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Taggart M, Tchir A, Van Dieren L, Chen H, Hassan M, Taveras C, Dinicu A, Lellouch AG, Toner M, Sandlin RD, Uygun K. Parallelized Droplet Vitrification for Single-Run Vitrification of Hepatocytes from an Entire Rat Liver. ACS APPLIED MATERIALS & INTERFACES 2025; 17:16507-16519. [PMID: 40066620 DOI: 10.1021/acsami.4c19419] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/19/2025]
Abstract
Drug discovery pipelines rely on the availability of isolated primary hepatocytes for investigating potential hepatotoxicity prior to clinical application. These hepatocytes are isolated from livers rejected for transplantation and subsequently cryopreserved for later usage. The gold standard cryopreservation technique, slow-freezing, is a labor-intensive process with significant poststorage viability loss. In this work, we introduce parallelized droplet vitrification, a technique for high-volumetric, rapid vitrification of suspended cells. We show the utility of this technique through the single-run vitrification of the whole rat liver hepatocyte yield, resulting in the vitrification of 250 million cells in 40 mL of a vitrification solution at 10 mL/min. Additionally, we showed that these implementations maintained improved postpreservation outcomes in primary rat hepatocytes.
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Affiliation(s)
- McLean Taggart
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Alexandra Tchir
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
- Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States
| | - Loïc Van Dieren
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
- Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States
- Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States
- Faculty of Medicine and Health Sciences, University of Antwerp, Wilrjk 2000, Belgium
| | - Huyun Chen
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Madeeha Hassan
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Christopher Taveras
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Antonia Dinicu
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Alexandre G Lellouch
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
- Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States
- Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, United States
- INSERM UMRS 1140 Innovation Thérapeutique en Hémostase, University of Paris, Paris 75006, France
| | - Mehmet Toner
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
| | - Rebecca D Sandlin
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
| | - Korkut Uygun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States
- Shriners Children's Boston, Boston, Massachusetts 02114, United States
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Taggart MS, Tchir A, Van Dieren L, Chen H, Hassan M, Taveras C, Lellouch AG, Toner M, Sandlin RD, Uygun K. Parallelized Droplet Vitrification Enables Single-Run Vitrification of the Whole Rat Liver Hepatocyte Yield. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.07.14.603471. [PMID: 39071342 PMCID: PMC11275928 DOI: 10.1101/2024.07.14.603471] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/30/2024]
Abstract
Drug discovery pipelines rely on the availability of isolated primary hepatocytes for investigating potential hepatotoxicity prior to clinical application. These hepatocytes are typically isolated from livers rejected for transplantation and subsequently cryopreserved for later usage. The gold-standard cryopreservation technique, slow-freezing, is a labor-intensive process, with significant post-storage viability loss. In this work, we introduce parallelized droplet vitrification, a technique for high-volumetric, rapid vitrification of suspended cells. We show the utility of this technique through the single-run vitrification of the whole-rate liver hepatocyte yield, resulting in a 1600% increase in single-batch vitrification and a 500% increase in droplet generation rate compared to previous droplet vitrification approaches. Additionally, we showed that these implementations maintained improved post-preservation outcomes in primary rat hepatocytes.
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Affiliation(s)
- M S Taggart
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
| | - A Tchir
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
- Massachusetts Institute of Technology, Boston, MA
| | - L Van Dieren
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
- Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
- Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
| | - H Chen
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
| | - M Hassan
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
| | - C Taveras
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
| | - A G Lellouch
- Shriners Children's Boston, Boston, MA, USA
- Vascularized Composite Allotransplantation Laboratory, Center for Transplantation Sciences, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
- Division of Plastic and Reconstructive Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
- INSERM UMRS 1140 Innovation Thérapeutique en Hémostase, University of Paris, Paris, France
| | - M Toner
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
| | - R D Sandlin
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
| | - K Uygun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA
- Shriners Children's Boston, Boston, MA, USA
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Gokaltun A, Asik E, Byrne D, Yarmush ML, Usta OB. Supercooled preservation of cultured primary rat hepatocyte monolayers. Front Bioeng Biotechnol 2024; 12:1429412. [PMID: 39076209 PMCID: PMC11284110 DOI: 10.3389/fbioe.2024.1429412] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Accepted: 06/27/2024] [Indexed: 07/31/2024] Open
Abstract
Supercooled preservation (SCP) is a technology that involves cooling a substance below its freezing point without initiating ice crystal formation. It is a promising alternative to prolong the preservation time of cells, tissues, engineered tissue products, and organs compared to the current practices of hypothermic storage. Two-dimensional (2D) engineered tissues are extensively used in in vitro research for drug screening and development and investigation of disease progression. Despite their widespread application, there is a lack of research on the SCP of 2D-engineered tissues. In this study, we presented the effects of SCP at -2 and -6°C on primary rat hepatocyte (PRH) monolayers for the first time and compared cell viability and functionality with cold storage (CS, + 4°C). We preserved PRH monolayers in two different commercially available solutions: Hypothermosol-FRS (HTS-FRS) and the University of Wisconsin (UW) with and without supplements (i.e., polyethylene glycol (PEG) and 3-O-Methyl-Α-D-Glucopyranose (3-OMG)). Our findings revealed that UW with and without supplements were inadequate for the short-term preservation of PRH monolayers for both SCP and CS with high viability, functionality, and monolayer integrity. The combination of supplements (PEG and 3-OMG) in the HTS-FRS solution outperformed the other groups and yielded the highest viability and functional capacity. Notably, PRH monolayers exhibited superior viability and functionality when stored at -2°C through SCP for up to 3 days compared to CS. Overall, our results demonstrated that SCP is a feasible approach to improving the short-term preservation of PRH monolayers and enables readily available 2D-engineered tissues to advance in vitro research. Furthermore, our findings provide insights into preservation outcomes across various biological levels, from cells to tissues and organs, contributing to the advancement of bioengineering and biotechnology.
