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Hu W, Wang Y, Han J, Zhang W, Chen J, Li X, Wang L. Microfluidic organ-on-a-chip models for the gut-liver axis: from structural mimicry to functional insights. Biomater Sci 2025; 13:1624-1656. [PMID: 40019226 DOI: 10.1039/d4bm01273a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/01/2025]
Abstract
The gut-liver axis plays a crucial role in maintaining metabolic balance and overall human health. It orchestrates various processes, such as blood flow, nutrient transfer, metabolite processing, and immune cell communication between the two organs. Traditional methods, such as animal models and two-dimensional (2D) cell cultures, are insufficient in fully replicating the intricate functions of the gut-liver axis. The emergence of microfluidic technology has revolutionized this field, facilitating the development of organ-on-a-chip (OOC) systems. These systems are capable of mimicking the complex structures and dynamic environments of the gut and liver in vitro and incorporating sensors for real-time monitoring. In this article, we review the latest progress in gut-on-a-chip (GOC) and liver-on-a-chip (LOC) systems, as well as the integrated gut-liver-on-a-chip (GLOC) models. Our focus lies in the simulation of physiological parameters, three-dimensional (3D) structural mimicry, microbiome integration, and multicellular co-culture. All these aspects are essential for constructing accurate in vitro models of the gut and liver. Furthermore, we explore the current applications of OOC technology in the study of the gut and liver, including its use in disease modeling, toxicity testing, and drug screening. Finally, we discuss the challenges that remain and outline potential future directions for advancing GOC and LOC development in vitro.
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Affiliation(s)
- Wanlin Hu
- School of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China.
- Shandong Institute of Mechanical Design and Research, Jinan 250353, China
| | - Yushen Wang
- School of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China.
- Shandong Institute of Mechanical Design and Research, Jinan 250353, China
| | - Junlei Han
- School of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China.
- Shandong Institute of Mechanical Design and Research, Jinan 250353, China
| | - Wenhong Zhang
- College of Mechanical Engineering, Donghua University, Shanghai 201620, China
| | - Jun Chen
- School of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China.
- Shandong Institute of Mechanical Design and Research, Jinan 250353, China
| | - Xinyu Li
- Department of Minimally Invasive Comprehensive Treatment of Cancer, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong, 250021, China.
| | - Li Wang
- School of Mechanical Engineering, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China.
- Shandong Institute of Mechanical Design and Research, Jinan 250353, China
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Maity S, Deb VK, Mondal S, Chakraborty A, Pramanick K, Adhikari S. Leveraging supramolecular systems in biomedical breakthroughs. Biofactors 2025; 51:e70005. [PMID: 39902766 DOI: 10.1002/biof.70005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/05/2024] [Accepted: 01/21/2025] [Indexed: 02/06/2025]
Abstract
Supramolecular systems, intricate assemblies of molecular subunits organized through various intermolecular interactions, offer versatile platforms for diverse applications, including gene therapy, antimicrobial therapy, and cellular engineering. These systems are cost-effective and environmentally friendly, contributing to their attractiveness in biomaterial design. Furthermore, supramolecular biomaterials based on acyclic, macrocyclic compounds and lipid-based assembly offer potential applications in distinct types of biomedical approaches. In this context, they can transport several therapeutic agents very effectively to the target site. Supramolecular hydrogels exhibit potent antimicrobial activity by disrupting microbial membranes, offering promising solutions to combat drug-resistant pathogens. Additionally, supramolecular luminescent nanoparticles enable targeted cell imaging, facilitating disease diagnosis and treatment with high specificity and sensitivity. In cellular engineering, supramolecular assemblies of small molecules demonstrate biological activities, overcoming challenges in cancer treatment by inhibiting signaling pathways and inducing apoptosis in cancer cells. This review emphasizes the applications of supramolecular systems from gene therapy to cellular imaging, tissue engineering, and antimicrobial therapy, showcasing their potential to drive innovation and address pressing healthcare challenges.
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Affiliation(s)
- Shreya Maity
- Integrative Biology Research Unit, Department of Life Sciences, Presidency University, Kolkata, India
| | | | - Sayani Mondal
- Integrative Biology Research Unit, Department of Life Sciences, Presidency University, Kolkata, India
| | - Akansha Chakraborty
- Integrative Biology Research Unit, Department of Life Sciences, Presidency University, Kolkata, India
| | - Kousik Pramanick
- Integrative Biology Research Unit, Department of Life Sciences, Presidency University, Kolkata, India
| | - Suman Adhikari
- Department of Chemistry, Government Degree College, Dharmanagar, India
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Lee JH, Park HJ, Kim YA, Lee DH, Noh JK, Jung JG, Yang MS, Lee JE, Lee SH, Yoon HH, Lee SK, Lee S. Selecting serum-free hepatocyte cryopreservation stage and storage temperature for the application of an "off-the-shelf" bioartificial liver system. Sci Rep 2024; 14:12168. [PMID: 38806510 PMCID: PMC11133438 DOI: 10.1038/s41598-024-60711-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2023] [Accepted: 04/26/2024] [Indexed: 05/30/2024] Open
Abstract
The bioartificial liver (BAL) system can potentially rescue acute liver failure (ALF) patients by providing partial liver function until a suitable donor liver can be found or the native liver has self-regenerated. In this study, we established a suitable cryopreservation process for the development of an off-the-shelf BAL system. The viability of hepatocyte spheroids cryopreserved in liquid nitrogen was comparable to that of fresh primary hepatocyte spheroids. When hepatocyte spheroids were subjected to cryopreservation in a deep freezer, no statistically significant differences were observed in ammonia removal rate or urea secretion rate based on the cryopreservation period. However, the functional activity of the liver post-cryopreservation in a deep freezer was significantly lower than that observed following liquid nitrogen cryopreservation. Moreover, cryopreserving spheroid hydrogel beads in a deep freezer resulted in a significant decrease (approximately 30%) in both ammonia removal and urea secretion rates compared to the group cryopreserved in liquid nitrogen. The viabilities of spheroid hydrogel beads filled into the bioreactor of a BAL system were similar across all four groups. However, upon operating the BAL system for 24 h, the liver function activity was significantly higher in the group comprising hydrogel beads generated after thawing hepatocyte spheroids cryopreserved in liquid nitrogen. Consequently, the manufacturing of beads after the cryopreservation of hepatocyte spheroids is deemed the most suitable method, considering efficiency, economic feasibility, and liver function activity, for producing a BAL system.
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Affiliation(s)
- Ji-Hyun Lee
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Republic of Korea
| | - Hey-Jung Park
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Republic of Korea
| | - Young-A Kim
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul, Republic of Korea
| | - Doo-Hoon Lee
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Jeong-Kwon Noh
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Jong-Gab Jung
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Mal Sook Yang
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Jong Eun Lee
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Se Hoon Lee
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Hee-Hoon Yoon
- Research Institute, HLB Cell, Co. Ltd., Hwaseong, Republic of Korea
| | - Suk-Koo Lee
- Department of Surgery, Myongji Hospital, Goyang, Republic of Korea.
| | - Sanghoon Lee
- Department of Surgery, School of Medicine, Samsung Medical Center, Sungkyunkwan University, 50 Irwon-Dong, Gangnam-Gu, Seoul, 06354, Republic of Korea.
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Feng L, Wang Y, Fu Y, Li T, He G. Stem Cell-Based Strategies: The Future Direction of Bioartificial Liver Development. Stem Cell Rev Rep 2024; 20:601-616. [PMID: 38170319 DOI: 10.1007/s12015-023-10672-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 12/21/2023] [Indexed: 01/05/2024]
Abstract
Acute liver failure (ALF) results from severe liver damage or end-stage liver disease. It is extremely fatal and causes serious health and economic burdens worldwide. Once ALF occurs, liver transplantation (LT) is the only definitive and recommended treatment; however, LT is limited by the scarcity of liver grafts. Consequently, the clinical use of bioartificial liver (BAL) has been proposed as a treatment strategy for ALF. Human primary hepatocytes are an ideal cell source for these methods. However, their high demand and superior viability prevent their widespread use. Hence, finding alternatives that meet the seed cell quality and quantity requirements is imperative. Stem cells with self-renewing, immunogenic, and differentiative capacities are potential cell sources. MSCs and its secretomes encompass a spectrum of beneficial properties, such as anti-inflammatory, immunomodulatory, anti-ROS (reactive oxygen species), anti-apoptotic, pro-metabolomic, anti-fibrogenesis, and pro-regenerative attributes. This review focused on the recent status and future directions of stem cell-based strategies in BAL for ALF. Additionally, we discussed the opportunities and challenges associated with promoting such strategies for clinical applications.
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Affiliation(s)
- Lei Feng
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Affiliated Hospital of Guizhou Medical University, Guiyang, 550000, Guizhou, China.
| | - Yi Wang
- Shanxi Cancer Hospital/Shanxi Hospital Affiliated to Cancer Hospital, Chinese Academy of Medical Sciences/Cancer Hospital Affiliated to Shanxi Medical University, Taiyuan, 030013, Shanxi, China
| | - Yu Fu
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China
| | - Ting Li
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
- Department of Hepatobiliary Surgery, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510140, Guangdong, China.
| | - Guolin He
- Department of Hepatobiliary Surgery II, Zhujiang Hospital, Southern Medical University, Guangzhou, 510282, Guangdong, China.
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Yin Y, Hu Z, Guan Z, Lv S, Wang Y, Su W, Yan C, Wang Y, Ma L, Jiang J, Feng S, Li L. Immunological characteristics of human umbilical cord mesenchymal stem cells after hepatogenic differentiation. Biomed Mater Eng 2023; 34:1-11. [PMID: 35180101 DOI: 10.3233/bme-211331] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
BACKGROUND Acute liver failure is one of the most intractable clinical problems. The use of bioartificial livers may solve donor shortage problems. Human umbilical cord mesenchymal stem cells (hUCMSCs) are an excellent seed cell choice for artificial livers because they change their characteristics to resemble hepatocyte-like cells (HLCs) following artificial liver transplantation. OBJECTIVE This study aimed to determine whether the immunological characteristics of hUCMSCs are changed after being transformed into hepatocyte-like cells. METHODS HUCMSCs were isolated by the adherent method. The following hUCMSC surface markers were detected using flow cytometry: CD45, CD90, CD105, CD34, and octamer-binding transcription factor 4 (OCT-4). Functional detection of adipogenic differentiation was performed. The hUCMSCs were cultured in complete medium (control group) or induction medium (induction group), and flow cytometry was used to detect cell surface markers. Peritoneal lavage fluid was collected after intraperitoneal injection of 1 × 106 cells/mouse over 40 minutes. The leukocyte count, labeled CD45, CD3, CD4 and CD8 antibodies, and flow detection of T lymphocyte subsets were determined using the peritoneal lavage fluid. RESULTS Using phenotypic and functional identification, hUCMSCs were successfully isolated using a two-step induction method. The surface markers of the hUCMSCs cells changed after HLC induction. In vivo immune results showed that hUCMSCs and HLsC induced leukocyte production. CONCLUSION Hepatic induction of hUCMSCs changes their cell surface markers. Both HLCs and hUCMSCs cause leukocytosis in vivo, but the immune response induced by HLCs is slightly stronger.
