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1
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Di Stefano J, Di Marco F, Cicalini I, FitzGerald U, Pieragostino D, Verhoye M, Ponsaerts P, Van Breedam E. Generation, interrogation, and future applications of microglia-containing brain organoids. Neural Regen Res 2025; 20:3448-3460. [PMID: 39665813 PMCID: PMC11974650 DOI: 10.4103/nrr.nrr-d-24-00921] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2024] [Revised: 10/29/2024] [Accepted: 11/05/2024] [Indexed: 12/13/2024] Open
Abstract
Brain organoids encompass a large collection of in vitro stem cell-derived 3D culture systems that aim to recapitulate multiple aspects of in vivo brain development and function. First, this review provides a brief introduction to the current state-of-the-art for neuro-ectoderm brain organoid development, emphasizing their biggest advantages in comparison with classical two-dimensional cell cultures and animal models. However, despite their usefulness for developmental studies, a major limitation for most brain organoid models is the absence of contributing cell types from endodermal and mesodermal origin. As such, current research is highly investing towards the incorporation of a functional vasculature and the microglial immune component. In this review, we will specifically focus on the development of immune-competent brain organoids. By summarizing the different approaches applied to incorporate microglia, it is highlighted that immune-competent brain organoids are not only important for studying neuronal network formation, but also offer a clear future as a new tool to study inflammatory responses in vitro in 3D in a brain-like environment. Therefore, our main focus here is to provide a comprehensive overview of assays to measure microglial phenotype and function within brain organoids, with an outlook on how these findings could better understand neuronal network development or restoration, as well as the influence of physical stress on microglia-containing brain organoids. Finally, we would like to stress that even though the development of immune-competent brain organoids has largely evolved over the past decade, their full potential as a pre-clinical tool to study novel therapeutic approaches to halt or reduce inflammation-mediated neurodegeneration still needs to be explored and validated.
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Affiliation(s)
- Julia Di Stefano
- Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Wilrijk, Belgium
- Bio-Imaging Lab, University of Antwerp, Wilrijk, Belgium
| | - Federica Di Marco
- Center for Advanced Studies and Technology (CAST), G. d’Annunzio University of Chieti-Pescara, Chieti, Italy
- Department of Innovative Technologies in Medicine and Dentistry, University “G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
| | - Ilaria Cicalini
- Center for Advanced Studies and Technology (CAST), G. d’Annunzio University of Chieti-Pescara, Chieti, Italy
- Department of Innovative Technologies in Medicine and Dentistry, University “G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
| | - Una FitzGerald
- CÚRAM, Center for Research in Medical Devices, Biomedical Engineering, University of Galway, Ireland
- Galway Neuroscience Center, University of Galway, Ireland
| | - Damiana Pieragostino
- Center for Advanced Studies and Technology (CAST), G. d’Annunzio University of Chieti-Pescara, Chieti, Italy
- Department of Innovative Technologies in Medicine and Dentistry, University “G. d’Annunzio” of Chieti-Pescara, Chieti, Italy
| | - Marleen Verhoye
- Bio-Imaging Lab, University of Antwerp, Wilrijk, Belgium
- μNEURO Research Center of Excellence, University of Antwerp, Wilrijk, Belgium
| | - Peter Ponsaerts
- Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Wilrijk, Belgium
| | - Elise Van Breedam
- Laboratory of Experimental Hematology, Vaccine and Infectious Disease Institute (Vaxinfectio), University of Antwerp, Wilrijk, Belgium
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Igarashi R, Oda M, Okada R, Yano T, Takahashi S, Pastuhov S, Matano M, Masuda N, Togasaki K, Ohta Y, Sato S, Hishiki T, Suematsu M, Itoh M, Fujii M, Sato T. Generation of human adult hepatocyte organoids with metabolic functions. Nature 2025:10.1038/s41586-025-08861-y. [PMID: 40240606 DOI: 10.1038/s41586-025-08861-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2024] [Accepted: 03/04/2025] [Indexed: 04/18/2025]
Abstract
Proliferating hepatocytes often undergo ductal metaplasia to balance the energy trade-off between cellular functions and replication, hindering the expansion of human adult hepatocytes with functional competency1. Here we demonstrate that the combined activation of Wnt and STAT3 signalling enables long-term self-renewal of human adult hepatocyte organoids. YAP activation facilitates hepatocyte proliferation but commits it towards the biliary duct lineage. By contrast, STAT3 activation by oncostatin M induces hepatocyte proliferation while counteracting ductal metaplasia and maintaining the hepatic identity. Xenotransplanted hepatocyte organoids repopulate the recipient mouse liver and reconstitute the metabolic zonation structure. Upon niche factor removal and hormone supplementation, hepatocyte organoids form cord-like structures with bile canalicular networks and exhibit major liver metabolic functions comparable to those of in vivo hepatocytes. Hepatocyte organoids are amenable to gene editing, prompting functional modelling of inherent metabolic liver diseases. The new culture system offers a promising avenue for developing therapeutic strategies against human liver diseases.
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Affiliation(s)
- Ryo Igarashi
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Mayumi Oda
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Ryo Okada
- JSR-Keio University Medical and Chemical Innovation Center (JKiC), JSR Corporation, Tokyo, Japan
| | - Tomoki Yano
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Sirirat Takahashi
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Strahil Pastuhov
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Mami Matano
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Norio Masuda
- JSR-Keio University Medical and Chemical Innovation Center (JKiC), JSR Corporation, Tokyo, Japan
| | - Kazuhiro Togasaki
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Yuki Ohta
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Saeko Sato
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Takako Hishiki
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
- Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Makoto Suematsu
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
- Department of Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Manabu Itoh
- JSR-Keio University Medical and Chemical Innovation Center (JKiC), JSR Corporation, Tokyo, Japan
| | - Masayuki Fujii
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan
| | - Toshiro Sato
- Department of Organoid Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo, Japan.
- Department of Integrated Medicine and Biochemistry, Keio University School of Medicine, Tokyo, Japan.
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3
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Fukunaga I, Takebe T. In vitro liver models for toxicological research. Drug Metab Pharmacokinet 2025; 62:101478. [PMID: 40203632 DOI: 10.1016/j.dmpk.2025.101478] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2024] [Revised: 02/25/2025] [Accepted: 03/04/2025] [Indexed: 04/11/2025]
Abstract
Drug-induced liver injury (DILI) presents a major challenge not only in new drug development but also in post-marketing withdrawals and the safety of food, cosmetics, and chemicals. Experimental model organisms such as the rodents have been widely used for preclinical toxicological testing. However, the tension exists associated with the ethical and sustainable use of animals in part because animals do not necessarily inform the human-specific ADME (adsorption, dynamics, metabolism and elimination) profiling. To establish alternative models in humans, in vitro hepatic tissue models have been proposed, ranging from primary hepatocytes, immortal hepatocytes, to the development of new cell resources such as stem cell-derived hepatocytes. Given the evolving number of novel alternative methods, understanding possible combinations of cell sources and culture methods will be crucial to develop the context-of-use assays. This review primarily focuses on 3D liver organoid models for conducting. We will review the relevant cell sources, bioengineering methods, selection of training compounds, and biomarkers towards the rationale design of in vitro toxicology testing.
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Affiliation(s)
- Ichiro Fukunaga
- Center for Genomic and Regenerative Medicine, Juntendo University Graduate School of Medicine, 2-1-1, Hongo, Bunkyo-ku, Tokyo, 113-8431, Japan.
| | - Takanori Takebe
- Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan; Department of Genome Biology, Graduate School of Medicine, Osaka University, Suita, Osaka, 565-0871, Japan; Divisions of Gastroenterology, Hepatology & Nutrition, Developmental Biology and Biomedical Informatics, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229-3039, USA; Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University, Suita, Osaka, 565-0871, Japan
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4
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Dong Y, Zhou X, Ding Y, Luo Y, Zhao H. Advances in tumor microenvironment: Applications and challenges of 3D bioprinting. Biochem Biophys Res Commun 2024; 730:150339. [PMID: 39032359 DOI: 10.1016/j.bbrc.2024.150339] [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: 01/08/2024] [Revised: 06/27/2024] [Accepted: 07/01/2024] [Indexed: 07/23/2024]
Abstract
The tumor microenvironment (TME) assumes a pivotal role in the treatment of oncological diseases, given its intricate interplay of diverse cellular components and extracellular matrices. This dynamic ecosystem poses a serious challenge to traditional research methods in many ways, such as high research costs, inefficient translation, poor reproducibility, and low modeling success rates. These challenges require the search for more suitable research methods to accurately model the TME, and the emergence of 3D bioprinting technology is transformative and an important complement to these traditional methods to precisely control the distribution of cells, biomolecules, and matrix scaffolds within the TME. Leveraging digital design, the technology enables personalized studies with high precision, providing essential experimental flexibility. Serving as a critical bridge between in vitro and in vivo studies, 3D bioprinting facilitates the realistic 3D culturing of cancer cells. This comprehensive article delves into cutting-edge developments in 3D bioprinting, encompassing diverse methodologies, biomaterial choices, and various 3D tumor models. Exploration of current challenges, including limited biomaterial options, printing accuracy constraints, low reproducibility, and ethical considerations, contributes to a nuanced understanding. Despite these challenges, the technology holds immense potential for simulating tumor tissues, propelling personalized medicine, and constructing high-resolution organ models, marking a transformative trajectory in oncological research.
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Affiliation(s)
- Yingying Dong
- The First School of Climical Medicine of Zhejiang Chinese Medical University, Hangzhou, 310053, China.
| | - Xue Zhou
- School of Mechanical Engineering, Zhejiang University, Hangzhou, 310058, China; State Key Laboratory of Fluid Power & Mechatronic Systems, Zhejiang University, Hangzhou, 310058, China.
| | - Yunyi Ding
- Department of Emergency Medicine, The Second Affiliated Hospital of Zhejiang University, School, Hangzhou, 310009, China.
| | - Yichen Luo
- School of Mechanical Engineering, Zhejiang University, Hangzhou, 310058, China; State Key Laboratory of Fluid Power & Mechatronic Systems, Zhejiang University, Hangzhou, 310058, China.
| | - Hong Zhao
- The First School of Climical Medicine of Zhejiang Chinese Medical University, Hangzhou, 310053, China; Department of Breast Surgery, The First Affiliated Hospital of Zhejiang University of Traditional Chinese Medicine, (Zhejiang Provincial Hospital of Traditional Chinese Medicine), Hangzhou, 310060, China.
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5
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Salas G, Litta AA, Medeot AC, Schuck VS, Andermatten RB, Miszczuk GS, Ciriaci N, Razori MV, Barosso IR, Sánchez Pozzi EJ, Roma MG, Basiglio CL, Crocenzi FA. NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis. Biochimie 2024; 223:41-53. [PMID: 38608750 DOI: 10.1016/j.biochi.2024.04.002] [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: 01/08/2024] [Revised: 04/04/2024] [Accepted: 04/08/2024] [Indexed: 04/14/2024]
Abstract
The endogenous metabolite of estradiol, estradiol 17β-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.
