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Tonooka Y, Takaku T, Toyoshima M, Takahashi Y, Kitamoto S. Suppression of the Epithelial-Mesenchymal Transition and Maintenance of the Liver Functions in Primary Hepatocytes through Dispersion Culture within a Dome-Shaped Collagen Matrix. Biol Pharm Bull 2024; 47:1241-1247. [PMID: 38945897 DOI: 10.1248/bpb.b24-00180] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/02/2024]
Abstract
Primary hepatocytes are valuable for studying liver diseases, drug-induced liver injury, and drug metabolism. However, when cultured in a two-dimensional (2D) environment, primary hepatocytes undergo rapid dedifferentiation via an epithelial-mesenchymal transition (EMT) and lose their liver-specific functions. On the other hand, a three-dimensional (3D) culture of primary hepatocyte organoids presents challenges for analyzing cellular functions and molecular behaviors due to strong cell-cell adhesion among heterogeneous cells. In this study, we developed a novel dispersion culture method of hepatocytes within a dome-shaped collagen matrix, overcoming conventional limitations. The expression levels of EMT-related genes were lower in rat primary hepatocytes cultured using this method for 4 d than in cells cultured using the 2D method. Furthermore, albumin production, a marker of liver function, declined sharply in rat primary hepatocytes cultured in two dimensions from 6.40 µg/mL/48 h on day 4 to 1.35 µg/mL/48 h on day 8, and declined gradually from 4.92 µg/mL/48 h on day 8 to 3.89 µg/mL/48 h on day 14 in rat primary hepatocytes cultured using our new method. These findings indicate that the newly developed culture method can suppress EMT and maintain liver functions for 14 d in rat primary hepatocytes, potentially expanding the utility of primary hepatocyte cultured by using conventional 3D methods.
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Affiliation(s)
- Yoshino Tonooka
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
| | - Tomoyuki Takaku
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
| | - Manabu Toyoshima
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
| | | | - Sachiko Kitamoto
- Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd
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Zhao Y, Wang EY, Lai FBL, Cheung K, Radisic M. Organs-on-a-chip: a union of tissue engineering and microfabrication. Trends Biotechnol 2023; 41:410-424. [PMID: 36725464 PMCID: PMC9985977 DOI: 10.1016/j.tibtech.2022.12.018] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2022] [Revised: 12/23/2022] [Accepted: 12/27/2022] [Indexed: 02/03/2023]
Abstract
We review the emergence of the new field of organ-on-a-chip (OOAC) engineering, from the parent fields of tissue engineering and microfluidics. We place into perspective the tools and capabilities brought into the OOAC field by early tissue engineers and microfluidics experts. Liver-on-a-chip and heart-on-a-chip are used as two case studies of systems that heavily relied on tissue engineering techniques and that were amongst the first OOAC models to be implemented, motivated by the need to better assess toxicity to human tissues in preclinical drug development. We review current challenges in OOAC that often stem from the same challenges in the parent fields, such as stable vascularization and drug absorption.
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Affiliation(s)
- Yimu Zhao
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada
| | - Erika Yan Wang
- David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
| | - Fook B L Lai
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada
| | - Krisco Cheung
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada
| | - Milica Radisic
- Institute of Biomedical Engineering, University of Toronto, Toronto, Ontario M5S 3G9, Canada; Toronto General Hospital Research Institute, University Health Network, Toronto, Ontario M5G 2C4, Canada; Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada.
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Ezzat T, Dhar DK, Malago M, Damink SWMO. Dynamic tracking of stem cells in an acute liver failure model. World J Gastroenterol 2012; 18:507-16. [PMID: 22363116 PMCID: PMC3280395 DOI: 10.3748/wjg.v18.i6.507] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/01/2011] [Revised: 09/02/2011] [Accepted: 10/28/2011] [Indexed: 02/06/2023] Open
Abstract
AIM: To investigate a dual labeling technique, which would enable real-time monitoring of transplanted embryonic stem cell (ESC) kinetics, as well as long-term tracking.