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Affiliation(s)
- Yanfeng Yin
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Zongqiang Hu
- Department of Hepatobiliary Surgery, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Zheng Guan
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Sha Lv
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Yiyin Wang
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Wenjun Su
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Chuntao Yan
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Yiting Wang
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Li Ma
- Biomedical Research Center, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Jie Jiang
- Department of Hepatobiliary Surgery, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Shiming Feng
- Department of Hepatobiliary Surgery, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
| | - Li Li
- Department of Hepatobiliary Surgery, The First Hospital of Kunming (the Affiliated Calmette Hospital of Kunming Medical University), Kunming, China
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Commercially Available Textiles as a Scaffolding Platform for Large-Scale Cell Culture. Int J Biomater 2023; 2023:2227509. [PMID: 36909982 PMCID: PMC9995198 DOI: 10.1155/2023/2227509] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Revised: 12/29/2022] [Accepted: 02/15/2023] [Indexed: 03/05/2023] Open
Abstract
The present study outlines the evaluation of textile materials that are currently in the market for cell culture applications. By using normal LaserJet printing techniques, we created the substrates, which were then characterized physicochemically and biologically. In particular, (i) we found that the weave pattern and (ii) the chemical nature of the textiles significantly influenced the behaviour of the cells. Textiles with closely knitted fibers and cell adhesion motifs, exhibited better cell adhesion and proliferation over a period of 7 days. All the substrates supported good viability of cells (>80%). We believe that these aspects make commercially available textiles as a potential candidate for large-scale culture of adherent cells.
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Lee JH, Park HJ, Kim YA, Lee DH, Noh JK, Jung JG, Yoon HH, Lee SK, Lee S. Establishment of a Serum-Free Hepatocyte Cryopreservation Process for the Development of an "Off-the-Shelf" Bioartificial Liver System. Bioengineering (Basel) 2022; 9:738. [PMID: 36550944 PMCID: PMC9774268 DOI: 10.3390/bioengineering9120738] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2022] [Revised: 11/15/2022] [Accepted: 11/23/2022] [Indexed: 12/05/2022] Open
Abstract
To use hepatocytes immediately when necessary for hepatocyte transplantation and bioartificial liver (BAL) systems, a serum-free cryopreservation protocol ensuring the high survival of hepatocytes and maintenance of their functions should be developed. We established a serum-free protocol for the cryopreservation of primary hepatocytes, hepatocyte spheroids, and hepatocyte spheroid beads in liquid nitrogen. The serum-free cryopreservation solutions showed a significantly higher performance in maintaining enhanced viability and ammonia removal, urea secretion, and the albumin synthesis of hepatocyte spheroids and spheroid beads. The serum-free thawing medium, containing human serum albumin (HSA) and N-acetylcysteine (NAC), was compared with a fetal bovine serum-containing thawing medium for the development of a serum-free thawing medium. Our results show that hepatocyte spheroids and spheroid beads thawed using a serum-free thawing medium containing HSA and NAC exhibited increased hepatocyte viability, ammonia removal, urea secretion, and albumin synthesis compared to those thawed using the serum-containing medium. Finally, we evaluated the liver functions of the cryopreserved BAL system-applied serum-free cryopreservation process compared to the fresh BAL system. The ammonia removal efficiency of the cryopreserved hepatocyte spheroids BAL was lower than or similar to that of the fresh BAL system. Additionally, the urea concentrations in the media of all three BAL systems were not significantly different during BAL system operation. This cryopreserved spheroid-based BAL system using a serum-free process will be a good candidate for the treatment of patients.
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Affiliation(s)
- Ji-Hyun Lee
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
| | - Hey-Jung Park
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
| | - Young-A Kim
- Stem Cell and Regenerative Medicine Center, Research Institute for Future Medicine, Samsung Medical Center, Seoul 06351, Republic of Korea
| | - Doo-Hoon Lee
- Research Institute, HLB Cell Co., Ltd., Hwaseong 18469, Republic of Korea
| | - Jeong-Kwon Noh
- Research Institute, HLB Cell Co., Ltd., Hwaseong 18469, Republic of Korea
| | - Jong-Gab Jung
- Research Institute, HLB Cell Co., Ltd., Hwaseong 18469, Republic of Korea
| | - Hee-Hoon Yoon
- Research Institute, HLB Cell Co., Ltd., Hwaseong 18469, Republic of Korea
| | - Suk-Koo Lee
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Republic of Korea
| | - Sanghoon Lee
- Department of Surgery, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul 06351, Republic of Korea
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Telles-Silva KA, Pacheco L, Komatsu S, Chianca F, Caires-Júnior LC, Araujo BHS, Goulart E, Zatz M. Applied Hepatic Bioengineering: Modeling the Human Liver Using Organoid and Liver-on-a-Chip Technologies. Front Bioeng Biotechnol 2022; 10:845360. [PMID: 35237587 PMCID: PMC8882846 DOI: 10.3389/fbioe.2022.845360] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2021] [Accepted: 01/19/2022] [Indexed: 12/19/2022] Open
Abstract
The liver is the most important metabolic hub of endo and xenobiotic compounds. Pre-clinical studies using rodents to evaluate the toxicity of new drugs and cosmetics may produce inconclusive results for predicting clinical outcomes in humans, moreover being banned in the European Union. Human liver modeling using primary hepatocytes presents low reproducibility due to batch-to-batch variability, while iPSC-derived hepatocytes in monolayer cultures (2D) show reduced cellular functionality. Here we review the current status of the two most robust in vitro approaches in improving hepatocyte phenotype and metabolism while mimicking the hepatic physiological microenvironment: organoids and liver-on-chip. Both technologies are reviewed in design and manufacturing techniques, following cellular composition and functionality. Furthermore, drug screening and liver diseases modeling efficiencies are summarized. Finally, organoid and liver-on-chip technologies are compared regarding advantages and limitations, aiming to guide the selection of appropriate models for translational research and the development of such technologies.
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Affiliation(s)
- Kayque Alves Telles-Silva
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
| | - Lara Pacheco
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
| | - Sabrina Komatsu
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
| | - Fernanda Chianca
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
| | - Luiz Carlos Caires-Júnior
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
| | - Bruno Henrique Silva Araujo
- Lieber Institute for Brain Development, School of Medicine, Johns Hopkins University, Baltimore, MD, United States
| | - Ernesto Goulart
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
- *Correspondence: Ernesto Goulart, ; Mayana Zatz,
| | - Mayana Zatz
- Human Genome and Stem-Cell Research Center (HUG-CEL), Institute of Biosciences, University of Sao Paulo (USP), Sao Paulo, Brazil
- *Correspondence: Ernesto Goulart, ; Mayana Zatz,
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Wang X, Guo C, Guo L, Wang M, Liu M, Song Y, Jiao H, Wei X, Zhao Z, Kaplan DL. Radially Aligned Porous Silk Fibroin Scaffolds as Functional Templates for Engineering Human Biomimetic Hepatic Lobules. ACS APPLIED MATERIALS & INTERFACES 2022; 14:201-213. [PMID: 34929079 DOI: 10.1021/acsami.1c18215] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/14/2023]
Abstract
Bioengineering functional hepatic tissue constructs that physiologically replicate the human native liver tissue in vitro is sought for clinical research and drug discovery. However, the intricate architecture and specific biofunctionality possessed by the native liver tissue remain challenging to mimic in vitro. In the present study, a versatile strategy to fabricate lobular-like silk protein scaffolds with radially aligned lamellar sheets, interconnected channels, and a converging central cavity was designed and implemented. A proof-of-concept study to bioengineer biomimetic hepatic lobules was conducted through coculturing human hepatocytes and primary endothelial cells on these lobular-like scaffolds. Relatively long-term viability of hepatocyte/endothelial cells was found along with cell alignment and organization in vitro. The hepatocytes showed special epithelial polarity and bile duct formation, similar to the liver plate, while the aligned endothelial cells generated endothelial networks, similar to natural hepatic sinuses. This endowed the three-dimensional (3D) tissue constructs with the capability to recapitulate hepatic-like parenchymal-mesenchymal growth patterns in vitro. More importantly, the cocultured hepatocytes outperformed monocultures or monolayer cultures, displaying significantly enhanced hepatocyte functions, including functional gene expression, albumin (ALB) secretion, urea synthesis, and metabolic activity. Thus, this functional unit with a biomimetic phenotype provides a novel technology for bioengineering biomimetic hepatic lobules in vitro, with potential utility as a building block for bioartificial liver (BAL) engineering or as a robust tool for drug metabolism investigation.
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Affiliation(s)
- Xiuli Wang
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
| | - Chengchen Guo
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
- School of Engineering, Westlake University, Hangzhou, Zhejiang 310023, China
| | - Lina Guo
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Mingqi Wang
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Ming Liu
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Yizhe Song
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Hui Jiao
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Xiaoqing Wei
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - Zinan Zhao
- Department of Histology & Embryology, College of Basic Medical Sciences, Dalian Medical University, Liaoning 116044, China
| | - David L Kaplan
- Biomedical Engineering Department, Tufts University, Medford, Massachusetts 02155, United States
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10
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Luo S, Xiao S, Ai Y, Wang B, Wang Y. Changes in the hepatic differentiation potential of human mesenchymal stem cells aged in vitro. ANNALS OF TRANSLATIONAL MEDICINE 2021; 9:1628. [PMID: 34926672 PMCID: PMC8640908 DOI: 10.21037/atm-21-4918] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 08/31/2021] [Accepted: 10/13/2021] [Indexed: 11/15/2022]
Abstract
Background Due to their multipotency and ability for self-renewal, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great promise for generating hepatocytes. Previous research has successfully generated hepatocytes from early-passage [i.e., passage (P)3] hUC-MSCs; however, the populations of early-passage cells are limited, and these cells cannot produce sufficient functional hepatocytes for large-scale application in clinical therapy. Thus, a thorough investigation of the hepatic differentiation potential of in vitro-aged hUC-MSCs is needed. Methods hUC-MSCs were passaged in vitro and subcultured every 3 days up to P8, and their morphology, proliferative capacity, liver-specific marker expression, and liver function at the end of each passage were analyzed. The efficiency of the hepatogenic differentiation of hUC-MSCs driven by a functional hit 1 (FH1)-based strategy at different passages was also evaluated. Results The in vitro-aged hUC-MSCs gradually displayed morphological inhomogeneity, had reduced proliferative capability, and exhibited senescent properties while maintaining adipogenic and osteogenic differentiation potential. Additionally, senescence also decreased the expression of messenger RNA (mRNA) levels in albumin (ALB) and alpha 1-antitrpsin (A1AT) in these cells and their relative protein expression, which is the marker of a mature hepatocyte. The liver function of the in vitro-aged hUC-MSCs also deteriorated gradually. Finally, the percentage of hepatocyte-like cells (HLCs) generated from in vitro-aged hUC-MSCs reduced significantly, and the mature hepatocyte functions, such as ALB secretion, glycogen synthesis, low-density lipoprotein (LDL) intake, and indocyanine green (ICG) uptake, also changed. Conclusions hUC-MSCs possess mature hepatocytes’ specific markers and functions, which change gradually as they undergo cell senescence. Due to the loss of these properties within in vitro subcultures, the hepatic differentiation efficiency of in vitro-aged hUC-MSCs decreased dramatically in the late passage (P8). The current study provides valuable information can inform future research on liver disease.