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Affiliation(s)
- Aslihan Gokaltun
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Chemical and Biological Engineering, Tufts University, Medford, MA, United States
- Department of Chemical Engineering, Hacettepe University, Ankara, Türkiye
| | - Eda Asik
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Bioengineering, Hacettepe University, Ankara, Türkiye
| | - Delaney Byrne
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
| | - Martin L. Yarmush
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
- Department of Biomedical Engineering, Rutgers University, Newark, NJ, United States
| | - O. Berk Usta
- Center for Engineering in Medicine and Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States
- Shriners Hospitals for Children, Boston, MA, United States
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Gantier M, Rispal R, Fourrier A, Ménoret S, Delbos F, Anegon I, Nguyen TH. Cryopreserved cGMP-compliant human pluripotent stem cell-derived hepatic progenitors rescue mice from acute liver failure through rapid paracrine effects on liver cells. Stem Cell Res Ther 2024; 15:71. [PMID: 38475825 DOI: 10.1186/s13287-024-03673-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2023] [Accepted: 02/20/2024] [Indexed: 03/14/2024] Open
Abstract
BACKGROUND Liver transplantation remains the only curative treatment for end-stage liver diseases. Unfortunately, there is a drastic organ donor shortage. Hepatocyte transplantation emerged as a viable alternative to liver transplantation. Considering their unique expansion capabilities and their potency to be driven toward a chosen cell fate, pluripotent stem cells are extensively studied as an unlimited cell source of hepatocytes for cell therapy. It has been previously shown that freshly prepared hepatocyte-like cells can cure mice from acute and chronic liver failure and restore liver function. METHODS Human PSC-derived immature hepatic progenitors (GStemHep) were generated using a new protocol with current good manufacturing practice compliant conditions from PSC amplification and hepatic differentiation to cell cryopreservation. The therapeutic potential of these cryopreserved cells was assessed in two clinically relevant models of acute liver failure, and the mode of action was studied by several analytical methods, including unbiased proteomic analyses. RESULTS GStemHep cells present an immature hepatic phenotype (alpha-fetoprotein positive, albumin negative), secrete hepatocyte growth factor and do not express major histocompatibility complex. A single dose of thawed GStemHep rescue mice from sudden death caused by acetaminophen and thioacetamide-induced acute liver failure, both in immunodeficient and immunocompetent animals in the absence of immunosuppression. Therapeutic biological effects were observed as soon as 3 h post-cell transplantation with a reduction in serum transaminases and in liver necrosis. The swiftness of the therapeutic effect suggests a paracrine mechanism of action of GStemHep leading to a rapid reduction of inflammation as well as a rapid cytoprotective effect with as a result a proteome reprograming of the host hepatocytes. The mode of action of GStemHep relie on the alleviation of inhibitory factors of liver regeneration, an increase in proliferation-promoting factors and a decrease in liver inflammation. CONCLUSIONS We generated cryopreserved and current good manufacturing practice-compliant human pluripotent stem cell-derived immature hepatic progenitors that were highly effective in treating acute liver failure through rapid paracrine effects reprogramming endogenous hepatocytes. This is also the first report highlighting that human allogeneic cells could be used as cryopreserved cells and in the absence of immunosuppression for human PSC-based regenerative medicine for acute liver failure.
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Affiliation(s)
- Malika Gantier
- GoLiver Therapeutics, 44007, Nantes, France.
- Nantes Université, Inserm, Center for Research in Transplantation and Translational Immunology, UMR 1064, 44000, Nantes, France.
| | - Raphaël Rispal
- Nantes Université, Inserm, Center for Research in Transplantation and Translational Immunology, UMR 1064, 44000, Nantes, France
| | | | - Séverine Ménoret
- Nantes Université, CHU Nantes, Inserm, CNRS, SFR Santé, Inserm UMS 016 CNRS UMS 3556, 44000, Nantes, France
| | | | - Ignacio Anegon
- Nantes Université, Inserm, Center for Research in Transplantation and Translational Immunology, UMR 1064, 44000, Nantes, France
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Lomba L, García CB, Benito L, Sangüesa E, Santander S, Zuriaga E. Advances in Cryopreservatives: Exploring Safer Alternatives. ACS Biomater Sci Eng 2024; 10:178-190. [PMID: 38141007 DOI: 10.1021/acsbiomaterials.3c00859] [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: 12/24/2023]
Abstract
Cryopreservation of cells, tissues, and organs is widely used in the biomedical and research world. There are different cryopreservatives that are used for this process; however, many of them, such as DMSO, are used despite the problems they present, mainly due to the toxicity it presents to certain types of samples. The aim of this Review is to highlight the different types of substances used in the cryopreservation process. It has been shown that some of these substances are well-known, as in the case of the families of alcohols, sugars, sulfoxides, etc. However, in recent years, other compounds have appeared, such as ionic liquids, deep eutectic solvents, or certain polymers, which open the door to new cryopreservation methods and are also less toxic to frozen samples.