METHODS: Liver damage was induced in C57/BL6 male mice (n = 40) by acetaminophen (APAP) 300 mg/kg administered intraperitoneally. Green fluorescence protein (GFP) positive C57/BL6 mouse ESCs were stained with the near-infrared fluorescent lipophilic tracer 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) immediately before transplantation into the spleen. Each of the animals in the cell therapy group (n = 20) received 5 × 106 ESCs 4 h following treatment with APAP. The control group (n = 20) received the vehicle only. The distribution and dynamics of the cells were monitored in real-time with the IVIS Lumina-2 at 30 min post transplantation, then at 3, 12, 24, 48 and 72 h, and after one and 2 wk. Immunohistochemical examination of liver tissue was used to identify expression of GFP and albumin. Plasma alanine aminotransferase (ALT) was measured as an indication of liver damage.
RESULTS: DiR-stained ESCs were easily tracked with the IVIS using the indocyanine green filter due to its high background passband with minimal background autofluorescence. The transplanted cells were confined inside the spleen at 30 min post-transplantation, gradually moved into the splenic vein, and were detectable in parts of the liver at the 3 h time-point. Within 24 h of transplantation, homing of almost 90% of cells was confirmed in the liver. On day three, however, the DiR signal started to fade out, and ex vivo IVIS imaging of different organs allowed signal detection at time-points when the signal could not be detected by in vivo imaging, and confirmed that the highest photon emission was in the liver (P < 0.0001). At 2 wk, the DiRsignal was no longer detectable in vivo; however, immunohistochemistry analysis of constitutively-expressed GFP was used to provide an insight into the distribution of the cells. GFP +ve cells were detected in tissue sections resembling hepatocytes and were dispersed throughout the hepatic parenchyma, with the presence of a larger number of GFP +ve cells incorporated within the sinusoidal endothelial lining. Very faint albumin expression was detected in the transplanted GFP +ve cells at 72 h; however at 2 wk, few cells that were positive for GFP were also strongly positive for albumin. There was a significant improvement in serum levels of ALT, albumin and bilirubin in both groups at 2 wk when compared with the 72 h time-point. In the cell therapy group, serum ALT was significantly (P = 0.016) lower and albumin (P = 0.009) was significantly higher when compared with the control group at the 2 wk time-point; however there was no difference in mortality between the two groups.
CONCLUSION: Dual labeling is an easy to use and cheap method for longitudinal monitoring of distribution, survival and engraftment of transplanted cells, and could be used for cell therapy models.
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Sharma NS, Nagrath D, Yarmush ML. Metabolic profiling based quantitative evaluation of hepatocellular metabolism in presence of adipocyte derived extracellular matrix. PLoS One 2011; 6:e20137. [PMID: 21603575 PMCID: PMC3095641 DOI: 10.1371/journal.pone.0020137] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2011] [Accepted: 04/26/2011] [Indexed: 12/05/2022] Open
Abstract
The elucidation of the effect of extracellular matrices on hepatocellular metabolism is critical to understand the mechanism of functional upregulation. We have developed a system using natural extracellular matrices [Adipogel] for enhanced albumin synthesis of rat hepatocyte cultures for a period of 10 days as compared to collagen sandwich cultures. Primary rat hepatocytes isolated from livers of female Lewis rats recover within 4 days of culture from isolation induced injury while function is stabilized at 7 days post-isolation. Thus, the culture period can be classified into three distinct stages viz. recovery stage [day 0–4], pre-stable stage [day 5–7] and the stable stage [day 8–10]. A Metabolic Flux Analysis of primary rat hepatocytes cultured in Adipogel was performed to identify the key metabolic pathways modulated as compared to collagen sandwich cultures. In the recovery stage [day 4], the collagen-soluble Adipogel cultures shows an increase in TriCarboxylic Acid [TCA] cycle fluxes; in the pre-stable stage [day 7], there is an increase in PPP and TCA cycle fluxes while in the stable stage [day 10], there is a significant increase in TCA cycle, urea cycle fluxes and amino acid uptake rates concomitant with increased albumin synthesis rate as compared to collagen sandwich cultures throughout the culture period. Metabolic analysis of the collagen-soluble Adipogel condition reveals significantly higher transamination reaction fluxes, amino acid uptake and albumin synthesis rates for the stable vs. recovery stages of culture. The identification of metabolic pathways modulated for hepatocyte cultures in presence of Adipogel will be a useful step to develop an optimization algorithm to further improve hepatocyte function for Bioartificial Liver Devices. The development of this framework for upregulating hepatocyte function in Bioartificial Liver Devices will facilitate the utilization of an integrated experimental and computational approach for broader applications of Adipogel in tissue e engineering and regenerative medicine.