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Affiliation(s)
- Sang Luo
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Shuai Xiao
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Yang Ai
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Ben Wang
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
| | - Yefu Wang
- State Key Laboratory of Virology, School of Life Sciences, Wuhan University, Wuhan, China
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11
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Kim Y, Jeong J, Choi D. Generation and differentiation of chemically derived hepatic progenitors from mouse primary hepatocytes. STAR Protoc 2021; 2:100840. [PMID: 34585167 PMCID: PMC8455481 DOI: 10.1016/j.xpro.2021.100840] [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] [Indexed: 11/30/2022] Open
Abstract
This protocol describes the generation of bipotent chemically derived hepatic progenitors (mCdHs) from mouse primary hepatocytes and their subsequent differentiation into either hepatic or cholangiocytic lineages. The reprogrammed mCdHs have a high proliferation capacity and express progenitor markers in long-term passages. Differentiated mCdHs show the characteristics of either hepatic or cholangiocytic genes. This protocol has potential application for regenerative medicine, including ex vivo gene therapy, disease modeling, drug screening, and personalized medicine. For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).
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Affiliation(s)
- Yohan Kim
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Republic of Korea.,HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Republic of Korea
| | - Jaemin Jeong
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Republic of Korea.,HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Republic of Korea
| | - Dongho Choi
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Republic of Korea.,HY Indang Center of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Republic of Korea.,Department of HY-KIST Bio-convergence, Hanyang University, Seoul 04763, Republic of Korea
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12
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Li WJ, Zhu XJ, Yuan TJ, Wang ZY, Bian ZQ, Jing HS, Shi X, Chen CY, Fu GB, Huang WJ, Shi YP, Liu Q, Zeng M, Zhang HD, Wu HP, Yu WF, Zhai B, Yan HX. An extracorporeal bioartificial liver embedded with 3D-layered human liver progenitor-like cells relieves acute liver failure in pigs. Sci Transl Med 2021; 12:12/551/eaba5146. [PMID: 32641490 DOI: 10.1126/scitranslmed.aba5146] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2019] [Accepted: 06/03/2020] [Indexed: 12/12/2022]
Abstract
Clinical advancement of the bioartificial liver is hampered by the lack of expandable human hepatocytes and appropriate bioreactors and carriers to encourage hepatic cells to function during extracorporeal circulation. We have recently developed an efficient approach for derivation of expandable liver progenitor-like cells from human primary hepatocytes (HepLPCs). Here, we generated immortalized and functionally enhanced HepLPCs by introducing FOXA3, a hepatocyte nuclear factor that enables potentially complete hepatic function. When cultured on macroporous carriers in an air-liquid interactive bioartificial liver (Ali-BAL) support device, the integrated cells were alternately exposed to aeration and nutrition and grew to form high-density three-dimensional constructs. This led to highly efficient mass transfer and supported liver functions such as albumin biosynthesis and ammonia detoxification via ureagenesis. In a porcine model of drug overdose-induced acute liver failure (ALF), extracorporeal Ali-BAL treatment for 3 hours prevented hepatic encephalopathy and led to markedly improved survival (83%, n = 6) compared to ALF control (17%, n = 6, P = 0.02) and device-only (no-cell) therapy (0%, n = 6, P = 0.003). The blood ammonia concentrations, as well as the biochemical and coagulation indices, were reduced in Ali-BAL-treated pigs. Ali-BAL treatment attenuated liver damage, ameliorated inflammation, and enhanced liver regeneration in the ALF porcine model and could be considered as a potential therapeutic avenue for patients with ALF.
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Affiliation(s)
- Wei-Jian Li
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Xue-Jing Zhu
- Shanghai Celliver Biotechnology Co. Ltd., Shanghai 210201, China
| | - Tian-Jie Yuan
- Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China.,Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou 221004, China
| | - Zhen-Yu Wang
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Zheng-Qian Bian
- Training Center, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Hong-Shu Jing
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Xiao Shi
- Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Cai-Yang Chen
- Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Gong-Bo Fu
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200433, China
| | - Wei-Jian Huang
- International Cooperation Laboratory on Signal Transduction, Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai 200433, China
| | - Yao-Ping Shi
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Qian Liu
- Department of Laboratory Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China
| | - Min Zeng
- Shanghai Celliver Biotechnology Co. Ltd., Shanghai 210201, China
| | - Hong-Dan Zhang
- Shanghai Celliver Biotechnology Co. Ltd., Shanghai 210201, China
| | - Hong-Ping Wu
- Shanghai Celliver Biotechnology Co. Ltd., Shanghai 210201, China
| | - Wei-Feng Yu
- Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China. .,Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical University, Xuzhou 221004, China
| | - Bo Zhai
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China.
| | - He-Xin Yan
- Department of Interventional Oncology, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China. .,Shanghai Celliver Biotechnology Co. Ltd., Shanghai 210201, China.,Department of Anesthesiology and Critical Care Medicine, Renji Hospital, Jiaotong University School of Medicine, Shanghai 200127, China.,Shanghai Cancer Institute, Renji Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200127, China
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13
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Abstract
Abstract
The rapid development of nanotechnology paved the way for further expansion of polymer chemistry and the fabrication of advanced polymeric membranes. Such modifications allowed enhancing or adding some unique properties, including mechanical strength, excellent biocompatibility, easily controlled degradability, and biological activity. This chapter discusses various applications of polymeric membranes in three significant areas of biomedicine, including tissue engineering, drug delivery systems, and diagnostics. It is intended to highlight here possible ways of improvement the properties of polymeric membranes, by modifying with other polymers, functional groups, compounds, drugs, bioactive components, and nanomaterials.
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Affiliation(s)
- Marta J. Woźniak-Budych
- NanoBioMedical Centre , Adam Mickiewicz University , Wszechnicy Piastowskiej 3 , Poznań 61-614 , Poland
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14
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Deguchi S, Takayama K, Mizuguchi H. Generation of Human Induced Pluripotent Stem Cell-Derived Hepatocyte-Like Cells for Cellular Medicine. Biol Pharm Bull 2020; 43:608-615. [PMID: 32238703 DOI: 10.1248/bpb.b19-00740] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Liver transplantation and hepatocyte transplantation are effective treatments for severe liver injuries, but the donor shortage is a serious problem. Therefore, hepatocyte-like cells generated from human induced pluripotent stem (iPS) cells with unlimited proliferative ability are expected to be a promising new transplantation resource. The technology for hepatic differentiation from human iPS cells has made great progress in this decade. The efficiency of hepatic differentiation now exceeds 90%, making it possible to produce nearly homogeneous hepatocyte-like cells from human iPS cells. Because there is little contamination of undifferentiated cells, there is a lower risk of teratoma formation. To date, the transplantation of human iPS cell-derived hepatocyte-like cells has been shown to have therapeutic effects using various liver injury model mice. Currently, studies are underway using model animals larger than mice. The day when human iPS cell-derived hepatocyte-like cells can be used as cellular medicine is surely approaching. In this review, we introduce the forefront of regenerative medicine applications using human iPS cell-derived hepatocyte-like cells.
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Affiliation(s)
- Sayaka Deguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University
| | - Kazuo Takayama
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University.,PRESTO, Japan Science and Technology Agency.,Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University.,Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health and Nutrition.,Global Center for Medical Engineering and Informatics, Osaka University.,Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives (OTRI), Osaka University
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15
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van der Mark VA, Adam AAA, Chang JC, Oude Elferink RP, Chamuleau RAFM, Hoekstra R. Overexpression of the constitutive androstane receptor and shaken 3D-culturing increase biotransformation and oxidative phosphorylation and sensitivity to mitochondrial amiodarone toxicity of HepaRG cells. Toxicol Appl Pharmacol 2020; 399:115055. [PMID: 32428594 DOI: 10.1016/j.taap.2020.115055] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 05/11/2020] [Accepted: 05/13/2020] [Indexed: 02/06/2023]
Abstract
The liver cell line HepaRG is one of the preferred sources of human hepatocytes for in vitro applications. However, mitochondrial energy metabolism is relatively low, which affects hepatic functionality and sensitivity to hepatotoxins. Culturing in a bioartificial liver (BAL) system with high oxygen, medium perfusion, low substrate stiffness, and 3D conformation increases HepaRG functionality and mitochondrial activity compared to conventional monolayer culturing. In addition, drug metabolism has been improved by overexpression of the constitutive androstane receptor (CAR), a regulator of drug and energy metabolism in the new HepaRG-CAR line. Here, we investigated the effect of BAL culturing on the HepaRG-CAR line by applying a simple and downscaled BAL culture procedure based on shaking 3D cultures, named Bal-in-a-dish (BALIAD). We compared monolayer and BALIAD cultures of HepaRG and HepaRG-CAR cells. CAR overexpression and BALIAD culturing synergistically or additively increased transcript levels of CAR and three of the seven tested CAR target genes in biotransformation. Additionally, Cytochrome P450 3A4 activity was 35-fold increased. The mitochondrial energy metabolism was enhanced; lactate production and glucose consumption switched into lactate elimination and glucose production. BALIAD culturing alone reduced glycogen content and increased oxygen consumption and mitochondrial content. Both CAR overexpression and BALIAD culturing decreased mitochondrial superoxide levels. HepaRG-CAR BALIADs were most sensitive to mitochondrial toxicity induced by the hepatotoxin amiodarone, as indicated by oxygen consumption and mitochondrial superoxide accumulation. These data show that BALIAD culturing of HepaRG-CAR cells induces high mitochondrial energy metabolism and xenobiotic metabolism, increasing its potential for drug toxicity studies.
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Affiliation(s)
- Vincent A van der Mark
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands
| | - Aziza A A Adam
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Jung-Chin Chang
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Ronald P Oude Elferink
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Robert A F M Chamuleau
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
| | - Ruurdtje Hoekstra
- Amsterdam UMC, University of Amsterdam, Tytgat Institute for Liver and Intestinal Research, AG&M, Meibergdreef 69-71, 1105 BK, Amsterdam, the Netherlands.