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Affiliation(s)
- Laura Lomba
- Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 50830 Villanueva de Gállego, Zaragoza, Spain
| | - Cristina B García
- Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 50830 Villanueva de Gállego, Zaragoza, Spain
| | - Lucía Benito
- Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 50830 Villanueva de Gállego, Zaragoza, Spain
| | - Estela Sangüesa
- Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 50830 Villanueva de Gállego, Zaragoza, Spain
| | - Sonia Santander
- Faculty of Health and Sports Sciences, University of Zaragoza, Campus of Huesca, 22002 Huesca, Spain
| | - Estefanía Zuriaga
- Facultad de Ciencias de la Salud, Universidad San Jorge. Campus Universitario, Autov A23 km 299, 50830 Villanueva de Gállego, Zaragoza, Spain
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Sun Z, Yuan X, Wu J, Wang C, Zhang K, Zhang L, Hui L. Hepatocyte transplantation: The progress and the challenges. Hepatol Commun 2023; 7:e0266. [PMID: 37695736 PMCID: PMC10497249 DOI: 10.1097/hc9.0000000000000266] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Accepted: 07/26/2023] [Indexed: 09/13/2023] Open
Abstract
Numerous studies have shown that hepatocyte transplantation is a promising approach for liver diseases, such as liver-based metabolic diseases and acute liver failure. However, it lacks strong evidence to support the long-term therapeutic effects of hepatocyte transplantation in clinical practice. Currently, major hurdles include availability of quality-assured hepatocytes, efficient engraftment and repopulation, and effective immunosuppressive regimens. Notably, cell sources have been advanced recently by expanding primary human hepatocytes by means of dedifferentiation in vitro. Moreover, the transplantation efficiency was remarkably improved by the established preparative hepatic irradiation in combination with hepatic mitogenic stimuli regimens. Finally, immunosuppression drugs, including glucocorticoid and inhibitors for co-stimulating signals of T cell activation, were proposed to prevent innate and adaptive immune rejection of allografted hepatocytes. Despite remarkable progress, further studies are required to improve in vitro cell expansion technology, develop clinically feasible preconditioning regimens, and further optimize immunosuppression regimens or establish ex vivo gene correction-based autologous hepatocyte transplantation.
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Affiliation(s)
- Zhen Sun
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
| | - Xiang Yuan
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Jingqi Wu
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Chenhua Wang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Kun Zhang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Ludi Zhang
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
| | - Lijian Hui
- State Key Laboratory of Cell Biology, CAS Center for Excellence in Molecular Cell Science, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai, China
- School of Life Science and Technology, ShanghaiTech University, Shanghai, China
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Ietto G, Iori V, Gritti M, Inversini D, Costantino A, Izunza Barba S, Jiang ZG, Carcano G, Dalla Gasperina D, Pettinato G. Multicellular Liver Organoids: Generation and Importance of Diverse Specialized Cellular Components. Cells 2023; 12:1429. [PMID: 37408262 PMCID: PMC10217024 DOI: 10.3390/cells12101429] [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: 03/02/2023] [Revised: 05/11/2023] [Accepted: 05/17/2023] [Indexed: 07/07/2023] Open
Abstract
Over 40,000 patients in the United States are estimated to suffer from end-stage liver disease and acute hepatic failure, for which liver transplantation is the only available therapy. Human primary hepatocytes (HPH) have not been employed as a therapeutic tool due to the difficulty in growing and expanding them in vitro, their sensitivity to cold temperatures, and tendency to dedifferentiate following two-dimensional culture. The differentiation of human-induced pluripotent stem cells (hiPSCs) into liver organoids (LO) has emerged as a potential alternative to orthotropic liver transplantation (OLT). However, several factors limit the efficiency of liver differentiation from hiPSCs, including a low proportion of differentiated cells capable of reaching a mature phenotype, the poor reproducibility of existing differentiation protocols, and insufficient long-term viability in vitro and in vivo. This review will analyze various methodologies being developed to improve hepatic differentiation from hiPSCs into liver organoids, paying particular attention to the use of endothelial cells as supportive cells for their further maturation. Here, we demonstrate why differentiated liver organoids can be used as a research tool for drug testing and disease modeling, or employed as a bridge for liver transplantation following liver failure.
