect the molecular phenotype and functionality of the human liver. As a result, multiple drugs that passed preclinical safety evaluations failed due to liver toxicity in clinical trials or postmarketing stages in recent years. To improve the selection of molecules that are taken forward into the clinics, the development of more predictive in vitro systems that enable high-throughput screening of hepatotoxic liabilities and allow for investigative studies into DILI mechanisms has gained growing interest. Specifically, it became increasingly clear that the choice of cell types and culture method both constitute important parameters that affect the predictive power of test systems. In this review, we present current 3D culture paradigms for hepatotoxicity tests and critically evaluate their utility and performance for DILI prediction. In addition, we highlight possibilities of these emerging platforms for mechanistic evaluations of selected drug candidates and present current research directions towards the further improvement of preclinical liver safety tests. We conclude that organotypic and microphysiological liver systems have provided an important step towards more reliable DILI prediction. Furthermore, we expect that the increasing availability of comprehensive benchmarking studies will facilitate model dissemination that might eventually result in their regulatory acceptance.
Collapse
Affiliation(s)
| | | | - Volker M. Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
| |
Collapse
|
|
45
|
Katsuda T, Kawamata M, Inoue A, Yamaguchi T, Abe M, Ochiya T. Long‐term maintenance of functional primary human hepatocytes using small molecules. FEBS Lett 2019; 594:114-125. [DOI: 10.1002/1873-3468.13582] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Revised: 08/15/2019] [Accepted: 08/16/2019] [Indexed: 01/08/2023]
Affiliation(s)
- Takeshi Katsuda
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
| | - Masaki Kawamata
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
- Division of Organogenesis and Regeneration Medical Institute of Bioregulation Kyushu University Fukuoka Japan
| | - Ayako Inoue
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
| | - Tomoko Yamaguchi
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
- Department of Molecular and Cellular Medicine, Institute of Medical Science Tokyo Medical University Nishi-Shinjuku, Shinjuku-ku Tokyo Japan
| | - Maki Abe
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
- Department of Molecular and Cellular Medicine, Institute of Medical Science Tokyo Medical University Nishi-Shinjuku, Shinjuku-ku Tokyo Japan
| | - Takahiro Ochiya
- Division of Molecular and Cellular Medicine National Cancer Center Research Institute Tokyo Japan
- Department of Molecular and Cellular Medicine, Institute of Medical Science Tokyo Medical University Nishi-Shinjuku, Shinjuku-ku Tokyo Japan
| |
Collapse
|
|
46
|
Alhaque S, Themis M, Rashidi H. Three-dimensional cell culture: from evolution to revolution. Philos Trans R Soc Lond B Biol Sci 2019; 373:rstb.2017.0216. [PMID: 29786551 DOI: 10.1098/rstb.2017.0216] [Citation(s) in RCA: 49] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/19/2018] [Indexed: 02/06/2023] Open
Abstract
Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells along specific lineages have facilitated the development of high-fidelity modelling of several tissues in vitro Many of the novel approaches have employed self-organizing three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimensional platforms. Organoid technologies hold great promise for modelling diseases and predicting the outcome of drug responses in vitro Here, we outline the historical background and some of the recent advances in the field of three-dimensional organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using three-dimensional modelling technologies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.