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16
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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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17
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Santos-Vizcaino E, Orive G, Pedraz JL, Hernandez RM. Clinical Applications of Cell Encapsulation Technology. Methods Mol Biol 2020; 2100:473-491. [PMID: 31939144 DOI: 10.1007/978-1-0716-0215-7_32] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022]
Abstract
Cell encapsulation comprises immunoisolation three-dimensional systems for housing therapeutic cells that secrete bioactive compounds de novo and in a sustained manner. This allows transplantation of multiple allo- or xenogeneic cells without the aid of immunosuppressant drugs. Recent advances in the field have provided improvements to these cell-based drug delivery systems, which have gained the attention of the scientific community and inspired many biotechnological companies to develop their own product candidates. From micro- to macroencapsulation devices, this chapter describes some of the most important approaches that are being currently tested in late-stage clinical trials and are likely to reach the market as future game changers. Most studies involve the treatment of diabetes, eye disorders, and diseases of the central nervous system. However, many other pathologies are also amenable to benefit from this technology. Latest advances to overcome major pending challenges related to biosafety and efficacy are also discussed.
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Affiliation(s)
- Edorta Santos-Vizcaino
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain.,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain
| | - Gorka Orive
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain.,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain.,University Institute for Regenerative Medicine and Oral Implantology-UIRMI (UPV/EHU-Fundación Eduardo Anitua), Vitoria, Spain.,BTI Biotechnology Institute, Vitoria, Spain
| | - Jose Luis Pedraz
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain.,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain
| | - Rosa Maria Hernandez
- NanoBioCel Group, Laboratory of Pharmaceutics, School of Pharmacy, University of the Basque Country (UPV/EHU), Vitoria-Gasteiz, Spain. .,Biomedical Research Networking Centre in Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Vitoria-Gasteiz, Spain.
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18
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Li L, Meng H, Zou Q, Zhang J, Cai L, Yang B, Weng J, Lai L, Yang H, Gao Y. Establishment of gene-edited pigs expressing human blood-coagulation factor VII and albumin for bioartificial liver use. J Gastroenterol Hepatol 2019; 34:1851-1859. [PMID: 30884543 DOI: 10.1111/jgh.14666] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/26/2019] [Revised: 03/11/2019] [Accepted: 03/13/2019] [Indexed: 01/04/2023]
Abstract
BACKGROUND AND AIM Bioartificial livers (BALs) are considered as a solution to bridge patients with acute liver failure to liver transplantation or to assist in spontaneous recovery for patients with end-stage liver disease. Pig is the best donor of hepatocytes for BALs in clinical trials, because metabolic and detoxification function of its liver are close to human. However, using pig hepatocytes for BALs remains controversial for safety concern owing to nonhuman proteins secretion. Herein, we attempt to establish modified pigs expressing humanized liver proteins, blood-coagulation factor VII (F7), and albumin (ALB). These pigs should also be porcine endogenous retrovirus subtype C (PERV-C) free so that their ability of transmitting PERV to human could be diminished seriously. METHODS We devised both homology-dependent and independent knock-in approaches to insert a fusion of hF7 and hALB gene downstream the site of pig endogenous F7 promoter in pig fetal fibroblasts negative for PERV-C. The modified pigs were then generated through somatic cell nuclear transfer. RESULTS We obtained 14 and 10 cloned pigs by homology-dependent and independent approaches, respectively. Among them, 19 cloned pigs were with expected gene modification and 13 are alive to date. These modified pigs can successfully express hF7 and hALB in the liver and serum, and the expressed hF7 exhibits normal coagulation activity. CONCLUSIONS The gene-edited pigs expressing hF7 and hALB in the liver were generated successfully. We anticipate that our pigs could provide an alternative cell source for BALs as a promising treatment for patients with acute liver failure.
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Affiliation(s)
- Li Li
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Hongyi Meng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Qingjian Zou
- School of Chemical and Environmental Engineering, Wuyi University, Jiangmen, China
| | - Jianmin Zhang
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Lei Cai
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Bin Yang
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Jun Weng
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
| | - Liangxue Lai
- South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Huaqiang Yang
- College of Animal Science, South China Agricultural University, Guangzhou, China
| | - Yi Gao
- Department of Hepatobiliary Surgery II, Guangdong Provincial Research Center for Artificial Organ and Tissue Engineering, Institute of Regenerative Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, China
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19
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Zhang Y, Li L. State of the art—Artificial liver in China. Artif Organs 2019; 43:336-341. [DOI: 10.1111/aor.13448] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2019] [Accepted: 02/21/2019] [Indexed: 02/06/2023]
Affiliation(s)
- Yimin Zhang
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases the First Affiliated Hospital, College of Medicine, Zhejiang University Hangzhou China
| | - Lanjuan Li
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases the First Affiliated Hospital, College of Medicine, Zhejiang University Hangzhou China
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20
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Tong XF, Zhao FQ, Ren YZ, Zhang Y, Cui YL, Wang QS. Injectable hydrogels based on glycyrrhizin, alginate, and calcium for three-dimensional cell culture in liver tissue engineering. J Biomed Mater Res A 2018; 106:3292-3302. [DOI: 10.1002/jbm.a.36528] [Citation(s) in RCA: 35] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2018] [Revised: 07/23/2018] [Accepted: 08/14/2018] [Indexed: 01/03/2023]
Affiliation(s)
- Xiao-Fang Tong
- Tianjin State Key Laboratory of Modern Chinese Medicine, Research Center of Traditional Chinese Medicine; Tianjin University of Traditional Chinese Medicine; Tianjin 300193 China
| | - Fa-Quan Zhao
- Tianjin State Key Laboratory of Modern Chinese Medicine, Research Center of Traditional Chinese Medicine; Tianjin University of Traditional Chinese Medicine; Tianjin 300193 China
| | - Ying-Zong Ren
- Tianjin State Key Laboratory of Modern Chinese Medicine, Research Center of Traditional Chinese Medicine; Tianjin University of Traditional Chinese Medicine; Tianjin 300193 China
| | - Yi Zhang
- Tianjin State Key Laboratory of Modern Chinese Medicine, Research Center of Traditional Chinese Medicine; Tianjin University of Traditional Chinese Medicine; Tianjin 300193 China
| | - Yuan-Lu Cui
- Tianjin State Key Laboratory of Modern Chinese Medicine, Research Center of Traditional Chinese Medicine; Tianjin University of Traditional Chinese Medicine; Tianjin 300193 China
| | - Qiang-Song Wang
- Tianjin Key Laboratory of Biomedical Materials; Institute of Biomedical Engineering, Chinese Academy of Medical Science & Peking Union Medical College; Tianjin 300192 China
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21
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Wei G, Wang J, Lv Q, Liu M, Xu H, Zhang H, Jin L, Yu J, Wang X. Three-dimensional coculture of primary hepatocytes and stellate cells in silk scaffold improves hepatic morphology and functionality in vitro. J Biomed Mater Res A 2018; 106:2171-2180. [PMID: 29607608 DOI: 10.1002/jbm.a.36421] [Citation(s) in RCA: 21] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2018] [Revised: 03/11/2018] [Accepted: 03/21/2018] [Indexed: 11/08/2022]
Abstract
A vigorous in vitro model of liver that could recapitulate hepatic phenotype and functionality in vivo would exclusively improve the efficiency of bioartificial liver, drug discovery, or even transplantation therapy. Owing to the indispensable role of three-dimensional (3D) microenvironment in supporting viability and function of hepatocytes in vitro, much effort recently has been focused on improving reproducibility and standardization of primary hepatocyte cultures with a paradigm shift to 3D culture system, In the present study, an improved 3D coculture system of hepatocytes was established in which rat primary hepatocytes were cocultured with hepatic stellate cells in silk porous scaffolds. Silk scaffolds with incorporated extracellular matrix provided a suitable microenvironment for maintaining the viability, morphology and gene expression of the primary hepatocyte in vitro. The presence of stromal cells promoted primary hepatocyte to generate cellular aggregates with well-organized 3D architecture after 3 days of coculture in vitro. These aggregates exhibited proper morphology similar to liver tissue in vivo. Consistent with their phenotypic appearance, well-maintained functionality of hepatocytes was also observed in the cocultures, where albumin secretion/expression, urea synthesis as well as messenger ribonucleic acid expression of multiple cytochrome Ps (CYPs) enzymes increased significantly compared to either the 3D monocultures or monolayer cultures. Additionally, this 3D multicellular coculture model displayed an improved metabolic activity of CYPs enzymes to the probe drugs treatment. Thus, this culture system would not only contribute to the construction of micro-organoid tissue of liver but also potentially provide a robust tool for drug metabolism evaluation in vitro. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2171-2180, 2018.
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Affiliation(s)
- Guofeng Wei
- Second Hospital of Dalian Medical University, Dalian, 116023, People's Republic of China
| | - Jiwen Wang
- Second Hospital of Dalian Medical University, Dalian, 116023, People's Republic of China
| | - Qiang Lv
- National Engineering Laboratory for Modern Silk, Soochow University, Suzhou, 215123, People's Republic of China
| | - Ming Liu
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
| | - Hong Xu
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
| | - He Zhang
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
| | - Lingling Jin
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
| | - Jiachuan Yu
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
| | - Xiuli Wang
- College of Basic Medical Science, Dalian Medical University, Dalian, 116044, People's Republic of China
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22
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Talbot NC, Caperna TJ, Willard RR, Meekin JH, Garrett WM. Characterization of Two Subpopulations of the PICM-19 Porcine Liver Stem Cell Line for use in Cell-Based Extracorporeal Liver Assistance Devices. Int J Artif Organs 2018. [DOI: 10.1177/039139881003300603] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
Two cell lines, PICM-19H and PICM-19B, were derived from the bipotent PICM-19 pig liver stem cell line and assessed for their potential application in artificial liver devices (ALD). The study included assessments of growth rate and cell density in culture, morphological features, serum protein production, γ-glutamyltranspeptidase (GGT) activity and hepatocyte detoxification functions, i.e., inducible P450 activity, ammonia clearance, and urea production. The PICM-19H cell line was derived by temperature selection at 33–34°C. After each passage, PICM-19H cells grew to a nearly confluent monolayer of cells of hepatocyte morphology, i.e., cuboidal cells with centrally located nuclei joined by biliary canaliculi. No differentiation and self-organization into multi-cellular bile ductules, as observed in the parental PICM-19 cell line, occurred within the PICM-19H cell monolayers. The PICM-19H cells contained numerous mitochondria, Golgi apparatus, smooth and rough endoplasmic reticulum, vesicular bodies and occasional lipid vacuoles. The cells had a doubling time of 48–72 h and reached a final density of 1.5 x 105 cells/cm2 at ∼10 d post-passage from a 1:6 split ratio. PICM-19H cells displayed inducible P450 activity, cleared ammonia, and produced urea in a glutamine-free medium. The PICM-19B cells were colony-cloned after spontaneous generation from the PICM-19 parental cell line. PICM-19B cells grew as a tightly knit dome-forming monolayer with no visible biliary canaliculi. Their doubling time was 48–72 h with a final cell density of 2.6 x 105 cells/cm2. Ultrastructural analysis of the PICM-19B monolayers showed the roughly cuboidal cells displayed basal-apical polarization and were joined by tight junction-like complexes. Other ultrastructure features were similar to those of PICM-19H cells except that they possessed numerous cell bodies resembling mucus vacuoles. The PICM-19B cells had relatively high levels of GGT activity, but did retain some inducible P450 activity, and some ammonia clearance and urea synthesis ability. PICM-19B cells produced markedly less serum proteins than PICM-19H cells. These data indicated that both cell lines, either together or alone, may be useful as the cellular substrate for an ALD.