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Affiliation(s)
- Gimena Salas
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Alen A Litta
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Anabela C Medeot
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Virginia S Schuck
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Romina B Andermatten
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Gisel S Miszczuk
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Nadia Ciriaci
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Ma Valeria Razori
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Ismael R Barosso
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Enrique J Sánchez Pozzi
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Marcelo G Roma
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Cecilia L Basiglio
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Fernando A Crocenzi
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
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6
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Mehta V, Karnam G, Madgula V. Liver-on-chips for drug discovery and development. Mater Today Bio 2024; 27:101143. [PMID: 39070097 PMCID: PMC11279310 DOI: 10.1016/j.mtbio.2024.101143] [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: 02/28/2024] [Revised: 06/07/2024] [Accepted: 07/01/2024] [Indexed: 07/30/2024] Open
Abstract
Recent FDA modernization act 2.0 has led to increasing industrial R&D investment in advanced in vitro 3D models such as organoids, spheroids, organ-on-chips, 3D bioprinting, and in silico approaches. Liver-related advanced in vitro models remain the prime area of interest, as liver plays a central role in drug clearance of compounds. Growing evidence indicates the importance of recapitulating the overall liver microenvironment to enhance hepatocyte maturity and culture longevity using liver-on-chips (LoC) in vitro. Hence, pharmaceutical industries have started exploring LoC assays in the two of the most challenging areas: accurate in vitro-in vivo extrapolation (IVIVE) of hepatic drug clearance and drug-induced liver injury. We examine the joint efforts of commercial chip manufacturers and pharmaceutical companies to present an up-to-date overview of the adoption of LoC technology in the drug discovery. Further, several roadblocks are identified to the rapid adoption of LoC assays in the current drug development framework. Finally, we discuss some of the underexplored application areas of LoC models, where conventional 2D hepatic models are deemed unsuitable. These include clearance prediction of metabolically stable compounds, immune-mediated drug-induced liver injury (DILI) predictions, bioavailability prediction with gut-liver systems, hepatic clearance prediction of drugs given during pregnancy, and dose adjustment studies in disease conditions. We conclude the review by discussing the importance of PBPK modeling with LoC, digital twins, and AI/ML integration with LoC.
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Affiliation(s)
- Viraj Mehta
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
| | - Guruswamy Karnam
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
| | - Vamsi Madgula
- Organoid Technology Lab, DMPK Department, Sai Life Sciences, Hyderabad, 500078, India
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7
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Osonoi S, Takebe T. Organoid-guided precision hepatology for metabolic liver disease. J Hepatol 2024; 80:805-821. [PMID: 38237864 PMCID: PMC11828489 DOI: 10.1016/j.jhep.2024.01.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 12/28/2023] [Accepted: 01/02/2024] [Indexed: 03/09/2024]
Abstract
Metabolic dysfunction-associated steatotic liver disease affects millions of people worldwide. Progress towards a definitive cure has been incremental and treatment is currently limited to lifestyle modification. Hepatocyte-specific lipid accumulation is the main trigger of lipotoxic events, driving inflammation and fibrosis. The underlying pathology is extraordinarily heterogenous, and the manifestations of steatohepatitis are markedly influenced by metabolic communications across non-hepatic organs. Synthetic human tissue models have emerged as powerful platforms to better capture the mechanistic diversity in disease progression, while preserving person-specific genetic traits. In this review, we will outline current research efforts focused on integrating multiple synthetic tissue models of key metabolic organs, with an emphasis on organoid-based systems. By combining functional genomics and population-scale en masse profiling methodologies, human tissues derived from patients can provide insights into personalised genetic, transcriptional, biochemical, and metabolic states. These collective efforts will advance our understanding of steatohepatitis and guide the development of rational solutions for mechanism-directed diagnostic and therapeutic investigation.
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Affiliation(s)
- Sho Osonoi
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Gastroenterology, Hepatology and Nutrition, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA; Department of Endocrinology and Metabolism, Hirosaki University Graduate School of Medicine, Hirosaki, 036-8562, Japan
| | - Takanori Takebe
- Center for Stem Cell and Organoid Medicine (CuSTOM), Division of Gastroenterology, Hepatology and Nutrition, Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229, USA; WPI Premium Institute for Human Metaverse Medicine (WPI-PRIMe) and Department of Genome Biology, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan; Institute of Research, Tokyo Medical and Dental University (TMDU), Tokyo 113-8510, Japan; Communication Design Center, Advanced Medical Research Center, Yokohama City University, Yokohama 236-0004, Japan.
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8
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Willems C, Qi F, Trutschel ML, Groth T. Functionalized Gelatin/Polysaccharide Hydrogels for Encapsulation of Hepatocytes. Gels 2024; 10:231. [PMID: 38667650 PMCID: PMC11048940 DOI: 10.3390/gels10040231] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2024] [Revised: 03/22/2024] [Accepted: 03/25/2024] [Indexed: 04/28/2024] Open
Abstract
Liver diseases represent a considerable burden to patients and healthcare systems. Hydrogels play an important role in the engineering of soft tissues and may be useful for embedding hepatocytes for different therapeutic interventions or the development of in vitro models to study the pathogenesis of liver diseases or testing of drugs. Here, we developed two types of hydrogels by crosslinking hydrazide-functionalized gelatin with either oxidized dialdehyde hyaluronan or alginate through the formation of hydrazone bonds. Gel formulations were studied through texture analysis and rheometry, showing mechanical properties comparable to those of liver tissue while also demonstrating long-term stability. The biocompatibility of hydrogels and their ability to host hepatocytes was studied in vitro in comparison to pure gelatin hydrogels crosslinked by transglutaminase using the hepatocellular line HepG2. It was found that HepG2 cells could be successfully embedded in the hydrogels, showing no signs of gel toxicity and proliferating in a 3D environment comparable to pure transglutaminase cross-linked gelatin hydrogels used as control. Altogether, hydrazide gelatin in combination with oxidized polysaccharides makes stable in situ gelling systems for the incorporation of hepatocytes, which may pave the way for use in liver tissue engineering and drug testing.
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Affiliation(s)
- Christian Willems
- Department of Biomedical Materials, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany; (C.W.); (F.Q.)
| | - Fangdi Qi
- Department of Biomedical Materials, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany; (C.W.); (F.Q.)
| | - Marie-Luise Trutschel
- Department of Pharmaceutical Technology, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany
| | - Thomas Groth
- Department of Biomedical Materials, Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany; (C.W.); (F.Q.)
- Interdisciplinary Center of Materials Science, Martin-Luther University Halle-Wittenberg, 06120 Halle, Germany
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9
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Wistner SC, Rashad L, Slaughter G. Advances in tissue engineering and biofabrication for in vitro skin modeling. BIOPRINTING (AMSTERDAM, NETHERLANDS) 2023; 35:e00306. [PMID: 38645432 PMCID: PMC11031264 DOI: 10.1016/j.bprint.2023.e00306] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/23/2024]
Abstract
The global prevalence of skin disease and injury is continually increasing, yet conventional cell-based models used to study these conditions do not accurately reflect the complexity of human skin. The lack of inadequate in vitro modeling has resulted in reliance on animal-based models to test pharmaceuticals, biomedical devices, and industrial and environmental toxins to address clinical needs. These in vivo models are monetarily and morally expensive and are poor predictors of human tissue responses and clinical trial outcomes. The onset of three-dimensional (3D) culture techniques, such as cell-embedded and decellularized approaches, has offered accessible in vitro alternatives, using innovative scaffolds to improve cell-based models' structural and histological authenticity. However, these models lack adequate organizational control and complexity, resulting in variations between structures and the exclusion of physiologically relevant vascular and immunological features. Recently, biofabrication strategies, which combine biology, engineering, and manufacturing capabilities, have emerged as instrumental tools to recreate the heterogeneity of human skin precisely. Bioprinting uses computer-aided design (CAD) to yield robust and reproducible skin prototypes with unprecedented control over tissue design and assembly. As the interdisciplinary nature of biofabrication grows, we look to the promise of next-generation biofabrication technologies, such as organ-on-a-chip (OOAC) and 4D modeling, to simulate human tissue behaviors more reliably for research, pharmaceutical, and regenerative medicine purposes. This review aims to discuss the barriers to developing clinically relevant skin models, describe the evolution of skin-inspired in vitro structures, analyze the current approaches to biofabricating 3D human skin mimetics, and define the opportunities and challenges in biofabricating skin tissue for preclinical and clinical uses.
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Affiliation(s)
- Sarah C. Wistner
- Center for Bioelectronics, Old Dominion University, Norfolk, VA, 23508, USA
| | - Layla Rashad
- Center for Bioelectronics, Old Dominion University, Norfolk, VA, 23508, USA
| | - Gymama Slaughter
- Center for Bioelectronics, Old Dominion University, Norfolk, VA, 23508, USA
- Department of Electrical and Computer Engineering, Old Dominion University, Norfolk, VA, 23508, USA
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10
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Kaur I, Vasudevan A, Rawal P, Tripathi DM, Ramakrishna S, Kaur S, Sarin SK. Primary Hepatocyte Isolation and Cultures: Technical Aspects, Challenges and Advancements. Bioengineering (Basel) 2023; 10:131. [PMID: 36829625 PMCID: PMC9952008 DOI: 10.3390/bioengineering10020131] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/29/2022] [Revised: 01/11/2023] [Accepted: 01/13/2023] [Indexed: 01/20/2023] Open
Abstract
Hepatocytes are differentiated cells that account for 80% of the hepatic volume and perform all major functions of the liver. In vivo, after an acute insult, adult hepatocytes retain their ability to proliferate and participate in liver regeneration. However, in vitro, prolonged culture and proliferation of viable and functional primary hepatocytes have remained the major and the most challenging goal of hepatocyte-based cell therapies and liver tissue engineering. The first functional cultures of rat primary hepatocytes between two layers of collagen gel, also termed as the "sandwich cultures", were reported in 1989. Since this study, several technical developments including choice of hydrogels, type of microenvironment, growth factors and culture conditions, mono or co-cultures of hepatocytes along with other supporting cell types have evolved for both rat and human primary hepatocytes in recent years. All these improvements have led to a substantial improvement in the number, life-span and hepatic functions of these cells in vitro for several downstream applications. In the current review, we highlight the details, limitations and prospects of different technical strategies being used in primary hepatocyte cultures. We discuss the use of newer biomaterials as scaffolds for efficient culture of primary hepatocytes. We also describe the derivation of mature hepatocytes from other cellular sources such as induced pluripotent stem cells, bone marrow stem cells and 3D liver organoids. Finally, we also explain the use of perfusion-based bioreactor systems and bioengineering strategies to support the long-term function of hepatocytes in 3D conditions.