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Affiliation(s)
- Giuseppe Ietto
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Valentina Iori
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Mattia Gritti
- Department of General Surgery, Humanitas Clinical and Research Center, Rozzano, 20089 Milan, Italy
| | - Davide Inversini
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Angelita Costantino
- Department of Drug and Health Sciences, University of Catania, 95124 Catania, Italy;
| | - Sofia Izunza Barba
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Z. Gordon Jiang
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
| | - Giulio Carcano
- General, Emergency and Transplant Surgery Department, ASST-Sette Laghi, 21100 Varese, Italy
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
| | - Daniela Dalla Gasperina
- Department of Medicine and Innovation Technology (DiMIT), University of Insubria, 21100 Varese, Italy
- Department of Infectious Diseases, ASST-Sette Laghi, 21100 Varese, Italy
| | - Giuseppe Pettinato
- Division of Gastroenterology, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA
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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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9
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Jesus AR, Duarte ARC, Paiva A. Use of natural deep eutectic systems as new cryoprotectant agents in the vitrification of mammalian cells. Sci Rep 2022; 12:8095. [PMID: 35577888 PMCID: PMC9110728 DOI: 10.1038/s41598-022-12365-4] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/08/2022] [Accepted: 04/26/2022] [Indexed: 11/25/2022] Open
Abstract
In this work we present the potential of Natural Deep Eutectic Systems (NADES) as new vitrification media for the cryopreservation of mammalian cells. Several NADES composed of natural metabolites were prepared and tested as CPAs in two cell lines, L929 and HacaT cells. After the harvesting, cells were mixed with the eutectic systems, and frozen directly into liquid nitrogen to achieve a vitreous state. Then, the cells were thawed and it was observed that NADES were able to exert a significant cryoprotective effect in L929 cells, when compared with DMSO or in the absence of a CPA. For HacaT cells, only a eutectic system showed a slightly improvement in cell survival, while DMSO caused complete cell death. Moreover, the thermal behaviour of the best systems was studied for further understanding the protective properties of NADES as CPAs, and have shown a significant difference in terms of Tm and Tc when compared with DMSO and water. Additionally, the results obtained showed that NADES can be maintained in the growth media after the thawing step, without compromising cell viability. In summary, we have shown the great potential of NADES to be used as CPAs for the cryopreservation of different cell types, using the vitrification method.
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Affiliation(s)
- Ana Rita Jesus
- LAQV-REQUIMTE, Campus da Caparica, Monte da Caparica, 2825-149, Caparica, Portugal
| | - Ana Rita C Duarte
- LAQV-REQUIMTE, Campus da Caparica, Monte da Caparica, 2825-149, Caparica, Portugal
| | - Alexandre Paiva
- LAQV-REQUIMTE, Campus da Caparica, Monte da Caparica, 2825-149, Caparica, Portugal.
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10
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Hiramatsu S, Morizane A, Kikuchi T, Doi D, Yoshida K, Takahashi J. Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application. JOURNAL OF PARKINSON'S DISEASE 2022; 12:871-884. [PMID: 34958047 PMCID: PMC9108593 DOI: 10.3233/jpd-212934] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
BACKGROUND Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson's disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. OBJECTIVE To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. METHODS We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. RESULTS The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. CONCLUSION These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.
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Affiliation(s)
- Satoe Hiramatsu
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.,Regenerative and Cellular Medicine Kobe Center, Sumitomo Dainippon Pharma Co., Ltd, Kobe, Japan
| | - Asuka Morizane
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Tetsuhiro Kikuchi
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Daisuke Doi
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Kenji Yoshida
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan.,Regenerative and Cellular Medicine Kobe Center, Sumitomo Dainippon Pharma Co., Ltd, Kobe, Japan
| | - Jun Takahashi
- Department of Clinical Application, Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
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11
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Jindal A, Jagdish RK, Kumar A. Hepatic Regeneration in Cirrhosis. J Clin Exp Hepatol 2022; 12:603-616. [PMID: 35535091 PMCID: PMC9077225 DOI: 10.1016/j.jceh.2021.08.029] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Accepted: 08/31/2021] [Indexed: 01/03/2023] Open
Abstract
End-stage liver disease is characterized by massive hepatocyte death resulting in clinical decompensation and organ failures. Clinical consequences in cirrhosis are the results of the loss of functional hepatocytes and excessive scarring. The only curative therapy in advanced cirrhosis is orthotropic liver transplantation, but the clinical demand outweighs the availability of acceptable donor organs. Moreover, this also necessitates lifelong immunosuppression and carries associated risks. The liver has a huge capability for regeneration. Self-replication of quiescent differentiated hepatocytes and cholangiocytes occurs in patients with acute liver injury. Due to limited hepatocyte self-renewal capacity in advanced cirrhosis, great interest has therefore been shown in characterizing the possible role of hepatic progenitor cells and bone marrow-derived stem cells to therapeutically aid this process. Transplantation of cells from various sources that can be properly differentiated into functional liver cells or use of growth factors for ex-vivo expansion of progenitor cells is needed at utmost priority. Multiple researches over the last two decades have aided researchers in refining proliferation, differentiation, and storage techniques and understand the functionality of these cells for use in clinical practice. However, these cell-based therapies are still experimental and have to be used in trial settings.