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Affiliation(s)
- Neil C. Talbot
- US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland - USA
| | - Thomas J. Caperna
- US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland - USA
| | - Ryan R. Willard
- US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland - USA
| | - John H. Meekin
- HepaLife Technologies, Inc., Boston, Massachusetts - USA
| | - Wesley M. Garrett
- US Department of Agriculture, Agricultural Research Service, Animal and Natural Resources Institute, Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural Research Center, Beltsville, Maryland - USA
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23
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Nardo B, Montalti R, Puviani L, Pacilè V, Beltempo P, Bertelli R, Licursi M, Pariali M, Cianciavicchia D. An experimental pilot study on controlled portal vein arterialization with an extracorporeal device in the swine model of partial liver resection and ischemia. Int J Artif Organs 2018; 29:912-8. [PMID: 17033999 DOI: 10.1177/039139880602900912] [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: 11/16/2022]
Abstract
AIM To determine whether the physiologically oxygenated arterial blood reversed in the portal system by means of portal vein arterialization (PVA) through an extracorporeal device which we have called L.E.O2.NARDO (Liver Extracorporeal Oxygen. NARDO) is effective in treating swine with subtotal hepatectomy leading to acute liver failure (ALF). METHODS Ten swine with ALF induced by 85-90% liver resection and five minutes of ischemia-reperfusion injury were randomly divided into two groups: five animals received PVA extracorporeal treatment and five swine were not-treated (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system. An extracorporeal device was interposed between the outflow and the inflow in order to monitoring the hemodynamic parameters. Each treatment lasted 6 hours. Serum and liver samples were collected in both groups. The survival was assessed at 1 week. RESULTS The PVA-extracorporeal treatment yielded beneficial effects for subtotal hepatectomy-induced ALF swine with decreased serum ammonia, transaminases and total bilirubin as compared with the untreated group. INR recovered rapidly in the PVA-extracorporeal group remaining significantly lower than in untreated animals. The 7-day survival of PVA-extracorporeal group swine was significantly higher than that of untreated animals, with a statistically significant difference (p<0.05). Four swine in the PVA-extracorporeal group survived at 1 week while none of the swine in the control group were alive at that time; an average time of 144h+/-13h and 24.4h+/-5h was observed in the PVA-extracorporeal and control groups, respectively. CONCLUSIONS Arterial blood supply in the portal system through the extracorporeal device is easily applicable, efficacious, safe and may represent a novel approach for ALF swine induced by subtotal liver resection.
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Affiliation(s)
- B Nardo
- Department of Surgery, Intensive Care Unit and Transplantations, S. Orsola Hospital, University of Bologna, Bologna - Italy.
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24
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Liang S, Yu S, Zhou N, Deng J, Gao C. Controlling the selective and directional migration of hepatocytes by a complementary density gradient of glycosylated hyperbranched polymers and poly(ethylene glycol) molecules. Acta Biomater 2017; 56:161-170. [PMID: 27998813 DOI: 10.1016/j.actbio.2016.12.032] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2016] [Revised: 11/24/2016] [Accepted: 12/14/2016] [Indexed: 12/18/2022]
Abstract
Repair and regeneration of defected tissues and organs depends strongly on the directional migration of targeted cells, for example, the enhancement of directional migration of hepatocytes could be helpful in liver regeneration and transplantation. Herein a complementary gradient of galactose-modified hyperbranched polymers (LA-HPMA) and poly(ethylene glycol) (PEG) molecules was designed and prepared on a same substrate. Characterizations of X-ray photoelectron spectrometry and quartz crystal microbalance with dissipation (QCM-d) demonstrated the unidirectional change in grafting density of LA-HPMA and PEG molecules, respectively. On the LA-HPMA/PEG complementary gradient surface, the human hepatoma (HepG2) cells showed preferential orientation and enhanced directional migration toward the region of lower PEG density and higher LA-HPMA density. By contrast, the mouse embryonic fibroblasts (NIH3T3) showed random migration irrelevant to the gradient. The success of the complementary gradient relies on the specific interaction between galactose and asialoglycoprotein receptor (ASGPR) expressed on HepG2 cells. STATEMENT OF SIGNIFICANCE.
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25
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Takahashi M, Sakurai M, Enosawa S, Omasa T, Tsuruoka S, Matsumura T. Double-Compartment Cell Culture Apparatus: Construction and Biochemical Evaluation for Bioartificial Liver Support. Cell Transplant 2017; 15:945-52. [PMID: 17300000 DOI: 10.3727/000000006783981341] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Functional demands on a bioartificial liver support (BAL) device are not limited to biosynthetic activities, but must also encompass metabolic removal of potentially toxic substances. For most BALs, however, the concept and design are exclusively directed to biosynthetic support. To add the ability to metabolize and remove toxic substances, we designed a double-compartment cell culture apparatus (DCCA). Two compartments are separated from each other by a compact epithelial cell sheet spread over a synthetic microporous membrane. When a renal proximal convoluted tubular cell line that had been transduced with the human multidrug-resistant (MDR) gene, PCTL-MDR, was introduced into one of the compartments (hereafter referred to as the “inner” compartment) of the DCCA, a compact cellular monolayer was formed on the membrane. Ammonium ions passed across the membrane, but glucose and its metabolite lactate could not, indicating that the DCCA allowed selective transportation of cellular metabolites. In addition to PCTL-MDR, HepG2, a cell line of hepatic-origin, transduced with CYP3A4 (designated GS-3A4-HepG2), was seeded on the opposite side of the membrane, and the metabolism and transportation of lidocaine were studied. The lidocaine metabolite, monoethylglycinexylidide, was detected in the inner compartment across the PCTL-MDR cell layered membrane, indicating that metabolism and the selective transportation of metabolites between the two compartments occurred by cooperation of renal and hepatic cells. These results suggest that this type of DCCA represents a novel BAL that possesses biotransporting activities, as well as biosynthetic and metabolic activities.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics
- ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism
- Calcium/metabolism
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/metabolism
- Carcinoma, Hepatocellular/pathology
- Cell Culture Techniques/instrumentation
- Cell Culture Techniques/methods
- Cell Line, Tumor
- Chromatography, High Pressure Liquid
- Glucose/metabolism
- Humans
- Lactic Acid/metabolism
- Liver, Artificial
- Membranes, Artificial
- Models, Theoretical
- Quaternary Ammonium Compounds/metabolism
- Transfection
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Affiliation(s)
- Masahiro Takahashi
- Cell Technology Center, Roman Industries Co. Ltd., Yokohama 236-0004, Japan
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26
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Hoekstra R, Deurholt T, ten Bloemendaal L, Desille M, van Wijk ACWA, Clement B, Oude Elferink RPJ, van Gulik TM, Chamuleau RAFM. Assessment of in Vitro Applicability of Reversibly Immortalized NKNT-3 Cells and Clonal Derivatives. Cell Transplant 2017; 15:423-433. [DOI: 10.3727/000000006783981873] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
In vitro applications of human hepatocytes, such as bioartificial livers and toxicity assays, require thoroughly testing of human cell lines prior to using them as alternative cell sources. The reversibly immortalized NKNT-3 cell line was reported to show clear in vivo functionality. Here, NKNT-3 cells were tested for their in vitro applicability. Low-passage (P2) and high-passage (P28) NKNT-3 cells and clonal derivatives were characterized for reversion of immortalization, heterogeneity, and hepatic functionality. Reversion with reduced expression of immortalizing agent could be established. However, during culturing the cells lost the capacity to be selected for completed reversion. The phenotypic instability is probably associated with heterogeneity in the culture, as clonal derivatives of P2 cells varied in morphology, growth, and reversion characteristics. The mRNA levels of genes related with hepatic differentiation increased 4–20-fold after reversion. However, the levels never exceeded 0.1% of that detected in liver and no urea production nor ammonia elimination was detected. Additionally, activities of different cytochrome P450s were limited. In conclusion, the NKNT-3 culture is heterogeneous and unstable and the in vitro functionality is relatively low. These findings emphasize that in vivo testing of hepatic cell lines is little informative for predicting their value for in vitro applications.
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Affiliation(s)
- Ruurdtje Hoekstra
- Surgical Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands
- AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands
| | - Tanja Deurholt
- AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands
| | - Lysbeth ten Bloemendaal
- Surgical Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands
- AMC Liver Center, Academic Medical Center, University of Amsterdam, The Netherlands
| | - Mireille Desille
- INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, Rennes, France
| | | | - Bruno Clement
- INSERM U456, Detoxication and Tissue Repair Unit, University of Rennes I, Rennes, France
| | | | - Thomas M. van Gulik
- Surgical Laboratory, Academic Medical Center, University of Amsterdam, The Netherlands
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27
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Alzebdeh DA, Matthew HW. Metabolic Oscillations in Co-Cultures of Hepatocytes and Mesenchymal Stem Cells: Effects of Seeding Arrangement and Culture Mixing. J Cell Biochem 2017; 118:3003-3015. [PMID: 28252220 DOI: 10.1002/jcb.25962] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2016] [Accepted: 02/28/2017] [Indexed: 12/21/2022]
Abstract
In vitro assembly of functional liver tissue is a prerequisite for the transplantation of tissue-engineered livers. There is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability. Recent reports have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) can provide a number of cues promoting hepatocyte growth and development. In this study, the effects of BM-MSCs co-culture on hepatocyte metabolism were evaluated as a function of scaffold seeding arrangement. BM-MSCs were co-cultured with hepatocytes in porous chitosan-heparin scaffolds using several seeding arrangements. The seeded scaffolds were subjected to orbital shaking to enhance mass transfer. BM-MSC-hepatocyte co-cultures exhibited higher rates of hepatocyte-specific functions, compared to hepatocyte-only cultures, regardless of the seeding arrangement. Cells formed smaller-compact spheroids in the heterotypic systems compared to mono-cultures of hepatocytes only. The spheroids exhibited reduction in size with time in all conditions except for the condition where BM-MSCs were seeded one day after seeding hepatocytes. In this condition, spheroids increased in size due to BM-MSC proliferation. Spheroid size reduction was hypothesized to be the result of cyclic shear stresses generated by the orbital shaking. Furthermore, results suggested that BM-MSC seeding onto preformed hepatocyte spheroids provide a degree of shear-protection and trophic stimuli. Overall, the results indicate that co-culturing hepatocytes with BM-MSCs enhanced their metabolic functions for the first week of culture. J. Cell. Biochem. 118: 3003-3015, 2017. © 2017 Wiley Periodicals, Inc.