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Affiliation(s)
- Impreet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Ashwini Vasudevan
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Preety Rawal
- School of Biotechnology, Gautam Buddha University, Greater Noida 201312, India
| | - Dinesh M. Tripathi
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Seeram Ramakrishna
- Department of Mechanical Engineering, National University of Singapore, Singapore 117581, Singapore
| | - Savneet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | - Shiv K. Sarin
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi 110070, India
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11
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Kaur S, Kidambi S, Ortega-Ribera M, Thuy LTT, Nieto N, Cogger VC, Xie WF, Tacke F, Gracia-Sancho J. In Vitro Models for the Study of Liver Biology and Diseases: Advances and Limitations. Cell Mol Gastroenterol Hepatol 2022; 15:559-571. [PMID: 36442812 PMCID: PMC9868680 DOI: 10.1016/j.jcmgh.2022.11.008] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2022] [Revised: 11/23/2022] [Accepted: 11/23/2022] [Indexed: 11/27/2022]
Abstract
In vitro models of liver (patho)physiology, new technologies, and experimental approaches are progressing rapidly. Based on cell lines, induced pluripotent stem cells or primary cells derived from mouse or human liver as well as whole tissue (slices), such in vitro single- and multicellular models, including complex microfluidic organ-on-a-chip systems, provide tools to functionally understand mechanisms of liver health and disease. The International Society of Hepatic Sinusoidal Research (ISHSR) commissioned this working group to review the currently available in vitro liver models and describe the advantages and disadvantages of each in the context of evaluating their use for the study of liver functionality, disease modeling, therapeutic discovery, and clinical applicability.
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Affiliation(s)
- Savneet Kaur
- Department of Molecular and Cellular Medicine, Institute of Liver and Biliary Sciences, New Delhi, India
| | - Srivatsan Kidambi
- Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska
| | - Martí Ortega-Ribera
- Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts
| | - Le Thi Thanh Thuy
- Department of Hepatology, Graduate School of Medicine, Osaka Metropolitan University, Osaka, Japan
| | - Natalia Nieto
- Department of Pathology, University of Illinois at Chicago, Chicago, Illinois
| | - Victoria C Cogger
- Faculty of Medicine and Health, University of Sydney, Sydney, Australia
| | - Wei-Fen Xie
- Department of Gastroenterology, Changzheng Hospital, Naval Medical University, Shanghai, China
| | - Frank Tacke
- Department of Hepatology and Gastroenterology, Charité Universitätsmedizin Berlin, Berlin, Germany
| | - Jordi Gracia-Sancho
- Liver Vascular Biology, IDIBAPS Biomedical Research Institute, CIBEREHD, Barcelona, Spain; Department of Visceral Surgery and Medicine, Inselspital, Bern University Hospital, University of Bern, Switzerland; Department for BioMedical Research, Visceral Surgery and Medicine, University of Bern, Switzerland.
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12
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Temple J, Velliou E, Shehata M, Lévy R, Gupta P. Current strategies with implementation of three-dimensional cell culture: the challenge of quantification. Interface Focus 2022; 12:20220019. [PMID: 35992772 PMCID: PMC9372643 DOI: 10.1098/rsfs.2022.0019] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2022] [Accepted: 07/20/2022] [Indexed: 12/14/2022] Open
Abstract
From growing cells in spheroids to arranging them on complex engineered scaffolds, three-dimensional cell culture protocols are rapidly expanding and diversifying. While these systems may often improve the physiological relevance of cell culture models, they come with technical challenges, as many of the analytical methods used to characterize traditional two-dimensional (2D) cells must be modified or replaced to be effective. Here we review the advantages and limitations of quantification methods based either on biochemical measurements or microscopy imaging. We focus on the most basic of parameters that one may want to measure, the number of cells. Precise determination of this number is essential for many analytical techniques where measured quantities are only meaningful when normalized to the number of cells (e.g. cytochrome p450 enzyme activity). Thus, accurate measurement of cell number is often a prerequisite to allowing comparisons across different conditions (culturing conditions or drug and treatment screening) or between cells in different spatial states. We note that this issue is often neglected in the literature with little or no information given regarding how normalization was performed, we highlight the pitfalls and complications of quantification and call for more accurate reporting to improve reproducibility.
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Affiliation(s)
- Jonathan Temple
- Bioscience building, University of Liverpool, Liverpool L69 3BX, UK
| | - Eirini Velliou
- Centre for 3D Models of Health and Disease, University College London, London, UK
| | - Mona Shehata
- Hutchison-MRC Research Centre, University of Cambridge, Cambridge CB2 1TN, UK
| | - Raphaël Lévy
- Bioscience building, University of Liverpool, Liverpool L69 3BX, UK
- Laboratoire for Vascular Translational Science, Université Sorbonne Paris Nord, Bobigny, France
| | - Priyanka Gupta
- Centre for 3D Models of Health and Disease, University College London, London, UK
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13
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DiProspero TJ, Brown LG, Fachko TD, Lockett MR. HepaRG cells adopt zonal-like drug-metabolizing phenotypes under physiologically relevant oxygen tensions and Wnt/β-catenin signaling. Drug Metab Dispos 2022; 50:DMD-AR-2022-000870. [PMID: 35701181 PMCID: PMC9341261 DOI: 10.1124/dmd.122.000870] [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: 02/13/2022] [Revised: 05/19/2022] [Accepted: 05/26/2022] [Indexed: 11/22/2022] Open
Abstract
The cellular microenvironment plays an important role in liver zonation, the spatial distribution of metabolic tasks amongst hepatocytes lining the sinusoid. Standard tissue culture practices provide an excess of oxygen and a lack of signaling molecules typically found in the liver. We hypothesized that incorporating physiologically relevant environments would promote post-differentiation patterning of hepatocytes and result in zonal-like characteristics. To test this hypothesis, we evaluated the transcriptional regulation and activity of drug-metabolizing enzymes in HepaRG cells exposed to three different oxygen tensions, in the presence or absence of Wnt/β-catenin signaling. The drug-metabolizing activity of cells exposed to representative periportal (11% O2) or perivenous (5% O2) oxygen tensions were significantly less than cells exposed to ambient oxygen. A comparison of cytochrome P450 (CYP) 1A2, 2D6, and 3A4 activity at PP and PV oxygen tensions showed significant increases at the lower oxygen tension. The activation of the Wnt/β-catenin pathway only modestly impacted CYP activity at PV oxygen tension, despite a significant increase in CYP expression under this condition. Our results suggest oxygen tension is the major contributor to zonal patterning in HepaRG cells, with the Wnt/β-catenin signaling pathway playing a lesser albeit important role. Our datasets also highlight the importance of including activity-based assays, as transcript data alone does not provide an accurate picture of metabolic competence. Significance Statement This work investigates the post-differentiation patterning of HepaRG cells cultured at physiologically relevant oxygen tensions, in the presence and absence of Wnt/β-catenin signaling. HepaRG cells exposed to periportal (11% O2) or perivenous (5% O2) oxygen tensions display zonation-like patterning of both cytochrome P450 (CYP) and glucuronosyltransferase (UGT) enzymes. These datasets also suggest that oxygen is a primary regulator of post-differentiation patterning, with Wnt/β-catenin having a lesser effect on activity but a significant effect on transcriptional regulation of these enzymes.
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Affiliation(s)
| | - Lauren G Brown
- Chemistry, Univeristy of North Carolina at Chapel Hill, United States
| | - Trevor D Fachko
- Chemistry, University of North Carolina at Chapel Hill, United States
| | - Matthew R Lockett
- Chemistry, University of North Carolina at Chapel Hill, United States
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14
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Three-dimensional (3D) liver cell models - a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials. Drug Deliv Transl Res 2022; 12:2048-2074. [PMID: 35507131 PMCID: PMC9066991 DOI: 10.1007/s13346-022-01147-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/03/2022] [Indexed: 12/13/2022]
Abstract
Despite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them.
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15
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He T, Qiao S, Ma C, Peng Z, Wu Z, Ma C, Han L, Deng Q, Zhang T, Zhu Y, Pan G. FEK self-assembled peptide hydrogels facilitate primary hepatocytes culture and pharmacokinetics screening. J Biomed Mater Res B Appl Biomater 2022; 110:2015-2027. [PMID: 35301798 DOI: 10.1002/jbm.b.35056] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2021] [Revised: 09/11/2021] [Accepted: 10/05/2021] [Indexed: 11/10/2022]
Abstract
A FEFEFKFK (FEK, F, phenylalaninyl; E, glutamyl; K, lysinyl)-based self-assembling peptide hydrogel (FEK-SAPH) was developed to replace sandwich culture (SC) for improved culture of primary hepatocytes in vitro. Under neutral conditions, FEK self-assembles to form β-sheet nanofibers, which in turn form FEK-SAPH. For the culture of rat primary hepatocytes (RPH), the use of FEK-SAPH simplified operation steps and promoted excellent cell-cell interactions while maintaining the SC-related RPH polarity trend. Compared with SC, FEK-SAPH cultured RPH for 14 days, the bile duct network was formed, the secretion of albumin and urea was improved, and the metabolic clearance rate based on cytochrome P450 (CYPs) was comparable. In FEK-SAPH culture, the expression level of the biliary efflux transporter bile salt export pump increased by 230.7%, while the biliary excretion index value of deuterium-labeled sodium taurocholate (d8-TCA) differed slightly from the SC value (72% and 77%, respectively, p = .0195). The inhibitory effect of cholestasis drugs on FEK-SAPH was significantly higher than that of SC. In FEK-SAPH, hepatoprotective drugs were more effective in antagonizing hepatotoxicity induced by lithocholic acid (LCA). FEK-SAPH cultured RPH with hepatoprotective drugs can better recover from LCA-induced damage. In summary, FEK-SAPH can be used as a substitute for SC for pharmacokinetic screening to evaluate the drug absorption, disposition, metabolism, excretion, and toxicity (ADMET) in hepatocytes.
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Affiliation(s)
- Ting He
- School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, China.,Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China
| | - Shida Qiao
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Chen Ma
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Zhaoliang Peng
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Zhitao Wu
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China.,Nanjing University of Chinese Medicine, Nanjing, China
| | - Chenhui Ma
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Li Han
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Qiangqiang Deng
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Tianwei Zhang
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Yishen Zhu
- College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, China
| | - Guoyu Pan
- Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.,University of Chinese Academy of Sciences, Beijing, China
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16
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de Hoyos-Vega JM, Hong HJ, Stybayeva G, Revzin A. Hepatocyte cultures: From collagen gel sandwiches to microfluidic devices with integrated biosensors. APL Bioeng 2021; 5:041504. [PMID: 34703968 PMCID: PMC8519630 DOI: 10.1063/5.0058798] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2021] [Accepted: 09/21/2021] [Indexed: 02/06/2023] Open
Abstract
Hepatocytes are parenchymal cells of the liver responsible for drug detoxification, urea and bile production, serum protein synthesis, and glucose homeostasis. Hepatocytes are widely used for drug toxicity studies in bioartificial liver devices and for cell-based liver therapies. Because hepatocytes are highly differentiated cells residing in a complex microenvironment in vivo, they tend to lose hepatic phenotype and function in vitro. This paper first reviews traditional culture approaches used to rescue hepatic function in vitro and then discusses the benefits of emerging microfluidic-based culture approaches. We conclude by reviewing integration of hepatocyte cultures with bioanalytical or sensing approaches.