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Key Words
- Ang2, angiopoietin 2
- BM, Bone marrow
- BM-MNCs, bone marrow mononuclear cells
- BMSC, bone marrow stem cells
- DAMPs, Damage associated molecular patterns
- EPCs, endothelial progenitor cells
- ESRP2, epithelial splicing regulatory protein 2
- GCSF
- HGF, hepatocyte growth factor
- HPC, Hepatocyte progenitor cells
- HSCs, hematopoietic stem cells
- Hh, Hedgehog
- HybHP, hybrid periportal hepatocytes
- MMP, matrix metalloprotease
- MSCs, mesenchymal stromal cells
- OLT, Orthotropic liver transplantation
- PAMPs, Pathogen associated molecular patterns
- SAH, severe alcoholic hepatitis
- SDF1, stromal-derived factor 1
- TNFSF12, tumor necrosis factor ligand superfamily member 12
- Terthigh, high Telomerase reverse transcriptase
- [Hnf4a], Hepatocyte Nuclear Factor 4 Alpha
- [Mfsd2a], Major Facilitator Superfamily Domain containing 2A
- acute liver failure
- chronic liver diseases
- hepatocyte transplant
- liver regeneration
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Affiliation(s)
- Ankur Jindal
- Department of Hepatology, Institute of Liver and Biliary Sciences, New Delhi 110070, India
| | | | - Anupam Kumar
- Department of Research, Institute of Liver and Biliary Sciences, New Delhi 110070, India
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12
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Ashmore-Harris C, Fruhwirth GO. Generation of In Vivo Traceable Hepatocyte-Like Cells from Human iPSCs. Methods Mol Biol 2022; 2544:15-49. [PMID: 36125708 DOI: 10.1007/978-1-0716-2557-6_2] [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] [Indexed: 06/15/2023]
Abstract
In this chapter, we describe a protocol for differentiation of human-induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) and their transduction with a lentivirus for gene transfer. Here, we engineer them to express the human sodium iodide symporter, which can be exploited as a radionuclide reporter gene, thereby enabling these cells to be tracked in vivo by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Differentiation of HLCs from iPSCs involves three steps: induction of iPSCs to definitive endoderm, differentiation to a hepatic progenitor cell population, and maturation of immature HLCs. Once proliferation of hepatic progenitors has ceased and an immature HLC population is generated, lentiviral transduction can be performed. The immature hepatic gene expression profile/morphology at the stage of transduction will be compatible with further maturation following transgene expression either in vitro or in vivo, with expression of the transgene retained. We detail how transgenic cells can be imaged in vivo. While we provide a protocol for the NIS reporter gene, the cell engineering aspects of this protocol are transferable for use with other (reporter) genes if desired.
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Affiliation(s)
- Candice Ashmore-Harris
- Imaging Therapies and Cancer Group, Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, UK
- Centre for Regenerative Medicine, Institute for Regeneration and Repair, University of Edinburgh, Edinburgh, UK
| | - Gilbert O Fruhwirth
- Imaging Therapies and Cancer Group, Comprehensive Cancer Centre, School of Cancer and Pharmaceutical Sciences, King's College London, Guy's Cancer Centre, London, UK.
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13
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Askarizadeh N, Banijamali S, Irani S, Bakhtiar H. Effect of two different concentrations of 1α,25-dihydroxyvitamin D3 on odontogenic differentiation of stem cells from human exfoliated deciduous teeth. Dent Res J (Isfahan) 2022; 19:4. [PMID: 35308448 PMCID: PMC8927951 DOI: 10.4103/1735-3327.336689] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Revised: 06/04/2020] [Accepted: 09/19/2020] [Indexed: 11/04/2022] Open
Abstract
Background: Stem cells from human exfoliated deciduous teeth (SHEDs) can transform into odontoblasts in vitro and in vivo. The role of 1α, 25-dihydroxyvitamin D3 (1α,25 vitD3) has been reported in the mineralization of hard tissues and teeth, as well as osteoblastic differentiation. This study aimed to assess the effect of different concentrations of 1α,25 vitD3 on odontogenic differentiation of SHEDs. Materials and Methods: In this experimental study, second-passage SHEDs were exposed to odontogenic medium along with 0, 10, 50, 100, and 150 nmol concentrations of in 1α, 25 vitD3 to determine its optimal concentration for odontogenic differentiation. The methyl thiazolyl tetrazolium (MTT) assay was performed. Odontogenic differentiation was evaluated by QRT- polymerase chain reaction for dentin matrix protein (DMP1) and dentin sialophosphoprotein (DSPP) genes. Morphology of differentiated cells was studied by Scanning Electron Microscopy. Data were analyzed using the Kruskal–Wallis, Mann–Whitney, Friedman, and Chi-square test. P < 0.05 is considered statistically significant. Results: MTT test result showed the two groups of odontogenic medium + 10 nm 1α,25 vitD3 and odontogenic medium + 150 nm 1α,25 vitD3 provided the most suitable conditions for cell viability at 72 h. Expression of both genes significantly increased in the presence of 1α,25 vitD3 (P < 0.001). Expression of both genes was significantly higher at 14 days compared with 7 days (P < 0.01). At both time points, expression of both genes was significantly higher in the presence of 150 nm 1α,25 vitD3 compared with 10 nm (P < 0.01). The accumulation of cells with odontoblastic morphology, cell interactions, and calcifications were evident. Conclusion: 1α,25 vitD3 upregulates DMP1 and DSPP and results in odontogenic differentiation of SHEDs in odontogenic medium. This upregulation increases with time and by an increase in concentration of 1α,25 vitD3.