Collapse
Affiliation(s)
- Sharmin Alhaque
- Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK.,Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, Middlesex, UK
| | - Michael Themis
- Division of Biosciences, Department of Life Sciences, College of Health and Life Sciences, Brunel University London, Uxbridge, Middlesex, UK
| | - Hassan Rashidi
- Scottish Centre for Regenerative Medicine, University of Edinburgh, Edinburgh, UK
| |
Collapse
|
|
47
|
Yasukawa T, Morishima A, Suzuki M, Yoshioka J, Yoshimoto K, Mizutani F. Rapid Formation of Aggregates with Uniform Numbers of Cells Based on Three-dimensional Dielectrophoresis. ANAL SCI 2019; 35:895-901. [PMID: 31006719 DOI: 10.2116/analsci.19p074] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
We applied a fabrication method for the formation of island organization of cells based on a three-dimensional (3D) device for negative dielectrophoresis (n-DEP) to produce cell aggregates with uniform numbers of cells rapidly and simply. The intersections formed by rotating the interdigitated array (IDA) with two combs of band electrodes on the upper substrate by 90° relative to the IDA with two combs on the lower substrate were prepared in the device. The AC voltage was applied to a comb on the upper substrate and a comb on the lower substrate, while AC voltage with opposite phase was applied to another comb on the upper substrate and another comb on the lower substrate. Cells dispersed randomly were directed toward the intersections with relatively lower electric fields due to n-DEP, which formed by AC voltage applied bands with the identical phase, resulting in the formation of island patterns of cells. The cells accumulated at intersections were promoted to form the cell aggregates due to the close contact together. The production of cell aggregations adhered together was easily found by the dispersion behavior after switching the applied frequency to convert the cellular pattern. When cells were accumulated at the intersections by n-DEP for 45 min, almost accumulations of cells were adhered together, and hence a formations of cell aggregations. By using the present method, we can rapidly and simply fabricate cell aggregations with a uniform number of cells.
Collapse
Affiliation(s)
| | - Asa Morishima
- Graduate School of Material Science, University of Hyogo
| | - Masato Suzuki
- Graduate School of Material Science, University of Hyogo
| | - Junya Yoshioka
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo
| | - Keitaro Yoshimoto
- Department of Life Sciences, Graduate School of Arts and Sciences, The University of Tokyo
| | - Fumio Mizutani
- Graduate School of Material Science, University of Hyogo
| |
Collapse
|
|
48
|
Gijbels E, Vilas-Boas V, Deferm N, Devisscher L, Jaeschke H, Annaert P, Vinken M. Mechanisms and in vitro models of drug-induced cholestasis. Arch Toxicol 2019; 93:1169-1186. [PMID: 30972450 DOI: 10.1007/s00204-019-02437-2] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Accepted: 04/02/2019] [Indexed: 12/12/2022]
Abstract
Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.
Collapse
Affiliation(s)
- Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Vânia Vilas-Boas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Neel Deferm
- Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2, Herestraat 49, Bus 921, 3000, Leuven, Belgium
| | - Lindsey Devisscher
- Basic and Applied Medical Sciences, Gut-Liver Immunopharmacology Unit, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000, Ghent, Belgium
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 1018, Kansas City, KS, 66160, USA
| | - Pieter Annaert
- Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2, Herestraat 49, Bus 921, 3000, Leuven, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
| |
Collapse
|
|
49
|
Li Y, Chen HS, Shaheen M, Joo DJ, Amiot BP, Rinaldo P, Nyberg SL. Cold storage of porcine hepatocyte spheroids for spheroid bioartificial liver. Xenotransplantation 2019; 26:e12512. [PMID: 30968460 DOI: 10.1111/xen.12512] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2018] [Revised: 02/11/2019] [Accepted: 03/07/2019] [Indexed: 02/07/2023]
Abstract