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Affiliation(s)
| | - Howard William Matthew
- Department of Biomedical Engineering, Wayne State University, Detroit 48201, Michigan.,Department of Chemical Engineering and Materials Science, Wayne State University, Detroit 48202, Michigan
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28
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Pizarro MD, Mediavilla MG, Quintana AB, Scandizzi ÁL, Rodriguez JV, Mamprin ME. Performance of cold-preserved rat liver Microorgans as the biological component of a simplified prototype model of bioartificial liver. World J Hepatol 2016; 8:1442-1451. [PMID: 27957242 PMCID: PMC5124715 DOI: 10.4254/wjh.v8.i33.1442] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/31/2016] [Revised: 07/28/2016] [Accepted: 09/18/2016] [Indexed: 02/06/2023] Open
Abstract
AIM To develop a simplified bioartificial liver (BAL) device prototype, suitable to use freshly and preserved liver Microorgans (LMOs) as biological component.
METHODS The system consists of 140 capillary fibers through which goat blood is pumped. The evolution of hematocrit, plasma and extra-fiber fluid osmolality was evaluated without any biological component, to characterize the prototype. LMOs were cut and cold stored 48 h in BG35 and ViaSpan® solutions. Fresh LMOs were used as controls. After preservation, LMOs were loaded into the BAL and an ammonia overload was added. To assess LMOs viability and functionality, samples were taken to determine lactate dehydrogenase (LDH) release and ammonia detoxification capacity.
RESULTS The concentrations of ammonia and glucose, and the fluids osmolalities were matched after the first hour of perfusion, showing a proper exchange between blood and the biological compartment in the minibioreactor. After 120 min of perfusion, LMOs cold preserved in BG35 and ViaSpan® were able to detoxify 52.9% ± 6.5% and 53.6% ± 6.0%, respectively, of the initial ammonia overload. No significant differences were found with Controls (49.3% ± 8.8%, P < 0.05). LDH release was 6.0% ± 2.3% for control LMOs, and 6.2% ± 1.7% and 14.3% ± 1.1% for BG35 and ViaSpan® cold preserved LMOs, respectively (n = 6, P < 0.05).
CONCLUSION This prototype relied on a simple design and excellent performance. It’s a practical tool to evaluate the detoxification ability of LMOs subjected to different preservation protocols.
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29
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Zhou X, Cui L, Zhou X, Yang Q, Wang L, Guo G, Hou Y, Cai W, Han Z, Shi Y, Han Y. Induction of hepatocyte-like cells from human umbilical cord-derived mesenchymal stem cells by defined microRNAs. J Cell Mol Med 2016; 21:881-893. [PMID: 27874233 PMCID: PMC5387126 DOI: 10.1111/jcmm.13027] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2016] [Accepted: 10/03/2016] [Indexed: 12/15/2022] Open
Abstract
Generating functional hepatocyte‐like cells (HLCs) from mesenchymal stem cells (MSCs) is of great urgency for bio‐artificial liver support system (BALSS). Previously, we obtained HLCs from human umbilical cord‐derived MSCs by overexpressing seven microRNAs (HLC‐7) and characterized their liver functions in vitro and in vivo. Here, we aimed to screen out the optimal miRNA candidates for hepatic differentiation. We sequentially removed individual miRNAs from the pool and examined the effect of transfection with remainder using RT‐PCR, periodic acid—Schiff (PAS) staining and low‐density lipoprotein (LDL) uptake assays and by assessing their function in liver injury models. Surprisingly, miR‐30a and miR‐1290 were dispensable for hepatic differentiation. The remaining five miRNAs (miR‐122, miR‐148a, miR‐424, miR‐542‐5p and miR‐1246) are essential for this process, because omitting any one from the five‐miRNA combination prevented hepatic trans‐differentiation. We found that HLCs trans‐differentiated from five microRNAs (HLC‐5) expressed high level of hepatic markers and functioned similar to hepatocytes. Intravenous transplantation of HLC‐5 into nude mice with CCl4‐induced fulminant liver failure and acute liver injury not only improved serum parameters and their liver histology, but also improved survival rate of mice in severe hepatic failure. These data indicated that HLC‐5 functioned similar to HLC‐7 in vitro and in vivo, which have been shown to resemble hepatocytes. Instead of using seven‐miRNA combination, a simplified five‐miRNA combination can be used to obtain functional HLCs in only 7 days. Our study demonstrated an optimized and efficient method for generating functional MSC‐derived HLCs that may serve as an attractive cell alternative for BALSS.
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Affiliation(s)
- Xia Zhou
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Lina Cui
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Xinmin Zhou
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Qiong Yang
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Lu Wang
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Guanya Guo
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Yu Hou
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Weile Cai
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Zheyi Han
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Yongquan Shi
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
| | - Ying Han
- State Key Laboratory of Cancer Biology, Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, Xi'an, Shaanxi Province, China
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30
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Ma S, Lin Y, Deng B, Zheng Y, Hao C, He R, Ding F. Endothelial bioreactor system ameliorates multiple organ dysfunction in septic rats. Intensive Care Med Exp 2016; 4:23. [PMID: 27447715 PMCID: PMC4958089 DOI: 10.1186/s40635-016-0097-y] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Accepted: 07/13/2016] [Indexed: 12/26/2022] Open
Abstract
BACKGROUND The endothelium is a potentially valuable target for sepsis therapy. We have previously studied an extracorporeal endothelial cell therapy system, called the endothelial bioreactor (EBR), which prolonged the survival time of endotoxemia sepsis in swine. To further study of the therapeutic effects and possible mechanisms, we established a miniature EBR system for septic rats induced by cecal ligation and puncture (CLP). METHODS In the miniature EBR system, the extracorporeal circulation first passed through a mini-hemofilter, and the ultrafiltrate (UF) was separated, then the UF passed through an EBR (a 1-mL cartridge containing approximately 2 × 10(6) endothelial cells grown on microcarriers) and interact with endothelial cells. Eighteen hours after CLP, the rats were treated for 4 h with this extracorporeal system containing either endothelial cells (EBR group) or no cells (sham EBR group). Physiologic and biochemical parameters, cytokines, endothelial functions, and 7-day survival time were monitored. In vitro, the pulmonary endothelial cells of the septic rats were treated with the EBR system and the resulting changes in their functions were monitored. RESULTS The EBR system ameliorated CLP-induced sepsis compared with the sham EBR system. After CLP, the 7-day survival rate of sham-treated rats was only 25.0 %, while in the EBR-treated group, it increased to 57.1 % (p = 0.04). The EBR system protected the liver and renal function and ameliorated the kidney and lung injury. Meanwhile, this therapy reduced pulmonary vascular leakage and alleviated the infiltration of inflammatory cells in the lungs, especially neutrophils. Furthermore, after the EBR treatment both in vivo and in vitro, the expression of intercellular adhesion molecule-1 and the secretion of CXCL1 and CXCL2 of pulmonary endothelium decreased, which helped to alleviate the adhesion and chemotaxis of neutrophils. In addition, the EBR system decreased CD11b expression and intracellular free calcium level of peripheral blood neutrophils, modulated the activation of these neutrophils. CONCLUSIONS The EBR system significantly ameliorated CLP-induced sepsis and improved survival and organ functions. Compared with the sham EBR system, this extracorporeal endothelial therapy may be involved in modulating the function of pulmonary endothelial cells, reducing the adhesion and chemotaxis of neutrophil, and modulating the activation of peripheral blood neutrophils.
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Affiliation(s)
- Shuai Ma
- Division of Nephrology & Unit of Critical Nephrology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, 639 Zhizaoju Road, Shanghai, 200011, China.,Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, 200040, China
| | - Yuli Lin
- Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, 200032, China
| | - Bo Deng
- Division of Nephrology & Unit of Critical Nephrology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, 639 Zhizaoju Road, Shanghai, 200011, China.,Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, 200040, China
| | - Yin Zheng
- Division of Nephrology & Unit of Critical Nephrology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, 639 Zhizaoju Road, Shanghai, 200011, China.,Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, 200040, China
| | - Chuanming Hao
- Division of Nephrology, Huashan Hospital, Fudan University, Shanghai, 200040, China
| | - Rui He
- Department of Immunology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
| | - Feng Ding
- Division of Nephrology & Unit of Critical Nephrology, Shanghai Ninth People's Hospital, School of Medicine, Shanghai Jiaotong University, 639 Zhizaoju Road, Shanghai, 200011, China.
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Abstract
Liver disease is a leading cause of morbidity and mortality. Liver transplantation remains the only proven treatment for end-stage liver failure but is limited by the availability of donor organs. Hepatocyte cell therapy, either with bioartificial liver devices or hepatocyte transplantation, may help address this by delaying or preventing liver transplantation. Early clinical studies have shown promising results, however in most cases, the benefit has been short lived and so further research into these therapies is required. Alternative sources of hepatocytes, including stem cell-derived hepatocytes, are being investigated as the isolation of primary human hepatocytes is limited by the same shortage of donor organs. This review summarises the current clinical experience of hepatocyte cell therapy together with an overview of possible alternative sources of hepatocytes. Current and future areas for research that might lead towards the realisation of the full potential of hepatocyte cell therapy are discussed.