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Whaley D, Damyar K, Witek RP, Mendoza A, Alexander M, Lakey JRT. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transplant 2021; 30:963689721999617. [PMID: 33757335 PMCID: PMC7995302 DOI: 10.1177/0963689721999617] [Citation(s) in RCA: 131] [Impact Index Per Article: 32.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2021] [Revised: 01/28/2021] [Accepted: 02/12/2021] [Indexed: 12/21/2022] Open
Abstract
The origins of low-temperature tissue storage research date back to the late 1800s. Over half a century later, osmotic stress was revealed to be a main contributor to cell death during cryopreservation. Consequently, the addition of cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol (GLY), ethylene glycol (EG), or propylene glycol (PG), although toxic to cells at high concentrations, was identified as a necessary step to protect against rampant cell death during cryopreservation. In addition to osmotic stress, cooling and thawing rates were also shown to have significant influence on cell survival during low temperature storage. In general, successful low-temperature cell preservation consists of the addition of a CPA (commonly 10% DMSO), alone or in combination with additional permeating or non-permeating agents, cooling rates of approximately 1ºC/min, and storage in either liquid or vapor phase nitrogen. In addition to general considerations, cell-specific recommendations for hepatocytes, pancreatic islets, sperm, oocytes, and stem cells should be observed to maximize yields. For example, rapid cooling is associated with better cryopreservation outcomes for oocytes, pancreatic islets, and embryonic stem cells while slow cooling is recommended for cryopreservation of hepatocytes, hematopoietic stem cells, and mesenchymal stem cells. Yields can be further maximized by implementing additional pre-cryo steps such as: pre-incubation with glucose and anti-oxidants, alginate encapsulation, and selecting cells within an optimal age range and functional ability. Finally, viability and functional assays are critical steps in determining the quality of the cells post-thaw and improving the efficiency of the current cryopreservation methods.
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Affiliation(s)
- David Whaley
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Kimia Damyar
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | | | | | - Michael Alexander
- Department of Surgery, University of California Irvine, Orange, CA, USA
| | - Jonathan RT Lakey
- Department of Surgery, University of California Irvine, Orange, CA, USA
- Department of Biomedical Engineering, University of California Irvine, Irvine, CA, USA
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Drummond NJ, Singh Dolt K, Canham MA, Kilbride P, Morris GJ, Kunath T. Cryopreservation of Human Midbrain Dopaminergic Neural Progenitor Cells Poised for Neuronal Differentiation. Front Cell Dev Biol 2020; 8:578907. [PMID: 33224948 PMCID: PMC7674628 DOI: 10.3389/fcell.2020.578907] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/01/2020] [Accepted: 10/16/2020] [Indexed: 01/09/2023] Open
Abstract
Human pluripotent stem cells can be differentiated into midbrain dopaminergic (mDA) neurons by directing cells through a floor plate progenitor stage. The developmental identity of mDA neurons produced using floor plate protocols is similar to substantia nigra neurons, and this has improved the ability to model Parkinson's disease (PD) in a dish. Combined with the unlimited growth potential of pluripotent stem cells, mDA neural progenitor cell production can provide a scalable source of human dopaminergic (DA) neurons for diverse applications. However, due to the complexity and length of the protocols and inherent differences between cell lines, considerable variability of the final population of neurons is often observed. One solution to this problem is to cryopreserve committed mDA neural progenitor cells in a ready-to-use format. Creating a bank of cryopreserved mDA neural progenitor cells poised for neuronal differentiation could significantly improve reproducibility and facilitate collaborations. Here we have compared six (6) different commercial cryopreservation media and different freezing conditions for mDA neural progenitor cells differentiated from human embryonic stem cell (hESC) lines. Significant differences in cell recovery were observed at 24 h post-thawing, but no differences were observed immediately upon thawing. The presence of ROCK inhibitors improved cell recovery at 24 h for all cryopreservation media tested. A faster cooling rate of 1-2°C/min was significantly better than 0.5°C/min for all conditions tested, while rapid thawing at 37°C was not always superior to slow thawing at 4°C. Importantly, cryopreservation of mDA neural progenitor cells did not alter their potential to resume differentiation into mDA neurons. Banks of cryopreserved committed mDA neural progenitor cells provide a method to generate human DA neurons with reduced batch-to-batch variability, and establish a mechanism to share lineage-primed cells for collaborative research.
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Affiliation(s)
- Nicola J. Drummond
- MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom
| | - Karamjit Singh Dolt
- MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom
| | - Maurice A. Canham
- MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom
| | | | | | - Tilo Kunath
- MRC Centre for Regenerative Medicine, Institute for Stem Cell Research, School of Biological Sciences, The University of Edinburgh, Edinburgh, United Kingdom,UK Centre for Mammalian Synthetic Biology, The University of Edinburgh, Edinburgh, United Kingdom,*Correspondence: Tilo Kunath,
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16
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Kong D, Xu H, Chen M, Yu Y, Qian Y, Qin T, Tong Y, Xia Q, Hang H. Co-encapsulation of HNF4α overexpressing UMSCs and human primary hepatocytes ameliorates mouse acute liver failure. Stem Cell Res Ther 2020; 11:449. [PMID: 33097090 PMCID: PMC7583302 DOI: 10.1186/s13287-020-01962-7] [Citation(s) in RCA: 17] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2020] [Accepted: 10/01/2020] [Indexed: 02/07/2023] Open
Abstract
BACKGROUND Acute liver failure (ALF) is a complicated condition that is characterized by global hepatocyte death and often requires immediate liver transplantation. However, this therapy is limited by shortage of donor organs. Mesenchymal stem cells (MSCs) and hepatocytes are two attractive sources of cell-based therapies to treat ALF. The combined transplantation of hepatocytes and MSCs is considered to be more effective for the treatment of ALF than single-cell transplantation. We have previously demonstrated that HNF4α-overexpressing human umbilical cord MSCs (HNF4α-UMSCs) promoted the expression of hepatic-specific genes. In addition, microencapsulation allows exchange of nutrients, forming a protective barrier to the transplanted cells. Moreover, encapsulation of hepatocytes improves the viability and synthetic ability of hepatocytes and circumvents immune rejection. This study aimed to investigate the therapeutic effect of microencapsulation of hepatocytes and HNF4α-UMSCs in ALF mice. METHODS Human hepatocytes and UMSCs were obtained separately from liver and umbilical cord, followed by co-encapsulation and transplantation into mice by intraperitoneal injection. LPS/D-gal was used to induce ALF by intraperitoneal injection 24 h after transplantation. In addition, Raw 264.7 cells (a macrophage cell line) were used to elucidate the effect of HNF4α-UMSCs-hepatocyte microcapsules on polarization of macrophages. The protein chip was used to define the important paracrine factors in the conditioned mediums (CMs) of UMSCs and HNF4α-UMSCs and investigate the possible mechanism of HNF4α-UMSCs for the treatment of ALF in mice. RESULTS HNF4α-UMSCs can enhance the function of primary hepatocytes in alginate-poly-L-lysine-alginate (APA) microcapsules. The co-encapsulation of both HNF4α-UMSCs and hepatocytes achieved better therapeutic effects in ALF mice by promoting M2 macrophage polarization and reducing inflammatory response mainly mediated by the paracrine factor HB-EGF secreted by HNF4α-UMSCs. CONCLUSIONS The present study confirms that the co-encapsulation of HNF4α-UMSC and hepatocytes could exert therapeutic effect on ALF mainly by HB-EGF secreted by HNF4α-UMSCs and provides a novel strategy for the treatment of ALF.