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Affiliation(s)
- Nripen S. Sharma
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and The Shriners Hospitals for Children, Boston, Massachusetts, United States of America
| | - Deepak Nagrath
- Department of Chemical and Biomolecular Engineering, Rice University, Houston, Texas, United States of America
| | - Martin L. Yarmush
- Center for Engineering in Medicine/Surgical Services, Massachusetts General Hospital, Harvard Medical School, and The Shriners Hospitals for Children, Boston, Massachusetts, United States of America
- * E-mail:
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Baxter MA, Rowe C, Alder J, Harrison S, Hanley KP, Park BK, Kitteringham NR, Goldring CE, Hanley NA. Generating hepatic cell lineages from pluripotent stem cells for drug toxicity screening. Stem Cell Res 2010; 5:4-22. [PMID: 20483202 PMCID: PMC3556810 DOI: 10.1016/j.scr.2010.02.002] [Citation(s) in RCA: 53] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/23/2009] [Revised: 02/24/2010] [Accepted: 02/25/2010] [Indexed: 02/06/2023] Open
Abstract
Hepatotoxicity is an enormous and increasing problem for the pharmaceutical industry. Early detection of problems during the drug discovery pathway is advantageous to minimize costs and improve patient safety. However, current cellular models are sub-optimal. This review addresses the potential use of pluripotent stem cells in the generation of hepatic cell lineages. It begins by highlighting the scale of the problem faced by the pharmaceutical industry, the precise nature of drug-induced liver injury and where in the drug discovery pathway the need for additional cell models arises. Current research is discussed, mainly for generating hepatocyte-like cells rather than other liver cell-types. In addition, an effort is made to identify where some of the major barriers remain in translating what is currently hypothesis-driven laboratory research into meaningful platform technologies for the pharmaceutical industry.
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Affiliation(s)
- Melissa A. Baxter
- Endocrinology & Diabetes, School of Biomedicine, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - Cliff Rowe
- Endocrinology & Diabetes, School of Biomedicine, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - Jane Alder
- School of Pharmacy and Pharmaceutical Science, University of Central Lancashire, Preston PR1 2HE, UK
| | - Sean Harrison
- Endocrinology & Diabetes, School of Biomedicine, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - Karen Piper Hanley
- Endocrinology & Diabetes, School of Biomedicine, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK
| | - B. Kevin Park
- MRC Centre for Drug Safety Science, Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK
| | - Neil R. Kitteringham
- MRC Centre for Drug Safety Science, Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK
| | - Chris E. Goldring
- MRC Centre for Drug Safety Science, Department of Pharmacology & Therapeutics, University of Liverpool, Sherrington Buildings, Ashton Street, Liverpool L69 3GE, UK
| | - Neil A. Hanley
- Endocrinology & Diabetes, School of Biomedicine, Manchester Academic Health Science Centre, University of Manchester, Oxford Road, Manchester, M13 9PT, UK
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Fukumitsu K, Ishii T, Yasuchika K, Amagai Y, Kawamura-Saito M, Kawamoto T, Kawase E, Suemori H, Nakatsuji N, Ikai I, Uemoto S. Establishment of a cell line derived from a mouse fetal liver that has the characteristic to promote the hepatic maturation of mouse embryonic stem cells by a coculture method. Tissue Eng Part A 2010; 15:3847-56. [PMID: 19558317 DOI: 10.1089/ten.tea.2009.0357] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Stromal cells residing in murine fetal livers have the ability to promote the hepatic maturation of murine embryonic stem cells (ESCs) and hepatic progenitor cells (HPCs) 3848 in vitro. These stromal cells were isolated as the CD49f(+/-)CD45(-)Thy1(+)gp38(+) cell fraction. The present study established a murine fetal liver stromal cell line that induced hepatic maturation in