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Affiliation(s)
- Jose M. de Hoyos-Vega
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Hye Jin Hong
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota 55902, USA
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17
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DiProspero TJ, Dalrymple E, Lockett MR. Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells. Toxicol In Vitro 2021; 74:105156. [PMID: 33811995 PMCID: PMC8111698 DOI: 10.1016/j.tiv.2021.105156] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2020] [Revised: 03/10/2021] [Accepted: 03/29/2021] [Indexed: 01/20/2023]
Abstract
This study evaluates the impact of physiologically relevant oxygen tensions on the response of HepG2 cells to known inducers and hepatotoxic drugs. We compared transcriptional regulation and CYP1A activity after a 48 h exposure at atmospheric culture conditions (20% O2) with representative periportal (8% O2) and perivenous (3% O2) oxygen tensions. We evaluated cellular responses in 2D and 3D cultures at each oxygen tension in parallel, using monolayers and a paper-based culture platform that supports cells suspended in a collagen-rich environment. Our findings highlight that the toxicity, potency, and mechanism of action of drugs are dependent on both culture format and oxygen tension. HepG2 cells in 3D environments at physiologic oxygen tensions better matched primary human hepatocyte data than HepG2 cells cultured under standard conditions. Despite altered transcriptional regulation with decreasing oxygen tensions, we did not observe the zonation patterns of drug-metabolizing enzymes found in vivo. Our approach demonstrates that oxygen is an important regulator of liver function but it is not the sole regulator. It also highlights the utility of the 3D paper-based culture platform for continued mechanistic studies of microenvironmental influences on cellular responses.
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Affiliation(s)
- Thomas J DiProspero
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States of America
| | - Erin Dalrymple
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States of America
| | - Matthew R Lockett
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States of America; Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, 450 West Drive, Chapel Hill, NC 27599-7295, United States of America.
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18
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Ghosh S, Börsch A, Ghosh S, Zavolan M. The transcriptional landscape of a hepatoma cell line grown on scaffolds of extracellular matrix proteins. BMC Genomics 2021; 22:238. [PMID: 33823809 PMCID: PMC8025518 DOI: 10.1186/s12864-021-07532-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2020] [Accepted: 03/14/2021] [Indexed: 11/10/2022] Open
Abstract
Background The behavior of cells in vivo is complex and highly dynamic, as it results from an interplay between intercellular matrix proteins with surface receptors and other microenvironmental cues. Although the effects of the cellular niche have been investigated for a number of cell types using different molecular approaches, comprehensive assessments of how the global transcriptome responds to 3D scaffolds composed of various extracellular matrix (ECM) constituents at different concentrations are still lacking. Results In this study, we explored the effects of two diverse extracellular matrix (ECM) components, Collagen I and Matrigel, on the transcriptional profile of cells in a cell culture system. Culturing Huh-7 cells on traditional cell culture plates (Control) or on the ECM components at different concentrations to modulate microenvironment properties, we have generated transcriptomics data that may be further explored to understand the differentiation and growth potential of this cell type for the development of 3D cultures. Our analysis infers transcription factors that are most responsible for the transcriptome response to the extracellular cues. Conclusion Our data indicates that the Collagen I substrate induces a robust transcriptional response in the Huh-7 cells, distinct from that induced by Matrigel. Enhanced hepatocyte markers (ALB and miR-122) reveal a potentially robust remodelling towards primary hepatocytes. Our results aid in defining the appropriate culture and transcription pathways while using hepatoma cell lines. As systems mimicking the in vivo structure and function of liver cells are still being developed, our study could potentially circumvent bottlenecks of limited availability of primary hepatocytes for preclinical studies of drug targets. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-07532-2.
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Affiliation(s)
- Souvik Ghosh
- Biozentrum, University of Basel, Basel, Switzerland.
| | - Anastasiya Börsch
- Biozentrum, University of Basel, Basel, Switzerland.,Swiss Institute of Bioinformatics, Lausanne, Switzerland
| | | | - Mihaela Zavolan
- Biozentrum, University of Basel, Basel, Switzerland. .,Swiss Institute of Bioinformatics, Lausanne, Switzerland.
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19
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Lim RHG, Liew JXK, Wee A, Masilamani J, Chang SKY, Phan TT. Safety Evaluation of Human Cord-Lining Epithelial Stem Cells Transplantation for Liver Regeneration in a Porcine Model. Cell Transplant 2021; 29:963689719896559. [PMID: 32166974 PMCID: PMC7444229 DOI: 10.1177/0963689719896559] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
We investigated the safety of using umbilical cord-lining stem cells for liver regeneration and tested a novel method for stem cell delivery. Stem cells are known by their ability to repair damaged tissues and have the potential to be used as regenerative therapies. The umbilical cord's outer lining membrane is known to be a promising source of multipotent stem cells and can be cultivated in an epithelial cell growth medium to produce cell populations which possess the properties of both epithelial cells and embryonic stem cells-termed cord-lining epithelial cells (CLEC). Hepatocytes are epithelial cells of the liver and their proliferation upon injury is the main mechanism in restoring the liver. Earlier studies conducted showed CLEC can be differentiated into functioning hepatocyte-like cells (HLC) and can survive in immunologically competent specimens. In this study, we chose a porcine model to investigate CLEC as a treatment modality for liver failure. We selected 16 immune competent Yorkshire-Dutch Landrace pigs, with a mean weight of 40.5 kg, for this study. We performed a 50% hepatectomy to simulate the liver insufficient disease model. After the surgery, four pigs were transplanted with a saline scaffold while seven pigs were transplanted with a HLC scaffold. Five pigs died on the surgical table and were omitted from the study analysis. This study addressed the safety of transplanting human CLEC in a large animal model. The transplant interfaces were evaluated and no signs of cellular rejection were observed in both groups.
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Affiliation(s)
| | | | - Aileen Wee
- Department of Pathology, Singapore National University Hospital, Singapore
| | | | - Stephen Kin Yong Chang
- Department of Surgery, Singapore National University Hospital, Singapore.,Glad Clinic Pte. Ltd, Singapore
| | - Toan Thang Phan
- Department of Surgery, Singapore National University Hospital, Singapore.,CellResearch Corporation Pte. Ltd, Singapore
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20
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Abstract
Accurate estimation of in vivo clearance in human is pivotal to determine the dose and dosing regimen for drug development. In vitro-in vivo extrapolation (IVIVE) has been performed to predict drug clearance using empirical and physiological scalars. Multiple in vitro systems and mathematical modeling techniques have been employed to estimate in vivo clearance. The models for predicting clearance have significantly improved and have evolved to become more complex by integrating multiple processes such as drug metabolism and transport as well as passive diffusion. This chapter covers the use of conventional as well as recently developed methods to predict metabolic and transporter-mediated clearance along with the advantages and disadvantages of using these methods and the associated experimental considerations. The general approaches to improve IVIVE by use of appropriate scalars, incorporation of extrahepatic metabolism and transport and application of physiologically based pharmacokinetic (PBPK) models with proteomics data are also discussed. The chapter also provides an overview of the advantages of using such dynamic mechanistic models over static models for clearance predictions to improve IVIVE.
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21
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Abstract
Microphysiological systems (MPS), often referred to as "organ-on-chips," are microfluidic-based in vitro models that aim to recapitulate the dynamic chemical and mechanical microenvironment of living organs. MPS promise to bridge the gap between in vitro and in vivo models and ultimately improve the translation from preclinical animal studies to clinical trials. However, despite the explosion of interest in this area in recent years, and the obvious rewards for such models that could improve R&D efficiency and reduce drug attrition in the clinic, the pharmaceutical industry has been slow to fully adopt this technology. The ability to extract robust, quantitative information from MPS at scale is a key requirement if these models are to impact drug discovery and the subsequent drug development process. Microscopy imaging remains a core technology that enables the capture of information at the single-cell level and with subcellular resolution. Furthermore, such imaging techniques can be automated, increasing throughput and enabling compound screening. In this review, we discuss a range of imaging techniques that have been applied to MPS of varying focus, such as organoids and organ-chip-type models. We outline the opportunities these technologies can bring in terms of understanding mechanistic biology, but also how they could be used in higher-throughput screens, widening the scope of their impact in drug discovery. We discuss the associated challenges of imaging these complex models and the steps required to enable full exploitation. Finally, we discuss the requirements for MPS, if they are to be applied at a scale necessary to support drug discovery projects.
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Affiliation(s)
- Samantha Peel
- Discovery Biology, Discovery Sciences, R&D, AstraZeneca, Cambridge, United Kingdom
| | - Mark Jackman
- Advanced Drug Delivery, Pharmaceutical Sciences, R&D, AstraZeneca, Cambridge, United Kingdom
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22
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Ponsoda X, Gómez-Lechón MJ, Castell JV. Toxicity and Cell Density Monitoring in Monolayer and Three-dimensional Cultures with the XTT Assay. Altern Lab Anim 2020. [DOI: 10.1177/026119299802600308] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
The application of viability criteria (MTT and XTT tests) to monolayer cultures and immobilised cells in three-dimensional systems was investigated in order to assess cell viability and cell proliferation. The suitability and accuracy of these tests were compared with the conventional criteria (cellular protein and DNA content) used in monolayer cultures for the same purpose. The colorimetric assay based on the metabolic reduction of the tetrazolium salt XTT to a water-soluble formazan proved to be very useful, rapid and sensitive. This automated spectrophotometric enzymatic method, due to its lack of toxicity, also permits repeated nondestructive assays on a single cellular culture for the long-term monitoring of cytotoxicity, cell survival and cell proliferation, and can be performed in 96-well plates with minimal handling. This method could offer a solution for cellular density evaluation in complex cell cultures that do not permit visual examination; it is also the best choice for protein-based, three-dimensional systems such as collagen gels.
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Affiliation(s)
- Xavier Ponsoda
- Departament de Parasitologia, i Biologia Cellular, Facultat de Ciències Biològiques, Universitat de València, Avda Dr Moliner 50, 46100 Burjassot, Spain
- Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain
| | - Maria Jose Gómez-Lechón
- Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain
| | - Jose V. Castell
- Unidad de Hepatología Experimental, Centro de Investigación, Hospital Universitario La Fe, Avda Campanar 21, 46009 Valencia, Spain
- Departament de Bioquimíca i Biologia Molecular, Facultat de Medicina, Universitat de València, Avda Blasco Ibáñez 10, 46010 Valencia, Spain
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23
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Choi JH, Loarca L, De Hoyos-Vega JM, Dadgar N, Loutherback K, Shah VH, Stybayeva G, Revzin A. Microfluidic confinement enhances phenotype and function of hepatocyte spheroids. Am J Physiol Cell Physiol 2020; 319:C552-C560. [PMID: 32697600 DOI: 10.1152/ajpcell.00094.2020] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
A number of cell culture approaches have been described for maintenance of primary hepatocytes. Forming hepatocytes into three-dimensional (3-D) spheroids is one well-accepted method for extending epithelial phenotype of these cells. Our laboratory has previously observed enhanced function of two-dimensional (2-D, monolayer) hepatocyte cultures in microfluidic devices due to increased production of several hepato-inductive growth factors, including hepatocyte growth factor (HGF). In the present study, we wanted to test a hypothesis that culturing hepatocyte spheroids (3-D) in microfluidic devices will also result in enhanced phenotype and function. To test this hypothesis, we fabricated devices with small and large volumes. Both types of devices included a microstructured floor containing arrays of pyramidal wells to promote assembly of hepatocytes into spheroids with individual diameters of ~100 µm. The hepatocyte spheroids were found to be more functional, as evidenced by higher level of albumin synthesis, bile acid production, and hepatic enzyme expression, in low-volume compared with large-volume devices. Importantly, high functionality of spheroid cultures correlated with elevated levels of HGF secretion. Although decay of hepatic function (albumin secretion) was observed over the course 3 wk, this behavior could be abrogated by inhibiting TGF-β1 signaling. With TGF-β1 inhibitor, microfluidic hepatocyte spheroid cultures maintained high and stable levels of albumin synthesis over the course of 4 wk. To further highlight utility of this culture platform for liver disease modeling, we carried out alcohol injury experiments in microfluidic devices and tested protective effects of interleukin-22: a potential therapy for alcoholic hepatitis.