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Affiliation(s)
- Defu Kong
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Huiming Xu
- State Key Laboratory of Oncogenes and Related Genes, Renji-MedX Clinical Stem Cell Research Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China
| | - Mo Chen
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Yeping Yu
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Yongbing Qian
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Tian Qin
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Ying Tong
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China
| | - Qiang Xia
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China.
| | - Hualian Hang
- Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, 160 Pujian Road, Shanghai, 200127, China.
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Dhawan A, Chaijitraruch N, Fitzpatrick E, Bansal S, Filippi C, Lehec SC, Heaton ND, Kane P, Verma A, Hughes RD, Mitry RR. Alginate microencapsulated human hepatocytes for the treatment of acute liver failure in children. J Hepatol 2020; 72:877-884. [PMID: 31843649 DOI: 10.1016/j.jhep.2019.12.002] [Citation(s) in RCA: 54] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/17/2019] [Revised: 12/03/2019] [Accepted: 12/05/2019] [Indexed: 12/11/2022]
Abstract
BACKGROUND & AIMS Liver transplantation (LT) is the most effective treatment for patients with acute liver failure (ALF), but is limited by surgical risks and the need for life-long immunosuppression. Transplantation of microencapsulated human hepatocytes in alginate is an attractive option over whole liver replacement. The safety and efficacy of hepatocyte microbead transplantation have been shown in animal models. We report our experience of this therapy in children with ALF treated on a named-patient basis. METHODS Clinical grade human hepatocyte microbeads (HMBs) and empty microbeads were tested in immunocompetent healthy rats. Subsequently, 8 children with ALF, who were awaiting a suitable allograft for LT, received intraperitoneal transplantation of HMBs. We monitored complications of the procedure, assessing the host immune response and residual function of the retrieved HMBs, either after spontaneous native liver regeneration or at the time of LT. RESULTS Intraperitoneal transplantation of HMBs in healthy rats was safe and preserved synthetic and detoxification functions, without the need for immunosuppression. Subsequently, 8 children with ALF received HMBs (4 neonatal haemochromatosis, 2 viral infections and 2 children with unknown cause at time of infusion) at a median age of 14.5 days, range 1 day to 6 years. The procedure was well tolerated without complications. Of the 8 children, 4 avoided LT while 3 were successfully bridged to LT following the intervention. HMBs retrieved after infusions (at the time of LT) were structurally intact, free of host cell adherence and contained viable hepatocytes with preserved functions. CONCLUSION The results demonstrate the feasibility and safety of an HMB infusion in children with ALF. LAY SUMMARY Acute liver failure in children is a rare but devastating condition. Liver transplantation is the most effective treatment, but it has several important limitations. Liver cell (hepatocyte) transplantation is an attractive option, as many patients only require short-term liver support while their own liver recovers. Human hepatocytes encapsulated in alginate beads can perform the functions of the liver while alginate coating protects the cells from immune attack. Herein, we demonstrated that transplantation of these beads was safe and feasible in children with acute liver failure.
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Affiliation(s)
- Anil Dhawan
- Paediatric Liver GI and Nutrition Center, King's College Hospital, London, United Kingdom; Dhawan Lab at the Mowat Labs, Institute of Liver Studies, King's College London at King's College Hospital, London, United Kingdom.