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Affiliation(s)
- David Christopher Bartlett
- a NIHR Centre for Liver Research and Biomedical Research Unit, University of Birmingham, Birmingham, UK.,b Liver Unit, University Hospital Birmingham NHS Foundation Trust, Birmingham, UK
| | - Philip N Newsome
- a NIHR Centre for Liver Research and Biomedical Research Unit, University of Birmingham, Birmingham, UK.,b Liver Unit, University Hospital Birmingham NHS Foundation Trust, Birmingham, UK
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32
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Mohanty S, Sanger K, Heiskanen A, Trifol J, Szabo P, Dufva M, Emnéus J, Wolff A. Fabrication of scalable tissue engineering scaffolds with dual-pore microarchitecture by combining 3D printing and particle leaching. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2016; 61:180-9. [DOI: 10.1016/j.msec.2015.12.032] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/13/2015] [Revised: 11/27/2015] [Accepted: 12/14/2015] [Indexed: 10/22/2022]
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33
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Jain E, Damania A, Shakya AK, Kumar A, Sarin SK, Kumar A. Fabrication of macroporous cryogels as potential hepatocyte carriers for bioartificial liver support. Colloids Surf B Biointerfaces 2015; 136:761-71. [PMID: 26519938 DOI: 10.1016/j.colsurfb.2015.10.012] [Citation(s) in RCA: 32] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2015] [Revised: 10/03/2015] [Accepted: 10/09/2015] [Indexed: 01/15/2023]
Abstract
Two different cryogels composed of copolymer of acrylonitrile (AN) and N-vinyl-2-pyrrolidone (NVP) (poly(AN-co-NVP)) and interpenetrated polymer networks (IPN) of chitosan and poly(N-isopropylacrylamide) (poly(NiPAAm)-chitosan) were fabricated by gelation at sub-zero temperatures. The two cryogels possess an interconnected network of macropores of size 20-100 μm and efficient transport properties as determined by physiochemical analysis. Both cryogels support in vitro growth and function of fibroblasts (COS-7) and human liver hepatocarcinoma cells (HepG2). The cryogels are hemocompatible as demonstrated by low albumin adsorption and platelet adherence. Furthermore, in vivo implantation of poly(NiPAAm)-chitosan cryogel in mice shows its biocompatibility with the surrounding tissue. Primary rat hepatocytes grown on poly(NiPAAm)-chitosan cryogel for 96 h formed cellular aggregates and maintained their functions in terms of, ammonia removal, ureagenesis and drug detoxification. Cryogel-based closed continuous bioreactor systems could maintain HepG2 cells at high density for 7 days. Off-line clinical evaluation of these cryogel-based bioreactors showed the ability of immobilized cells to detoxify circulating plasma obtained from patients with acute on chronic liver failure (ACLF). Altogether, the presented data suggests cryogels as a potential bioreactor matrix for bio-artificial liver support system.
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Affiliation(s)
- Era Jain
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Apeksha Damania
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Akhilesh Kumar Shakya
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India
| | - Anupam Kumar
- Institute of Liver and Biliary Sciences, New Delhi, India
| | - Shiv K Sarin
- Institute of Liver and Biliary Sciences, New Delhi, India
| | - Ashok Kumar
- Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur 208016, UP, India.
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Mohanty S, Larsen LB, Trifol J, Szabo P, Burri HVR, Canali C, Dufva M, Emnéus J, Wolff A. Fabrication of scalable and structured tissue engineering scaffolds using water dissolvable sacrificial 3D printed moulds. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2015; 55:569-78. [DOI: 10.1016/j.msec.2015.06.002] [Citation(s) in RCA: 109] [Impact Index Per Article: 10.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/23/2015] [Revised: 05/07/2015] [Accepted: 06/04/2015] [Indexed: 01/08/2023]
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35
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Teotia RS, Kalita D, Singh AK, Verma SK, Kadam SS, Bellare JR. Bifunctional Polysulfone-Chitosan Composite Hollow Fiber Membrane for Bioartificial Liver. ACS Biomater Sci Eng 2015; 1:372-381. [DOI: 10.1021/ab500061j] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/16/2023]
Affiliation(s)
| | | | | | | | - Sachin S. Kadam
- Defence Institute of Advanced Technology, (Deemed University), Girinagar, Pune 411025, India
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36
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Zhou Q, Li L, Li J. Stem cells with decellularized liver scaffolds in liver regeneration and their potential clinical applications. Liver Int 2015; 35:687-94. [PMID: 24797694 DOI: 10.1111/liv.12581] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/15/2014] [Accepted: 04/27/2014] [Indexed: 02/13/2023]
Abstract
End-stage hepatic failure is a potentially life-threatening condition for which orthotopic liver transplantation (OLT) is the only effective treatment. However, a shortage of available donor organs for transplantation each year results in the death of many patients waiting for liver transplantation. Cell-based therapies and hepatic tissue engineering have been considered as alternatives to liver transplantation. However, primary hepatocyte transplantation has rarely produced therapeutic effects because mature hepatocytes cannot be effectively expanded in vitro, and the availability of hepatocytes is often limited by shortages of donor organs. Decellularization is an attractive technique for scaffold preparation in stem cell-based liver engineering, as the resulting material can potentially retain the liver architecture, native vessel network and specific extracellular matrix (ECM). Thus, the reconstruction of functional and practical liver tissue using decellularized scaffolds becomes possible. This review focuses on the current understanding of liver tissue engineering, whole-organ liver decellularization techniques, cell sources for recellularization and potential clinical applications and challenges.
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Affiliation(s)
- Qian Zhou
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Rd., Hangzhou, 310003, China
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Duan B, Zheng X, Xia Z, Fan X, Guo L, Liu J, Wang Y, Ye Q, Zhang L. Highly biocompatible nanofibrous microspheres self-assembled from chitin in NaOH/urea aqueous solution as cell carriers. Angew Chem Int Ed Engl 2015; 54:5152-6. [PMID: 25712796 DOI: 10.1002/anie.201412129] [Citation(s) in RCA: 102] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/17/2014] [Revised: 01/22/2015] [Indexed: 12/29/2022]
Abstract
In this work, chitin microspheres (NCM) having a nanofibrous architecture were constructed using a "bottom-up" fabrication pathway. The chitin chains rapidly self-assembled into nanofibers in NaOH/urea aqueous solution by a thermally induced method and subsequently formed weaved microspheres. The diameter of the chitin nanofibers and the size of the NCM were tunable by controlling the temperature and the processing parameters to be in the range from 26 to 55 nm and 3 to 130 μm, respectively. As a result of the nanofibrous surface and the inherent biocompatibility of chitin, cells could adhere to the chitin microspheres and showed a high attachment efficiency, indicating the great potential of the NCM for 3D cell microcarriers.
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Affiliation(s)
- Bo Duan
- College of Chemistry & Molecule Science, Wuhan University, Wuhan, 430072 (China)
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Duan B, Zheng X, Xia Z, Fan X, Guo L, Liu J, Wang Y, Ye Q, Zhang L. Highly Biocompatible Nanofibrous Microspheres Self-Assembled from Chitin in NaOH/Urea Aqueous Solution as Cell Carriers. Angew Chem Int Ed Engl 2015. [DOI: 10.1002/ange.201412129] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
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Portal blood arterialization with an extracorporeal device to treat toxic acute hepatic failure in a swine model. Int J Artif Organs 2014; 37:847-53. [PMID: 25501739 DOI: 10.5301/ijao.5000367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 10/22/2014] [Indexed: 11/20/2022]
Abstract
PURPOSE This study aimed to determine whether a controlled portal blood arterialization by a liver extracorporeal device (L.E.O2 NARDO) is effective in treating acute hepatic failure (AHF) induced through CCl4 administration in a swine model. METHODS 20 swine with AHF induced by intraperitoneal injection of carbon tetrachloride (CCl4) in oil solution, were randomly divided into two groups: animals receiving L.E.O2 NARDO treatment 48 h after the intoxication (study group); animals sham operated 48 h after the intoxication (control group). Blood was withdrawn from the iliac artery and reversed in the portal venous system by an interposed extracorporeal device. Each treatment lasted 6 h. The survival was assessed at 5 days after L.E.O2 NARDO treatment or sham operation. In both groups blood samples were collected for biochemical analysis at different time points and liver biopsies were collected 48 h after intoxication and at sacrifice. RESULTS We observed decreased transaminases levels and a more rapid INR recovery in the study group, as compared to the control group. Eight animals of the study group vs. two animals of the control group survived at five days after surgery with a statistically significant difference (p<0.05). Liver biopsies performed at sacrifice showed a reduction of the damaged hepatic areas in the study group as compared to the control group. CONCLUSIONS Arterial blood supply in the portal system through the L.E.O2 NARDO device is easily applicable, efficacious, and safe in a swine model of AHF induced by CCl4 intoxication.
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Sarika PR, Sidhy Viha CV, Sajin Raj RG, Nirmala RJ, Anil Kumar PR. A non-adhesive hybrid scaffold from gelatin and gum Arabic as packed bed matrix for hepatocyte perfusion culture. MATERIALS SCIENCE & ENGINEERING. C, MATERIALS FOR BIOLOGICAL APPLICATIONS 2014; 46:341-7. [PMID: 25491996 DOI: 10.1016/j.msec.2014.10.044] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 03/14/2014] [Revised: 09/27/2014] [Accepted: 10/21/2014] [Indexed: 10/24/2022]
Abstract
Development of liver support systems has become one of the most investigated areas for the last 50 years because of the shortage of donor organs for orthotopic liver transplantations. Bioartificial liver (BAL) device is one of the alternatives for liver failure which provides a curing method and support patients to recover from certain liver failure diseases. The biological compartment of BAL is called the bioreactor where functionally active hepatocytes are maintained to support the liver specific functions. We have developed a packed bed bioreactor with a cytocompatible, polysaccharide-protein hybrid scaffold. The scaffold prepared from gelatin and gum Arabic acts as a packed bed matrix for hepatocyte culture. Quantitative evaluation of the hepatocytes cultured using packed bed bioreactor demonstrated that cells maintained liver specific functions like albumin and urea synthesis for seven days. These results indicated that the system can be scaled up to form the biological component of a bioartificial liver.
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Affiliation(s)
- P R Sarika
- Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547, India
| | - C V Sidhy Viha
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India
| | - R G Sajin Raj
- Device Testing Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India
| | - Rachel James Nirmala
- Department of Chemistry, Indian Institute of Space Science and Technology, Valiamala, Thiruvananthapuram, Kerala 695 547, India
| | - P R Anil Kumar
- Tissue Culture Laboratory, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura, Trivandrum, Kerala 695 012, India.
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Efficient large-scale generation of functional hepatocytes from mouse embryonic stem cells grown in a rotating bioreactor with exogenous growth factors and hormones. Stem Cell Res Ther 2014; 4:145. [PMID: 24294908 PMCID: PMC4054944 DOI: 10.1186/scrt356] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2013] [Revised: 09/30/2013] [Accepted: 10/21/2013] [Indexed: 12/19/2022] Open
Abstract
Introduction Embryonic stem (ES) cells are considered a potentially advantageous source of hepatocytes for both transplantation and the development of bioartificial livers. However, the efficient large-scale generation of functional hepatocytes from ES cells remains a major challenge, especially for those methods compatible with clinical applications. Methods In this study, we investigated whether a large number of functional hepatocytes can be differentiated from mouse ES (mES) cells using a simulated microgravity bioreactor. mES cells were cultured in a rotating bioreactor in the presence of exogenous growth factors and hormones to form embryoid bodies (EBs), which then differentiated into hepatocytes. Results During the rotating culture, most of the EB-derived cells gradually showed the histologic characteristics of normal hepatocytes. More specifically, the expression of hepatic genes and proteins was detected at a higher level in the differentiated cells from the bioreactor culture than in cells from a static culture. On further growing, the EBs on tissue-culture plates, most of the EB-derived cells were found to display the morphologic features of hepatocytes, as well as albumin synthesis. In addition, the EB-derived cells grown in the rotating bioreactor exhibited higher levels of liver-specific functions, such as glycogen storage, cytochrome P450 activity, low-density lipoprotein, and indocyanine green uptake, than did differentiated cells grown in static culture. When the EB-derived cells from day-14 EBs and the cells’ culture supernatant were injected into nude mice, the transplanted cells were engrafted into the recipient livers. Conclusions Large quantities of high-quality hepatocytes can be generated from mES cells in a rotating bioreactor via EB formation. This system may be useful in the large-scale generation of hepatocytes for both cell transplantation and the development of bioartificial livers.