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Affiliation(s)
- Jong Hoon Choi
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Lorena Loarca
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Jose M De Hoyos-Vega
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Neda Dadgar
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Kevin Loutherback
- Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota
| | - Vijay H Shah
- Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, Minnesota
| | - Gulnaz Stybayeva
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
| | - Alexander Revzin
- Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, Minnesota
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24
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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.
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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
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25
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Ishida S. Requirements for designing organ-on-a-chip platforms to model the pathogenesis of liver disease. ORGAN-ON-A-CHIP 2020:181-213. [DOI: 10.1016/b978-0-12-817202-5.00005-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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26
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Arez F, Rebelo SP, Fontinha D, Simão D, Martins TR, Machado M, Fischli C, Oeuvray C, Badolo L, Carrondo MJT, Rottmann M, Spangenberg T, Brito C, Greco B, Prudêncio M, Alves PM. Flexible 3D Cell-Based Platforms for the Discovery and Profiling of Novel Drugs Targeting Plasmodium Hepatic Infection. ACS Infect Dis 2019; 5:1831-1842. [PMID: 31479238 DOI: 10.1021/acsinfecdis.9b00144] [Citation(s) in RCA: 26] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The restricted pipeline of drugs targeting the liver stage of Plasmodium infection reflects the scarcity of cell models that mimic the human hepatic phenotype and drug metabolism, as well as Plasmodium hepatic infection. Using stirred-tank culture systems, spheroids of human hepatic cell lines were generated, sustaining a stable hepatic phenotype over 4 weeks of culture. Spheroids were employed in the establishment of 3D Plasmodium berghei infection platforms that relied on static or dynamic culture conditions. P. berghei invasion and development were recapitulated in the hepatic spheroids, yielding blood-infective merozoites. The translational potential of the 3D platforms was demonstrated by comparing the in vitro minimum inhibitory concentration of M5717, a compound under clinical development, with in vivo plasma concentrations that clear liver stage P. berghei in mice. Our results show that the 3D platforms are flexible and scalable and can predict the efficacy of antiplasmodial therapies, constituting a powerful tool for integration in drug discovery programs.
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Affiliation(s)
- Francisca Arez
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Sofia P. Rebelo
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Diana Fontinha
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | - Daniel Simão
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Tatiana R. Martins
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Marta Machado
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | - Christoph Fischli
- Swiss Tropical and Public Health Institute, Basel 4051, Switzerland
- University of Basel, Basel 4003, Switzerland
| | - Claude Oeuvray
- Global Health Institute of Merck, Ares Trading S.A., a subsidiary of Merck KGaA, Darmstadt, Germany, 1262 Eysins, Switzerland
| | - Lassina Badolo
- Discovery and Development Technologies, Merck Healthcare KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
| | - Manuel J. T. Carrondo
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Matthias Rottmann
- Swiss Tropical and Public Health Institute, Basel 4051, Switzerland
- University of Basel, Basel 4003, Switzerland
| | - Thomas Spangenberg
- Global Health Institute of Merck, Ares Trading S.A., a subsidiary of Merck KGaA, Darmstadt, Germany, 1262 Eysins, Switzerland
| | - Catarina Brito
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
| | - Beatrice Greco
- Global Health Institute of Merck, Ares Trading S.A., a subsidiary of Merck KGaA, Darmstadt, Germany, 1262 Eysins, Switzerland
| | - Miguel Prudêncio
- Instituto de Medicina Molecular João Lobo Antunes, Faculdade de Medicina, Universidade de Lisboa, 1649-028 Lisboa, Portugal
| | - Paula M. Alves
- iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
- Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Avenida da República, 2780-157 Oeiras, Portugal
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27
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Sato T, Semura K, Fujimoto I. Micro‑dimpled surface atelocollagen maintains primary human hepatocytes in culture and may promote their functionality compared with collagen coat culture. Int J Mol Med 2019; 44:960-972. [PMID: 31257473 PMCID: PMC6657980 DOI: 10.3892/ijmm.2019.4251] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2018] [Accepted: 06/04/2019] [Indexed: 11/11/2022] Open
Abstract
Primary human hepatocytes (PHHs) are the gold standard for drug development procedures; however, maintaining functional PHHs in vitro is challenging in conventional collagen-coated cultures. In the present study, we developed a new scaffold comprising high amounts (≥1 mg/cm2) of atelocollagen exposed to ultraviolet radiation to induce cross-linking and improve stability. Scanning and transmission electron microscopy revealed a micro-dimpled surface (MDS) scaffold composed of randomly arranged atelocollagen fibrils. The scaffold was therefore designated as MDS atelocollagen. PHHs cultured on MDS atelocollagen were round with a compact cytoplasm and exhibited enhanced levels of albumin (ALB) secretion and cytochrome P450 (CYP) 3A4 activity. The expression of hepatocyte-related genes, such as serum proteins, drug metabolism-related CYPs, and nuclear receptors, was enhanced in cells cultured on MDS atelocollagen, but not in those cultured on conventional atelocollagen. Moreover, the abnormal gene expression of cell adhesion molecules observed in conventional atelocollagen culture was suppressed when the cells were grown on MDS atelocollagen, thereby suggesting a cell behavior similar to that of in vivo hepatocytes. These results suggest that MDS atelocollagen functionally preserves PHHs while conserving the simplicity of conventional PHH atelocollagen-coated cultures.
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Affiliation(s)
- Tetsuro Sato
- Koken Research Center, Koken Co., Ltd., Tokyo 115‑0051, Japan
| | - Kayoko Semura
- Koken Research Center, Koken Co., Ltd., Tokyo 115‑0051, Japan
| | - Ichiro Fujimoto
- Koken Research Center, Koken Co., Ltd., Tokyo 115‑0051, Japan
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28
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Wu L, Ferracci G, Wang Y, Fan TF, Cho NJ, Chow PKH. Porcine hepatocytes culture on biofunctionalized 3D inverted colloidal crystal scaffolds as an in vitro model for predicting drug hepatotoxicity. RSC Adv 2019; 9:17995-18007. [PMID: 35520590 PMCID: PMC9064660 DOI: 10.1039/c9ra03225h] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2019] [Accepted: 05/27/2019] [Indexed: 01/03/2023] Open
Abstract
As drug-induced hepatotoxicity represents one of the most common causes of drug failure, three-dimensional (3D) in vitro liver platforms represent a fantastic toolbox to predict drug toxicity and thus reduce in vivo animal studies and lessen drug attrition rates. The aim of this study is to establish a functional porcine hepatocyte culture using a biofunctionalized 3D inverted colloidal crystal (ICC) hydrogel platform. The performances of non-adhesive bare poly(ethylene glycol)diacrylate (PEGDA) ICCs and PEGDA ICCs coated with either collagen type I or fibronectin have been investigated. Porcine hepatocytes viability, morphology, hepatic-specific functions and patterns of gene expression have been evaluated over a period of two weeks in culture to test diclofenac, a well-known hepatotoxic drug. Interestingly, cells in the fibronectin-functionalized scaffold exhibit different aggregation patterns and maintain better liver-specific function than those in bare ICCs and in collagen functionalized scaffold. We concluded that the 3D cell culture environment and the presence of extracellular matrix (ECM) proteins, especially fibronectin, facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro, and enable us to predict hepatotoxicity. As drug-induced hepatotoxicity represents one of the most common causes of drug failure, three-dimensional in vitro liver platforms represent a fantastic toolbox to predict drug toxicity and reduce in vivo studies and drug attrition rates.![]()
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Affiliation(s)
- Lingyan Wu
- Division of Surgical Oncology, National Cancer Centre Singapore 11 Hospital Drive 169610 Singapore
| | - Gaia Ferracci
- Interdisciplinary Graduate School, NTU Institute for Health Technologies, Nanyang Technological University 50 Nanyang Drive 637553 Singapore.,School of Materials Science and Engineering, Nanyang Technological University 50 Nanyang Avenue 639798 Singapore .,Centre for Biomimetic Sensor Science, Nanyang Technological University 50 Nanyang Drive 637553 Singapore
| | - Yan Wang
- School of Materials Science and Engineering, Nanyang Technological University 50 Nanyang Avenue 639798 Singapore .,Centre for Biomimetic Sensor Science, Nanyang Technological University 50 Nanyang Drive 637553 Singapore
| | - Teng Fei Fan
- School of Materials Science and Engineering, Nanyang Technological University 50 Nanyang Avenue 639798 Singapore .,Centre for Biomimetic Sensor Science, Nanyang Technological University 50 Nanyang Drive 637553 Singapore
| | - Nam-Joon Cho
- School of Materials Science and Engineering, Nanyang Technological University 50 Nanyang Avenue 639798 Singapore .,Centre for Biomimetic Sensor Science, Nanyang Technological University 50 Nanyang Drive 637553 Singapore.,School of Chemical and Biomedical Engineering, Nanyang Technological University 62 Nanyang Drive 637459 Singapore
| | - Pierce K H Chow
- Division of Surgical Oncology, National Cancer Centre Singapore 11 Hospital Drive 169610 Singapore .,Duke-NUS Medical School 8 College Road 169857 Singapore.,Department of Hepatopancreatobiliary and Transplant Surgery, Singapore General Hospital Outram Road 169608 Singapore
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29
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Evaluation of Drug Biliary Excretion Using Sandwich-Cultured Human Hepatocytes. Eur J Drug Metab Pharmacokinet 2019; 44:13-30. [PMID: 30167999 DOI: 10.1007/s13318-018-0502-x] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Evaluation of hepatobiliary transport of drugs is an important challenge, notably during the development of new molecular identities. In this context, sandwich-cultured human hepatocytes (SCHH) have been proposed as an interesting and integrated tool for predicting in vitro biliary excretion of drugs. The present review was therefore designed to summarize key findings about SCHH, including their establishment, their main functional features and their use for the determination of canalicular transport and the prediction of in vivo biliary clearance and hepatobiliary excretion-related drug-drug interactions. Reviewed data highlight the fact that SCHH represent an original and probably unique holistic in vitro approach to predict biliary clearance in humans, through taking into account sinusoidal drug uptake, passive drug diffusion, drug metabolism and sinusoidal and canalicular drug efflux. Limits and proposed refinements for SCHH-based analysis of drug biliary excretion, as well as putative human alternative in vitro models to SCHH are also discussed.