BACKGROUND AND AIMS Cell-based therapies for liver disease such as bioartificial liver rely on a large quantity and high quality of hepatocytes. Cold storage was previously shown to be a better way to preserve the viability and functionality of hepatocytes during transportation rather than freezing, but this was only proved at a lower density of rat hepatocytes spheroids. The purpose of this study was to optimize conditions for cold storage of high density of primary porcine hepatocyte spheroids. METHODS Porcine hepatocytes were isolated by a three-step perfusion method; hepatocyte spheroids were formed by a 24 hours rocked culture technique. Hepatocyte cell density was 5 × 106 /mL in 1000 mL spheroid forming medium. Spheroids were then maintained in rocked culture at 37°C (control condition) or cold stored at 4°C for 24, 48 or 72 hours in four different cold storage solutions: histidine-tryptophan-ketoglutarate (HTK) alone; HTK + 1 mM deferoxamine (DEF); HTK + 5 mM N-acetyl-L-cysteine (NAC); and HTK + 1 mM DEF + 5 mM NAC. The viability, ammonia clearance, albumin production, gene expression, and functional activity of cytochrome P450 enzymes were measured after recovery from the cold storage. RESULTS In this study, we observed that cold-induced injury was reduced by the addition of the iron chelator. Viability of HTK + DEF group hepatocyte spheroids was increased compared with other cold storage groups (P < 0.05). Performance metrics of porcine hepatocyte spheroids cold stored for 24 hours were similar to those in control conditions. The hepatocyte spheroids in control conditions started to lose their ability to clear ammonia while production of albumin was still active at 48 and 72 hours (P < 0.05). In contrast, the viability and functionality of hepatocyte spheroids including ammonia clearance and albumin secretion were preserved in HTK + DEF group at both 48- and 72-hour time points (P < 0.05). CONCLUSIONS The beneficial effects of HTK supplemented with DEF were more obvious after cold storage of high density of porcine hepatocyte spheroids for 72 hours. The porcine hepatocyte spheroids were above the cutoff criteria for use in a spheroid-based bioartificial liver.
Collapse
Affiliation(s)
- Yi Li
- Laboratory of Pathology, Key Laboratory of Transplant Engineering and Immunology, NHFPC, West China Hospital, Sichuan University, Chengdu, China.,Department of Surgery, Mayo Clinic, Rochester, Minnesota.,William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota
| | - Harvey S Chen
- Department of Surgery, Mayo Clinic, Rochester, Minnesota.,William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota
| | - Mohammed Shaheen
- Department of Surgery, Mayo Clinic, Rochester, Minnesota.,William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota
| | - Dong Jin Joo
- Department of Surgery, Mayo Clinic, Rochester, Minnesota.,William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota
| | - Bruce P Amiot
- Department of Surgery, Mayo Clinic, Rochester, Minnesota
| | - Piero Rinaldo
- Department Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
| | - Scott L Nyberg
- Department of Surgery, Mayo Clinic, Rochester, Minnesota.,William J. von Liebig Center for Transplantation and Clinical Regeneration, Mayo Clinic, Rochester, Minnesota
| |
Collapse
|
|
50
|
Characterisation of a functional rat hepatocyte spheroid model. Toxicol In Vitro 2018; 55:160-172. [PMID: 30578835 PMCID: PMC6361770 DOI: 10.1016/j.tiv.2018.12.014] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2018] [Revised: 12/13/2018] [Accepted: 12/18/2018] [Indexed: 12/28/2022]
Abstract
Many in vitro liver cell models, such as 2D systems, that are used to assess the hepatotoxic potential of xenobiotics suffer major limitations arising from a lack of preservation of physiological phenotype and metabolic competence. To circumvent some of these limitations there has been increased focus on producing more representative 3D models. Here we have used a novel approach to construct a size-controllable 3D hepatic spheroid model using freshly isolated primary rat hepatocytes (PRH) utilising the liquid-overlay technique whereby PRH spontaneously self-assemble in to 3D microtissues. This system produces viable spheroids with a compact in vivo-like structure for up to 21 days with sustained albumin production for the duration of the culture period. F-actin was seen throughout the spheroid body and P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) transporters had polarised expression on the canalicular membrane of hepatocytes within the spheroids upon formation (day 3). The MRP2 transporter was able to functionally transport 5 μM 5-chloromethylfluorescein diacetate (CMFDA) substrates into these canalicular structures. These PRH spheroids display in vivo characteristics including direct cell-cell contacts, cellular polarisation, 3D cellular morphology, and formation of functional secondary structures throughout the spheroid. Such a well-characterised system could be readily exploited for pre-clinical and non-clinical repeat-dose investigations and could make a significant contribution to replace, reduce and refine the use of animals for applied research.
Collapse
|