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Abstract
Bioartificial liver support (BAL) systems are potential new therapeutic approaches for use as liver support to prevent nutrient deficiencies, hypoxia, or ischemia before the acquisition of donated organs. To investigate whether islets are beneficial for hepatocyte function and survival, we cocultured BALB/c mouse islets with C57BL/6J hepatocytes to assess hepatocyte viability, function, and apoptosis. We observe cell viability to decrease progressively by 50% from day 0 to day 3 among isolated hepatocytes (group A) and hepatocytes cocultured with islets (group B). However, group A was prone to necrosis and reduced albumin secretion during culture. In contrast, at day 7 group B maintained albumin secretion (0.3351 ± 0.0581 vs 0.1451 ± 0.0329 μg/h/mL; P < .05). Early apoptosis was observed at day 3 among group A but at day 7 in group B. In addition, quantitative analysis of the apoptotic cells revealed group B to show a delayed phenotype of both early and late apoptosis compared with group A. Our results indicated that islets could retain hepatocyte function and delay apoptosis, suggesting that the coculture system is potentially applicable to develop a high-performance BAL.
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Induction of highly functional hepatocytes from human umbilical cord mesenchymal stem cells by HNF4α transduction. PLoS One 2014; 9:e104133. [PMID: 25137413 PMCID: PMC4138090 DOI: 10.1371/journal.pone.0104133] [Citation(s) in RCA: 22] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2014] [Accepted: 07/10/2014] [Indexed: 12/17/2022] Open
Abstract
Aim To investigate the differentiation potential of human umbilical mesenchymal stem cells (HuMSCs) and the key factors that facilitate hepatic differentiation. Methods HuMSCs were induced to become hepatocyte-like cells according to a previously published protocol. The differentiation status of the hepatocyte-like cells was examined by observing the morphological changes under an inverted microscope and by immunofluorescence analysis. Hepatocyte nuclear factor 4 alpha (HNF4α) overexpression was achieved by plasmid transfection of the hepatocyte-like cells. The expression of proteins and genes of interest was then examined by Western blotting and reverse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. Results Our results demonstrated that HuMSCs can easily be induced into hepatocyte-like cells using a published differentiation protocol. The overexpression of HNF4α in the induced HuMSCs significantly enhanced the expression levels of hepatic-specific proteins and genes. HNF4α overexpression may be associated with liver-enriched transcription factor networks and the Wnt/β-Catenin pathway. Conclusion The overexpression of HNF4α improves the hepatic differentiation of HuMSCs and is a simple way to improve cellular sources for clinical applications.
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Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy. Sci Rep 2014; 4:6044. [PMID: 25116435 PMCID: PMC4131220 DOI: 10.1038/srep06044] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2014] [Accepted: 07/18/2014] [Indexed: 02/07/2023] Open
Abstract
Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy.
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No DY, Jeong GS, Lee SH. Immune-protected xenogeneic bioartificial livers with liver-specific microarchitecture and hydrogel-encapsulated cells. Biomaterials 2014; 35:8983-91. [PMID: 25088727 DOI: 10.1016/j.biomaterials.2014.07.009] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2014] [Accepted: 07/10/2014] [Indexed: 12/18/2022]
Abstract
Development of a xenogeneic biological liver support is important in providing a bridge to transplantation or liver regeneration, thus helping to overcome the chronic shortage of liver donors. Among the critical factors in developing biological liver support are the creation of in vivo mimetic micro liver tissue (mLT), especially mLTs containing liver-specific ultrastructure, and an encapsulation method that can package massive numbers of cells while providing immune-protection from the host immune system. We describe here the development of mLTs that include liver microarchitecture and their in situ encapsulation in hydrogel composites. Concave microwells and the tri-culture of three types of primary liver cells were applied for the construction of mLTs showing excellent liver functions and long-term (>1 month) viability in vitro. Large quantities of rat mLTs were encapsulated in collagen-alginate composites, implanted into hepatic failure mice and sustained their survival during regeneration of the remaining liver. The proposed liver support system offers xenogeneic hepatic assistance by mimicking native liver microarchitecture and providing immune-protection without the need for complicated devices or processes, and as such represents a promising system for recovery of organ function.
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Affiliation(s)
- Da Yoon No
- Department of Biomedical Engineering, Korea University, Seoul 136-701, Republic of Korea
| | - Gi Seok Jeong
- Department of Biomedical Engineering, Korea University, Seoul 136-701, Republic of Korea
| | - Sang-Hoon Lee
- Department of Biomedical Engineering, Korea University, Seoul 136-701, Republic of Korea; KU-KIST Graduate School of Converging of Sciences & Technologies, Korea University, Seoul 136-703, Republic of Korea.
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Du C, Narayanan K, Leong MF, Wan AC. Induced pluripotent stem cell-derived hepatocytes and endothelial cells in multi-component hydrogel fibers for liver tissue engineering. Biomaterials 2014; 35:6006-14. [DOI: 10.1016/j.biomaterials.2014.04.011] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2014] [Accepted: 04/07/2014] [Indexed: 12/22/2022]
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Yin CH, Chen W, Hsiao CC, Kuo CY, Chen CL, Wu WT. Production of Mouse Embryoid Bodies with Hepatic Differentiation Potential by Stirred Tank Bioreactor. Biosci Biotechnol Biochem 2014; 71:728-34. [PMID: 17341832 DOI: 10.1271/bbb.60568] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
Embryonic stem (ES) cells can differentiate into functional hepatic lineage cells, which can potentially be used in biomedicine. To obtain hepatic lineage cells from ES cells, embryoid bodies (EBs) must be formed. In this study, we developed an EB formation system using a spinner flask for mass production of EBs. ES cells were inoculated into the spinner flask, where they formed EBs within 4 d. The EBs were then transferred into an attached culture for hepatic differentiation. To verify the hepatic lineage cells, albumin secretion and hepatic-specific gene expression were examined. We found that EBs formed by either the spinner flask or hanging drops exhibited similar albumin secretion potential and hepatic-specific gene expression. We conclude that the spinner flask method can be used to produce mouse EBs that can be used to mass produce hepatic lineage cells for use in biomedicine.
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Affiliation(s)
- Chih-Hsiu Yin
- Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan
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Podichetty JT, Bhaskar PR, Khalf A, Madihally SV. Modeling Pressure Drop Using Generalized Scaffold Characteristics in an Axial-Flow Bioreactor for Soft Tissue Regeneration. Ann Biomed Eng 2014; 42:1319-30. [DOI: 10.1007/s10439-014-1009-5] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2013] [Accepted: 03/31/2014] [Indexed: 10/25/2022]
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Tahmasbi Rad A, Ali N, Kotturi HSR, Yazdimamaghani M, Smay J, Vashaee D, Tayebi L. Conducting scaffolds for liver tissue engineering. J Biomed Mater Res A 2014; 102:4169-81. [DOI: 10.1002/jbm.a.35080] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2013] [Revised: 12/13/2013] [Accepted: 01/15/2014] [Indexed: 12/24/2022]
Affiliation(s)
- Armin Tahmasbi Rad
- School of Materials Science and Engineering; Helmerich Advanced Technology Research Center; Oklahoma State University; Tulsa Oklahoma 74105
| | - Naushad Ali
- Department of Internal Medicine; Section of Digestive Diseases and Nutrition, University of Oklahoma Health Sciences Center; 975 NE 10th Street Oklahoma City Oklahoma 73104
| | - Hari Shankar R. Kotturi
- Department of Biology; University of Central Oklahoma; 100 North University Drive; Edmond Oklahoma 73034
| | | | - Jim Smay
- School of Chemical Engineering; Oklahoma State University; Stillwater Oklahoma 74078
| | - Daryoosh Vashaee
- School of Electrical and Computer Engineering; Helmerich Advanced Technology Research Center; Oklahoma State University; Tulsa Oklahoma 74105
| | - Lobat Tayebi
- School of Materials Science and Engineering; Helmerich Advanced Technology Research Center; Oklahoma State University; Tulsa Oklahoma 74105
- School of Chemical Engineering; Oklahoma State University; Stillwater Oklahoma 74078
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Zhang S, Chen L, Liu T, Wang Z, Wang Y. Integration of single-layer skin hollow fibers and scaffolds develops a three-dimensional hybrid bioreactor for bioartificial livers. JOURNAL OF MATERIALS SCIENCE. MATERIALS IN MEDICINE 2014; 25:207-216. [PMID: 23963686 DOI: 10.1007/s10856-013-5033-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/16/2013] [Accepted: 08/14/2013] [Indexed: 06/02/2023]
Abstract
Bioartificial liver support systems are expected to be an effective therapy as a "bridge" for liver transplantation or reversible acute liver disease. A major roadblock in the application of bioartificial livers is the need for a bioreactor that fully meets the requirements of hepatocyte culture, mass transfer and immunobarriers. In this study, we developed a three-dimensional hybrid bioreactor (3DHB) on a base of single-layer skin polyethersulfone hollow fibers by integrating with polyurethane scaffolds. The mass transfer of bilirubin and albumin from the intracapillary space to the extracapillary space of the hollow fibers was not significantly different between 3DHBs and hollow fiber bioreactors (HFBs). Cell viability staining showed that high-density hepatocytes were uniformly found in different regions of the 3DHB after 7 days of culture. Liver-specific functions of human mature hepatocytes cultured in the 3DHB, such as albumin secretion, urea production, ammonia removal rate and cytochrome P450 activity, were maintained stably and were significantly higher compared with the HFB. These results indicated that the 3DHB has good mass transfer and improves cell distribution and liver-specific functions. Meanwhile, the ammonia and unconjugated bilirubin concentrations in plasma from patients with liver failure were significantly decreased during 6 h of circulation by hepatocytes cultured in the 3DHB. Most hepatocytes in the 3DHB were viable after 6 h exposure to the patient plasma. We further demonstrated that bioartificial liver systems with 3DHB can remove toxins from and endure the deleterious effects of the patient plasma. Therefore, the 3DHB has the potential to accomplish different actions for the clinical application of bioartificial livers.
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Affiliation(s)
- Shichang Zhang
- Institute of Infectious Diseases, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China,
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