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30
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Tan K, Keegan P, Rogers M, Lu M, Gosset JR, Charest J, Bale SS. A high-throughput microfluidic microphysiological system (PREDICT-96) to recapitulate hepatocyte function in dynamic, re-circulating flow conditions. LAB ON A CHIP 2019; 19:1556-1566. [PMID: 30855604 DOI: 10.1039/c8lc01262h] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/15/2023]
Abstract
Microphysiological systems (MPSs) are dynamic cell culture systems that provide micro-environmental and external cues to support physiologically relevant, organ-specific functions. Recent progresses in MPS fabrication technologies have enabled the development of advanced models to capture microenvironments with physiological relevance, while increasing throughput and reducing material-based artefacts. In addition to conventional cell culture systems, advanced MPSs are emerging as ideal contenders for disease modeling and incorporation into drug screening. Since liver is a central organ for drug metabolism, liver-on-chip models have been developed to recapitulate hepatic microenvironment with varying complexities, while allowing long-term culture. Recently, we have developed a novel thermoplastic, oxygen-permeable MPS for primary human hepatocyte (PHH) culture. Herein, we have adapted and extended the MPS to a) a 96 microfluidic array (PREDICT-96 array) and b) integrated a novel, ultra-low volume, re-circulating pumping system (PREDICT-96 pump) - collectively known as the PREDICT-96 platform. The PREDICT-96 platform conforms to the industrial standard 96-well footprint and enables media re-circulation. First, we demonstrate the introduction of PHHs into the PREDICT-96 array using standard handling procedures for multi-well plates and allow cells to stabilize in static conditions. Next, we introduce recirculating flow into the bottom channel (using PREDICT-96 pump) to mimic mass transport in vivo. Our results indicate an increase in metabolic and secretory functions of PHHs in the PREDICT-96 platform, and their maintenance over 10 days of flow. Furthermore, long-term culture with fluid flow allows for the periodic introduction of media components (e.g., fatty acids, cytokines) and capture cellular responses to chronic stimuli. The low-volume footprint of the pump and small media volume in the MPS allow for the interrogation of hepatic responses incorporating secretion feedback to a stimulus, which is essential for disease model development and drug interrogation. We envision future development of this liver model to incorporate key primary hepatic cells, multi-cellular co-cultures and adaptation, integration with high-throughput analytical tools.
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Affiliation(s)
- Kelly Tan
- Draper, 555 Technology Square, Cambridge, MA 02138, USA.
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31
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Canová N, Kmonícková E, Lincová D, Vítek L, Farghali H. Evaluation of a Flat Membrane Hepatocyte Bioreactor for Pharmacotoxicological Applications: Evidence that Inhibition of Spontaneously Produced Nitric Oxide Improves Cell Functionality. Altern Lab Anim 2019; 32:25-35. [PMID: 15603551 DOI: 10.1177/026119290403200106] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes. In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2-3 days. Hepatocytes cultured in the FMB, specifically in a "sandwich" configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production. Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB. The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that minor changes could be observed at diverse time intervals and in a non-destructive way. The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity. In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.
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Affiliation(s)
- Nikolina Canová
- Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Albertov 4, 12800 Prague 2, Czech Republic.
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32
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Bale SS, Borenstein JT. Microfluidic Cell Culture Platforms to Capture Hepatic Physiology and Complex Cellular Interactions. Drug Metab Dispos 2018; 46:1638-1646. [PMID: 30115643 DOI: 10.1124/dmd.118.083055] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2018] [Accepted: 08/14/2018] [Indexed: 02/13/2025] Open
Abstract
Animal models such as rats and primates provide body-wide information for drug and metabolite responses, including organ-specific toxicity and any unforeseen side effects on other organs. Although effective in the drug-screening process, their translatability to humans is limited because of the lack of high concordance and correlation among enzymatic mechanisms, cellular mechanisms, and resulting toxicities. A significant mode of failure for safety prediction in drug screening is hepatotoxicity, resulting in ∼30% of all safety-related drug failures and withdrawals from the market. The liver is a multifunctional organ with diverse metabolic, secretory, and inflammatory response roles and is essential for maintaining key body functions. Conventional cell culture platforms (such as multiwell plate cultures) and metabolic enzyme systems (microsomes, cytochrome P450 enzymes) have been routinely used to assess drug pharmacokinetics and metabolism. However, current in vitro models often fail to recapitulate the complexity and dynamic nature of human tissues, imposing a heavy reliance on in vivo testing using preclinical species that have metabolic processes, disease mechanisms, and modes of toxicity distinct from humans. Recently, microphysiological systems (MPS) have gained attention as powerful tools with the potential to generate human-relevant information that can supplant and fill the gap of knowledge between preclinical animal models and simpler, conventional in vitro cell culture systems. Developments in microfabrication technologies for generating complex microfluidic systems, along with the ability to establish and maintain multicellular models to capture dynamic, human-relevant behavior, have provided new avenues to generate such physiologically relevant systems. These MPS platforms, when designed and developed with in vivo-derived design parameters, have the potential to capture key aspects and better mimic organ functionality. In this review, we discuss developments in microtechnologies for fabricating, establishing, and maintaining hepatic cell culture systems, with a specific focus on models that aim to capture in vivo physiology in vitro. By designing microscale systems to impart specific in vivo physiologic parameters, it is possible to create a dynamic system that can capture multiple aspects of the hepatic microenvironment, bringing us closer to a comprehensive in vitro testing platform for hepatic responses and toxicities.
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Affiliation(s)
- Shyam Sundhar Bale
- Cellular and Tissue Engineering, and Synthetic Biology and Bio-Instrumentation, Draper, Cambridge, Massachusetts
| | - Jeffrey T Borenstein
- Cellular and Tissue Engineering, and Synthetic Biology and Bio-Instrumentation, Draper, Cambridge, Massachusetts
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33
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Gokduman K, Bestepe F, Li L, Yarmush ML, Usta OB. Dose-, treatment- and time-dependent toxicity of superparamagnetic iron oxide nanoparticles on primary rat hepatocytes. Nanomedicine (Lond) 2018; 13:1267-1284. [PMID: 29949471 PMCID: PMC6219434 DOI: 10.2217/nnm-2017-0387] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2017] [Accepted: 03/13/2018] [Indexed: 12/12/2022] Open
Abstract
AIM As a first study in literature, to investigate concentration-dependent (0-400 μg/ml) and exposure-dependent (single dosing vs cumulative dosing) effects of superparamagnetic iron oxide nanoparticles (d = 10 nm) on primary rat hepatocytes in a time-dependent manner. MATERIALS & METHODS Sandwich-cultured hepatocyte model was used to evaluate viability, hepatocyte specific functions and reactive oxygen species level. RESULTS In terms of all parameters, generally statistically more significant effects were observed in a concentration- and time-dependent manner. In terms of hepatocyte-specific functions, cumulative dosing caused significantly (p < 0.05) more deleterious effects at 48th hour. CONCLUSION A combination of various biomarkers should be employed for the evaluation of the effect of superparamagnetic iron oxide nanoparticles on liver, and each biomarker should be analyzed in a time- and exposure-dependent manner.
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Affiliation(s)
- Kurtulus Gokduman
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals, Boston, MA 02114, USA
| | - Furkan Bestepe
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals, Boston, MA 02114, USA
- School of Medicine, Ankara University, Ankara 06100, Turkey
| | - Lei Li
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals, Boston, MA 02114, USA
- Key Laboratory of Cryogenics, Technical Institute of Physics & Chemistry, Chinese Academy of Sciences, Beijing 100190, China
| | - Martin L Yarmush
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals, Boston, MA 02114, USA
- Department of Biomedical Engineering, Rutgers State University, Piscataway, NJ 08854, USA
| | - O Berk Usta
- Center for Engineering in Medicine, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals, Boston, MA 02114, USA
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34
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Brown JH, Das P, DiVito MD, Ivancic D, Tan LP, Wertheim JA. Nanofibrous PLGA electrospun scaffolds modified with type I collagen influence hepatocyte function and support viability in vitro. Acta Biomater 2018; 73:217-227. [PMID: 29454157 PMCID: PMC5985221 DOI: 10.1016/j.actbio.2018.02.009] [Citation(s) in RCA: 67] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2017] [Revised: 01/23/2018] [Accepted: 02/08/2018] [Indexed: 02/06/2023]
Abstract
A major challenge of maintaining primary hepatocytes in vitro is progressive loss of hepatocyte-specific functions, such as protein synthesis and cytochrome P450 (CYP450) catalytic activity. We developed a three-dimensional (3D) nanofibrous scaffold made from poly(l-lactide-co-glycolide) (PLGA) polymer using a newly optimized wet electrospinning technique that resulted in a highly porous structure that accommodated inclusion of primary human hepatocytes. Extracellular matrix (ECM) proteins (type I collagen or fibronectin) at varying concentrations were chemically linked to electrospun PLGA using amine coupling to develop an in vitro culture system containing the minimal essential ECM components of the liver micro-environment that preserve hepatocyte function in vitro. Cell-laden nanofiber scaffolds were tested in vitro to maintain hepatocyte function over a two-week period. Incorporation of type I collagen onto PLGA scaffolds (PLGA-Chigh: 100 µg/mL) led to 10-fold greater albumin secretion, 4-fold higher urea synthesis, and elevated transcription of hepatocyte-specific CYP450 genes (CYP3A4, 3.5-fold increase and CYP2C9, 3-fold increase) in primary human hepatocytes compared to the same cells grown within unmodified PLGA scaffolds over two weeks. These indices, measured using collagen-bonded scaffolds, were also higher than scaffolds coupled to fibronectin or an ECM control sandwich culture composed of type I collagen and Matrigel. Induction of CYP2C9 activity was also higher in these same type I collagen PLGA scaffolds compared to other ECM-modified or unmodified PLGA constructs and was equivalent to the ECM control at 7 days. Together, we demonstrate a minimalist ECM-based 3D synthetic scaffold that accommodates primary human hepatocyte inclusion into the matrix, maintains long-term in vitro survival and stimulates function, which can be attributed to coupling of type I collagen. STATEMENT OF SIGNIFICANCE Culturing primary hepatocytes within a three-dimensional (3D) structure that mimics the natural liver environment is a promising strategy for extending the function and viability of hepatocytes in vitro. In the present study we generate porous PLGA nanofibers, that are chemically modified with extracellular matrix proteins, to serve as 3D scaffolds for the in vitro culture of primary human hepatocytes. Our findings demonstrate that the use of ECM proteins, especially type I collagen, in a porous 3D environment helps to improve the synthetic function of primary hepatocytes over time. We believe the work presented within will provide insights to readers for drug toxicity and tissue engineering applications.
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Affiliation(s)
- Jessica H Brown
- Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States
| | - Prativa Das
- Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore
| | - Michael D DiVito
- Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States
| | - David Ivancic
- Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States
| | - Lay Poh Tan
- Interdisciplinary Graduate School, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798, Singapore; School of Materials Science and Engineering, Nanyang Technological University, Singapore 639798 Singapore..
| | - Jason A Wertheim
- Comprehensive Transplant Center, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Department of Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, United States; Chemistry of Life Processes Institute, Northwestern University, Evanston, IL 60208, United States; Department of Surgery, Jesse Brown VA Medical Center, Chicago, IL 60612, United States; Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, Chicago, IL 60611, United States; Department of Biomedical Engineering, Northwestern University, Evanston, IL 60208, United States.