mouse ESCs and HPCs. A transgene containing a temperature-sensitive SV40 large T antigen was transfected into the primary fetal liver stromal cells. These immortalized cells, which were named as the gp38-positive and Thy1-positive murine liver stromal (MLSgt) cells, induced both mouse ESCs and HPCs to differentiate into mature hepatocyte-like cells using a coculture method. Since MLSgt is not a cloned cell line, one clone, MLSgt20, was selected as a line with the characteristic to induce hepatic differentiation, which was comparable to its parental stromal cells. The ESC-derived endoderm cells cocultured with the MLSgt20 cells expressed mature hepatocyte-specific gene markers, including glucose-6-phosphatase, tyrosine aminotransferase, tryptophan 2,3-dioxgenase, and cytochrome P450 (CYP1a1, Cyp1b1, Cyp1a2, and Cyp3a11). In addition, these cells also exhibited hepatic functions, such as glycogen storage and ammonia metabolism. Transmission electron microscopy showed that the cocultured ESCs expressed the morphologic features of mature hepatocytes. In conclusion, a cell line was established that has the characteristic to promote the hepatic maturation of mouse ESCs and HPCs by a coculture method.
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Affiliation(s)
- Ken Fukumitsu
- Department of Surgery, Graduate School of Medicine Kyoto University, Kyoto, Japan
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Wallenstein EJ, Barminko J, Schloss RS, Yarmush ML. Transient gene delivery for functional enrichment of differentiating embryonic stem cells. Biotechnol Bioeng 2008; 101:859-72. [PMID: 18942772 DOI: 10.1002/bit.22027] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
There is a critical need for new sources of hepatocytes, both clinically to provide support for patients with liver failure and in drug discovery for toxicity, metabolic and pharmacokinetic screening of new drug entities. We have reported previously a variety of methods for differentiating murine embryonic stem (ES) cells into hepatocyte-like cells. One major challenge of our work and others in the field has been the ability to selectively purify and enrich these cells from a heterogeneous population. Traditional approaches for inserting new genes (e.g., stable transfection, knock-in, retroviral transduction) involve permanent alterations in the genome. These approaches can lead to mutations and involve the extra costs and time of developing, validating and maintaining new cell lines. We have developed a transient gene delivery system that uses fluorescent gene reporters for purification of the cells. Following a transient transfection, the cells are purified through a fluorescence-activated cell sorter (FACS), re-plated in secondary culture and subsequent phenotypic analysis is performed. In an effort to test the ability of the reporters to work in a transient environment for our differentiation system, we engineered two non-viral plasmid reporters, the first driven by the mouse albumin enhancer/promoter and the second by the mouse cytochrome P450 7A1 (Cyp7A1) promoter. We optimized the transfection efficiency of delivering these genes into spontaneously differentiated ES cells and sorted independent fractions positive for each reporter 17 days after inducing differentiation. We found that cells sorted based on the Cyp7A1 promoter showed significant enrichment in terms of albumin secretion, urea secretion and cytochrome P450 1A2 detoxification activity as compared to enrichment garnered by the albumin promoter-based cell sort. Development of gene reporter systems that allow us to identify, purify and assess homogeneous populations of cells is important in better understanding stem cell differentiation pathways. And engineering cellular systems without making permanent gene changes will be critical for the generation of clinically acceptable cellular material in the future.
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Affiliation(s)
- Eric J Wallenstein
- Department of Biomedical Engineering, Rutgers University, 599 Taylor Road, Piscataway, New Jersey 08854, USA.
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