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Yang W, Chen Q, Xia R, Zhang Y, Shuai L, Lai J, You X, Jiang Y, Bie P, Zhang L, Zhang H, Bai L. A novel bioscaffold with naturally-occurring extracellular matrix promotes hepatocyte survival and vessel patency in mouse models of heterologous transplantation. Biomaterials 2018; 177:52-66. [PMID: 29885586 DOI: 10.1016/j.biomaterials.2018.05.026] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2018] [Revised: 05/14/2018] [Accepted: 05/16/2018] [Indexed: 01/26/2023]
Abstract
BACKGROUND Naïve decellularized liver scaffold (nDLS)-based tissue engineering has been impaired by the lack of a suitable extracellular matrix (ECM) to provide "active micro-environmental" support. AIM The present study aimed to examine whether a novel, regenerative DLS (rDLS) with an active ECM improves primary hepatocyte survival and prevents thrombosis. METHODS rDLS was obtained from a 30-55% partial hepatectomy that was maintained in vivo for 3-5 days and then perfused with detergent in vitro. Compared to nDLS generated from normal livers, rDLS possesses bioactive molecules due to the regenerative period in vivo. Primary mouse hepatocyte survival was evaluated by staining for Ki-67 and Trypan blue exclusion. Thrombosis was assessed by immunohistochemistry and ex vivo diluted whole-blood perfusion. Hemocompatibility was determined by near-infrared laser-Doppler flowmetry and heterotopic transplantation. RESULTS After recellularization, rDLS contained more Ki-67-positive primary hepatocytes than nDLS. rDLS had a higher oxygen saturation and blood flow velocity and a lower expression of integrin αIIb and α4 than nDLS. Tumor necrosis factor-α, hepatocyte growth factor, interleukin-10, interleukin-6 and interleukin-1β were highly expressed throughout the rDLS, whereas expression of collagen-I, collagen-IV and thrombopoietin were lower in rDLS than in nDLS. Improved blood vessel patency was observed in rDLS both in vitro and in vivo. The results in mice were confirmed in large animals (pigs). CONCLUSION rDLS is an effective DLS with an "active microenvironment" that supports primary hepatocyte survival and promotes blood vessel patency. This is the first study to demonstrate a rDLS with a blood microvessel network that promotes hepatocyte survival and resists thrombosis.
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Affiliation(s)
- Wei Yang
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China; Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Quanyu Chen
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China; Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Renpei Xia
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Yujun Zhang
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Ling Shuai
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Jiejuan Lai
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Xiaolin You
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Yan Jiang
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Ping Bie
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China
| | - Leida Zhang
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China.
| | - Hongyu Zhang
- Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China.
| | - Lianhua Bai
- Key Laboratory of Freshwater Fish Reproduction and Development, Ministry of Education, Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Beibei, 400715 Chongqing, China; Hepatobiliary Institute, Southwest Hospital, The Army Medical University, Chongqing 400038, China.
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36
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Sun T, Li X, Shi Q, Wang H, Huang Q, Fukuda T. Microfluidic Spun Alginate Hydrogel Microfibers and Their Application in Tissue Engineering. Gels 2018; 4:gels4020038. [PMID: 30674814 PMCID: PMC6209268 DOI: 10.3390/gels4020038] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2018] [Revised: 03/22/2018] [Accepted: 03/24/2018] [Indexed: 01/22/2023] Open
Abstract
Tissue engineering is focusing on processing tissue micro-structures for a variety of applications in cell biology and the “bottom-up” construction of artificial tissue. Over the last decade, microfluidic devices have provided novel tools for producing alginate hydrogel microfibers with various morphologies, structures, and compositions for cell cultivation. Moreover, microfluidic spun alginate microfibers are long, thin, and flexible, and these features facilitate higher-order assemblies for fabricating macroscopic cellular structures. In this paper, we present an overview of the microfluidic spinning principle of alginate hydrogel microfibers and their application as micro-scaffolds or scaffolding elements for 3D assembly in tissue engineering.
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Affiliation(s)
- Tao Sun
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
| | - Xingfu Li
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
| | - Qing Shi
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
| | - Huaping Wang
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
| | - Qiang Huang
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
| | - Toshio Fukuda
- Beijing Advanced Innovation Center for Intelligent Robots and Systems, Beijing Institute of Technology, 5 South Zhongguancun Street, Haidian District, Beijing 10081, China.
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37
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Koebe H, Wick M, Cramer U, Lange V, Schildberg F. Collagen Gel Immobilisation Provides a Suitable Cell Matrix for Long Term Human Hepatocyte Cultures in Hybrid Reactors. Int J Artif Organs 2018. [DOI: 10.1177/039139889401700207] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
An easy to apply culture technique is presented that protects a monolayer configuration of liver cells within an extracellular matrix. The Immobilising Gel (IG)-Technique not only preserves hepatocyte morphology and supports a variety of differentiated cell functions over long term periods, but also offers higher resistance of IG-culture systems against shear forces of fluids in a hybrid reactor device, as compared to other culture techniques. Human hepatocyte cultures in IG-Technique: DNA-normalised levels for the total production of cholinesterase, albumin, urea and lactate remained high throughout the investigational period (50 days). Glutamic-Pyruvic-Transaminase (GPT) release decreased after peak values during early culture adaptation. Electron Microscopic (EM) findings after the shear forces experiment revealed undisturbed subcellular structures and a preserved intercellular morphology, including bile canaliculi and desmosomes. We conclude that the IG-technique is of considerable advantage as compared to other culture systems, especially in the field of dynamic applications, e.g. hybrid reactors for artificial organ development.
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Affiliation(s)
- H.G. Koebe
- Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Muenchen - Germany
| | - M. Wick
- Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Muenchen - Germany
| | - U. Cramer
- Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Muenchen - Germany
| | - V. Lange
- Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Muenchen - Germany
| | - F.W. Schildberg
- Department of Surgery, Klinikum Grosshadern, L.M. University of Munich, Muenchen - Germany
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38
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Stange J, Mitzner S. Hepatocyte Encapsulation - Initial Intentions and New Aspects for Its Use in Bioartificial Liver Support. Int J Artif Organs 2018. [DOI: 10.1177/039139889601900107] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Affiliation(s)
- J. Stange
- Department of Internal Medicine, University of Rostock, Rostock - Germany
| | - S. Mitzner
- Department of Internal Medicine, University of Rostock, Rostock - Germany
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3D Co-Culture with Vascular Cells Supports Long-Term Hepatocyte Phenotype and Function In Vitro. REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2018. [DOI: 10.1007/s40883-018-0046-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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40
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Beckwitt CH, Clark AM, Wheeler S, Taylor DL, Stolz DB, Griffith L, Wells A. Liver 'organ on a chip'. Exp Cell Res 2018; 363:15-25. [PMID: 29291400 PMCID: PMC5944300 DOI: 10.1016/j.yexcr.2017.12.023] [Citation(s) in RCA: 155] [Impact Index Per Article: 22.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/19/2017] [Revised: 12/21/2017] [Accepted: 12/27/2017] [Indexed: 12/14/2022]
Abstract
The liver plays critical roles in both homeostasis and pathology. It is the major site of drug metabolism in the body and, as such, a common target for drug-induced toxicity and is susceptible to a wide range of diseases. In contrast to other solid organs, the liver possesses the unique ability to regenerate. The physiological importance and plasticity of this organ make it a crucial system of study to better understand human physiology, disease, and response to exogenous compounds. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems. These aspects have impelled many to develop liver tissue systems for study in isolation outside the body. Herein, we discuss these biologically engineered organoids and microphysiological systems.
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Affiliation(s)
- Colin H Beckwitt
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Research and Development Service, VA Pittsburgh Health System, Pittsburgh, PA 15240, USA
| | - Amanda M Clark
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Sarah Wheeler
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - D Lansing Taylor
- Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Donna B Stolz
- Cell Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA
| | - Linda Griffith
- Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Alan Wells
- Department of Pathology, University of Pittsburgh, Pittsburgh, PA 15213, USA; Computational and Systems Biology, University of Pittsburgh, Pittsburgh, PA 15213, USA; The McGowan Institute of Regenerative Medicine University of Pittsburgh, Pittsburgh, PA 15213, USA; Research and Development Service, VA Pittsburgh Health System, Pittsburgh, PA 15240, USA.
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41
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Diekmann S, Glöckner P, Bader A. The Influence of Different Cultivation Conditions on the Metabolic Functionality of Encapsulated Primary Hepatocytes. Int J Artif Organs 2018; 30:192-8. [PMID: 17417757 DOI: 10.1177/039139880703000303] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The clinical application of bioartificial liver support systems (BALS) is still limited because of technical problems associated with the storage, transport and scale-up of common systems. The encapsulation of primary hepatocytes could solve these problems since the scale-up depends only on the number of the beads and encapsulation leads to protection of the cells during the process of freezing and thawing. Many efforts have been made to find an appropriate material for the encapsulation of primary hepatocytes in terms of mechanical resistance as well as appropriate bio- and hemo-compatibility This study focuses on the improvement of the metabolic functionality of encapsulated primary hepatocytes. A comparison between two different cultivation models showed that dynamic cultivation conditions lead to a 20.4-fold increase in the albumin production and a 5.21-fold increase in the urea synthesis of encapsulated hepatocytes. Furthermore, the influence of different ratios of the number of the cells to the volume of the media was analyzed. Encapsulated hepatocytes cultured with a high amount of medium were characterized by a significantly higher metabolic activity compared to encapsulated hepatocytes cultured with a low level of medium. Interestingly, the cell concentration per mL alginate has no significant influence on the metabolic activity of encapsulated hepatocytes. In conclusion, different optimization strategies are discussed and, finally, the functionality of encapsulated hepatocytes is compared to the standard model of hepatocyte culture, the collagen sandwich.
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Affiliation(s)
- S Diekmann
- Biotechnological-Biomedical Center, Cell Techniques and Applied Stem Cell Biology, University of Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
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Orsini M, Sperber S, Noor F, Hoffmann E, Weber SN, Hall RA, Lammert F, Heinzle E. Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture. J Cell Biochem 2017; 119:447-454. [PMID: 28594086 DOI: 10.1002/jcb.26202] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2017] [Accepted: 06/07/2017] [Indexed: 12/18/2022]
Abstract
Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
- Malina Orsini
- Biochemical Engineering, Saarland University, Saarbrücken 66123, Germany
| | - Saskia Sperber
- Biochemical Engineering, Saarland University, Saarbrücken 66123, Germany
| | - Fozia Noor
- Biochemical Engineering, Saarland University, Saarbrücken 66123, Germany
| | - Esther Hoffmann
- Biochemical Engineering, Saarland University, Saarbrücken 66123, Germany
| | - Susanne N Weber
- Department of Medicine II, Saarland University, Saarland University Medical Center, Homburg 66421, Germany
| | - Rabea A Hall
- Department of Medicine II, Saarland University, Saarland University Medical Center, Homburg 66421, Germany
| | - Frank Lammert
- Department of Medicine II, Saarland University, Saarland University Medical Center, Homburg 66421, Germany
| | - Elmar Heinzle
- Biochemical Engineering, Saarland University, Saarbrücken 66123, Germany
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Duval K, Grover H, Han LH, Mou Y, Pegoraro AF, Fredberg J, Chen Z. Modeling Physiological Events in 2D vs. 3D Cell Culture. Physiology (Bethesda) 2017; 32:266-277. [PMID: 28615311 PMCID: PMC5545611 DOI: 10.1152/physiol.00036.2016] [Citation(s) in RCA: 1034] [Impact Index Per Article: 129.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2016] [Revised: 02/24/2017] [Accepted: 04/05/2017] [Indexed: 02/06/2023] Open
Abstract
Cell culture has become an indispensable tool to help uncover fundamental biophysical and biomolecular mechanisms by which cells assemble into tissues and organs, how these tissues function, and how that function becomes disrupted in disease. Cell culture is now widely used in biomedical research, tissue engineering, regenerative medicine, and industrial practices. Although flat, two-dimensional (2D) cell culture has predominated, recent research has shifted toward culture using three-dimensional (3D) structures, and more realistic biochemical and biomechanical microenvironments. Nevertheless, in 3D cell culture, many challenges remain, including the tissue-tissue interface, the mechanical microenvironment, and the spatiotemporal distributions of oxygen, nutrients, and metabolic wastes. Here, we review 2D and 3D cell culture methods, discuss advantages and limitations of these techniques in modeling physiologically and pathologically relevant processes, and suggest directions for future research.
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Affiliation(s)
- Kayla Duval
- Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire
| | - Hannah Grover
- Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire
| | - Li-Hsin Han
- Department of Mechanical Engineering and Mechanics, Drexel University, Philadelphia, Pennsylvania
| | - Yongchao Mou
- Department of Bioengineering, University of Illinois-Chicago, Rockford, Illinois
| | - Adrian F Pegoraro
- Harvard School of Engineering and Applied Sciences, Cambridge, Massachusetts; and
| | - Jeffery Fredberg
- Harvard T.H. Chan School of Public Health, Boston, Massachusetts
| | - Zi Chen
- Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire;
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Borel Rinkes IH, Toner M, Sheeha SJ, Tompkins RG, Yarmush ML. Long-Term Functional Recovery of Hepatocytes after Cryopreservation in a Three-Dimensional Culture Configuration. Cell Transplant 2017; 1:281-92. [PMID: 1344301 DOI: 10.1177/096368979200100405] [Citation(s) in RCA: 49] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022] Open
Abstract
Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5°C/min and warming at ≥400°C/min), freezing to −40°C resulted in full recovery of albumin secretion within 2-3 days post-freezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to −80°C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze-thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.
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45
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Hepatogenic Differentiation of Human Induced Pluripotent Stem cells on Collagen-Coated Polyethersulfone Nanofibers. ASAIO J 2017; 63:316-323. [DOI: 10.1097/mat.0000000000000469] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
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46
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Siltanen C, Diakatou M, Lowen J, Haque A, Rahimian A, Stybayeva G, Revzin A. One step fabrication of hydrogel microcapsules with hollow core for assembly and cultivation of hepatocyte spheroids. Acta Biomater 2017; 50:428-436. [PMID: 28069506 DOI: 10.1016/j.actbio.2017.01.010] [Citation(s) in RCA: 55] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2016] [Revised: 12/15/2016] [Accepted: 01/05/2017] [Indexed: 12/16/2022]
Abstract
3D hepatic microtissues can serve as valuable liver analogues for cell-based therapies and for hepatotoxicity screening during preclinical drug development. However, hepatocytes rapidly dedifferentiate in vitro, and typically require 3D culture systems or co-cultures for phenotype rescue. In this work we present a novel microencapsulation strategy, utilizing coaxial flow-focusing droplet microfluidics to fabricate microcapsules with liquid core and poly(ethylene glycol) (PEG) gel shell. When entrapped inside these capsules, primary hepatocytes rapidly formed cell-cell contacts and assembled into compact spheroids. High levels of hepatic function were maintained inside the capsules for over ten days. The microencapsulation approach described here is compatible with difficult-to-culture primary epithelial cells, allows for tuning gel mechanical properties and diffusivity, and may be used in the future for high density suspension cell cultures. STATEMENT OF SIGNIFICANCE Our paper combines an interesting new way for making capsules with cultivation of difficult-to-maintain primary epithelial cells (hepatocytes). The microcapsules described here will enable high density suspension culture of hepatocytes or other cells and may be used as building blocks for engineering tissues.
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Sandwich-Cultured Hepatocytes as a Tool to Study Drug Disposition and Drug-Induced Liver Injury. J Pharm Sci 2016; 105:443-459. [PMID: 26869411 DOI: 10.1016/j.xphs.2015.11.008] [Citation(s) in RCA: 53] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2015] [Revised: 11/06/2015] [Accepted: 11/09/2015] [Indexed: 12/21/2022]
Abstract
Sandwich-cultured hepatocytes (SCH) are metabolically competent and have proper localization of basolateral and canalicular transporters with functional bile networks. Therefore, this cellular model is a unique tool that can be used to estimate biliary excretion of compounds. SCH have been used widely to assess hepatobiliary disposition of endogenous and exogenous compounds and metabolites. Mechanistic modeling based on SCH data enables estimation of metabolic and transporter-mediated clearances, which can be used to construct physiologically based pharmacokinetic models for prediction of drug disposition and drug-drug interactions in humans. In addition to pharmacokinetic studies, SCH also have been used to study cytotoxicity and perturbation of biological processes by drugs and hepatically generated metabolites. Human SCH can provide mechanistic insights underlying clinical drug-induced liver injury (DILI). In addition, data generated in SCH can be integrated into systems pharmacology models to predict potential DILI in humans. In this review, applications of SCH in studying hepatobiliary drug disposition and bile acid-mediated DILI are discussed. An example is presented to show how data generated in the SCH model were used to establish a quantitative relationship between intracellular bile acids and cytotoxicity, and how this information was incorporated into a systems pharmacology model for DILI prediction.
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48
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ECM proteins in a microporous scaffold influence hepatocyte morphology, function, and gene expression. Sci Rep 2016; 6:37427. [PMID: 27897167 PMCID: PMC5126637 DOI: 10.1038/srep37427] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2016] [Accepted: 10/24/2016] [Indexed: 01/06/2023] Open
Abstract
It is well known that a three-dimensional (3D) culture environment and the presence of extracellular matrix (ECM) proteins facilitate hepatocyte viability and maintenance of the liver-specific phenotype in vitro. However, it is not clear whether specific ECM components such as collagen or fibronectin differentially regulate such processes, especially in 3D scaffolds. In this study, a series of ECM-functionalized inverted colloidal crystal (ICC) microporous scaffolds were fabricated and their influence on Huh-7.5 cell proliferation, morphology, hepatic-specific functions, and patterns of gene expression were compared. Both collagen and fibronectin promoted albumin production and liver-specific gene expression of Huh-7.5 cells, compared with the bare ICC scaffold. Interestingly, cells in the fibronectin-functionalized scaffold exhibited different aggregation patterns to those in the collagen-functionalized scaffold, a variation that could be related to the distinct mRNA expression levels of cell adhesion-related genes. Based on these results, we can conclude that different ECM proteins, such as fibronectin and collagen, indeed play distinct roles in the phenotypic regulation of cells cultured in a 3D environment.
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49
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Xiao W, Perry G, Komori K, Sakai Y. New physiologically-relevant liver tissue model based on hierarchically cocultured primary rat hepatocytes with liver endothelial cells. Integr Biol (Camb) 2016; 7:1412-22. [PMID: 26304784 DOI: 10.1039/c5ib00170f] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
To develop an in vitro liver tissue equivalent, hepatocytes should be cocultured with liver non-parenchymal cells to mimic the in vivo physiological microenvironments. In this work, we describe a physiologically-relevant liver tissue model by hierarchically organizing layers of primary rat hepatocytes and human liver sinusoidal endothelial cells (TMNK-1) on an oxygen-permeable polydimethylsiloxane (PDMS) membrane, which facilitates direct oxygenation by diffusion through the membrane. This in vivo-mimicking hierarchical coculture was obtained by simply proceeding the overlay of TMNK-1 cells on the hepatocyte layer re-formed on the collagen immobilized PDMS membranes. The comparison of hepatic functionalities was achieved between coculture and sandwich culture with Matrigel, in the presence and absence of direct oxygenation. A complete double-layered structure of functional liver cells with vertical contact between hepatocytes and TMNK-1 was successfully constructed in the coculture with direct oxygen supply and was well-maintained for 14 days. The hepatocytes in this hierarchical culture exhibited improved survival, functional bile canaliculi formation, cellular level polarization and maintenance of metabolic activities including Cyp1A1/2 activity and albumin production. By contrast, the two cell populations formed discontinuous monolayers on the same surfaces in the non-oxygen-permeable cultures. These results demonstrate that (i) the direct oxygenation through the PDMS membranes enables very simple formation of a hierarchical structure consisting of a hepatocyte layer and a layer of TMNK-1 and (ii) we may include other non-parenchymal cells in this format easily, which can be widely applicable to other epithelial organs.
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Affiliation(s)
- Wenjin Xiao
- Fe505, Institute of Industrial Science (IIS), University of Tokyo, Tokyo, 153-8505, Japan.
| | - Guillaume Perry
- Fe505, Institute of Industrial Science (IIS), University of Tokyo, Tokyo, 153-8505, Japan. and LIMMS CNRS/IIS, University of Tokyo, Japan
| | - Kikuo Komori
- Fe505, Institute of Industrial Science (IIS), University of Tokyo, Tokyo, 153-8505, Japan.
| | - Yasuyuki Sakai
- Fe505, Institute of Industrial Science (IIS), University of Tokyo, Tokyo, 153-8505, Japan.
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Shirahama H, Kumar SK, Jeon WY, Kim MH, Lee JH, Ng SS, Tabaei SR, Cho NJ. Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering. J Vis Exp 2016. [PMID: 27684530 DOI: 10.3791/54331] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/21/2022] Open
Abstract
The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics' cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.
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Affiliation(s)
- Hitomi Shirahama
- School of Materials Science and Engineering, Nanyang Technological University
| | - Supriya K Kumar
- School of Materials Science and Engineering, Nanyang Technological University
| | - Won-Yong Jeon
- School of Materials Science and Engineering, Nanyang Technological University
| | - Myung Hee Kim
- School of Materials Science and Engineering, Nanyang Technological University
| | - Jae Ho Lee
- School of Materials Science and Engineering, Nanyang Technological University
| | - Soon Seng Ng
- School of Materials Science and Engineering, Nanyang Technological University
| | - Seyed R Tabaei
- School of Materials Science and Engineering, Nanyang Technological University
| | - Nam-Joon Cho
- School of Materials Science and Engineering, Nanyang Technological University; School of Chemical and Biomedical Engineering, Nanyang Technological University;
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