| | - Nataruks Chaijitraruch
- Paediatric Liver GI and Nutrition Center, King's College Hospital, London, United Kingdom; Paediatric Gastroenterology and Hepatology, Department of Paediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Emer Fitzpatrick
- Paediatric Liver GI and Nutrition Center, King's College Hospital, London, United Kingdom
| | - Sanjay Bansal
- Paediatric Liver GI and Nutrition Center, King's College Hospital, London, United Kingdom
| | - Celine Filippi
- Dhawan Lab at the Mowat Labs, Institute of Liver Studies, King's College London at King's College Hospital, London, United Kingdom
| | - Sharon C Lehec
- Dhawan Lab at the Mowat Labs, Institute of Liver Studies, King's College London at King's College Hospital, London, United Kingdom
| | - Nigel D Heaton
- Liver Transplant Surgery, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Pauline Kane
- Department of Radiology, King's College Hospital, London, United Kingdom
| | - Anita Verma
- Department of Infection Sciences and Microbiology, King's College Hospital, London, United Kingdom
| | - Robin D Hughes
- Dhawan Lab at the Mowat Labs, Institute of Liver Studies, King's College London at King's College Hospital, London, United Kingdom
| | - Ragai R Mitry
- Dhawan Lab at the Mowat Labs, Institute of Liver Studies, King's College London at King's College Hospital, London, United Kingdom
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Miyoshi H, Iwamoto A, Koyama T. Growth and albumin secretion of mouse fetal liver cells cryopreserved within porous polymer scaffolds as a viable cell source for bioartificial livers. J Biosci Bioeng 2020; 130:212-216. [PMID: 32312490 DOI: 10.1016/j.jbiosc.2020.03.013] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Revised: 03/09/2020] [Accepted: 03/26/2020] [Indexed: 01/11/2023]
Abstract
To clinically apply bioartificial livers (BALs), an effective liver cell cryopreservation method is required for a stable cell supply. In this study, we performed tissue-engineered construct (TEC) cryopreservation of fetal liver cells (FLCs) in which FLCs cultured within a porous polymer scaffold were cryopreserved. Growth and albumin secretion in TEC-cryopreserved FLCs after thawing were compared to freshly isolated FLCs (control experiments). The effect of preculture duration prior to cryopreservation (0-3 weeks) on these functions was also examined. In the three-dimensional cultures, the TEC-cryopreserved FLCs with preculturing showed constant growth, and this growth was comparable to controls. On the contrary, the TEC-cryopreserved FLCs without preculturing did not proliferate after thawing. Albumin secretion of TEC-cryopreserved FLCs with preculturing rapidly increased up to day 12 and high secretory activity comparable to controls was maintained thereafter in FLCs with 1- or 2-week preculturing, suggesting this as an appropriate preculture duration. Compared to conventionally cryopreserved FLCs, growth and albumin secretion in the TEC-cryopreserved FLCs were significantly higher, indicating their usefulness as a potent cell source for BALs.
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Affiliation(s)
- Hirotoshi Miyoshi
- Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
| | - Ayako Iwamoto
- Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
| | - Toshie Koyama
- Department of Biomedical Engineering, Faculty of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan
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Nguyen MP, Jain V, Iansante V, Mitry RR, Filippi C, Dhawan A. Clinical application of hepatocyte transplantation: current status, applicability, limitations, and future outlook. Expert Rev Gastroenterol Hepatol 2020; 14:185-196. [PMID: 32098516 DOI: 10.1080/17474124.2020.1733975] [Citation(s) in RCA: 24] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Introduction: Hepatocyte transplantation (HT) is a promising alternative to liver transplantation for the treatment of liver-based metabolic diseases and acute liver failure (ALF). However, shortage of good-quality liver tissues, early cell loss post-infusion, reduced cell engraftment and function restricts clinical application.Areas covered: A comprehensive literature search was performed to cover pre-clinical and clinical HT studies. The review discusses the latest developments to address HT limitations: cell sources from marginal/suboptimal donors to neonatal livers, differentiating pluripotent stem cells into hepatocyte-like cells, in vitro expansion, prevention of immune response to transplanted cells by encapsulation or using innate immunity-inhibiting agents, and enhancing engraftment through partial hepatectomy or irradiation.Expert opinion: To date, published data are highly encouraging specially the alginate-encapsulated hepatocyte treatment of children with ALF. Hepatocyte functions can be further improved through co-culturing with mesenchymal stromal cells. Moreover, ex-vivo genetic correction will enable the use of autologous cells in future personalized medicine.
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Affiliation(s)
- Minh Phuong Nguyen
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Vandana Jain
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Valeria Iansante
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Ragai R Mitry
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Celine Filippi
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
| | - Anil Dhawan
- Dhawan Lab. at the Mowat Labs, Institute of Liver Studies, King's College Hospital, London, United Kingdom
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Improved in vivo efficacy of clinical-grade cryopreserved human hepatocytes in mice with acute liver failure. Cytotherapy 2020; 22:114-121. [PMID: 31987755 DOI: 10.1016/j.jcyt.2019.12.005] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/22/2019] [Revised: 12/04/2019] [Accepted: 12/08/2019] [Indexed: 12/21/2022]
Abstract
Clinical hepatocyte transplantation short-term efficacy has been demonstrated; however, some major limitations, mainly due to the shortage of organs, the lack of quality of isolated cells and the low cell engraftment after transplantation, should be solved for increasing its efficacy in clinical applications. Cellular stress during isolation causes an unpredictable loss of attachment ability of the cells, which can be aggravated by cryopreservation and thawing. In this work, we focused on the use of a Good Manufacturing Practice (GMP) solution compared with the standard cryopreservation medium, the University of Wisconsin medium, for the purpose of improving the functional quality of cells and their ability to engraft in vivo, with the idea of establishing a biobank of cryopreserved human hepatocytes available for their clinical use. We evaluated not only cell viability but also specific hepatic function indicators of the functional performance of the cells such as attachment efficiency, ureogenic capability, phase I and II enzymes activities and the expression of specific adhesion molecules in vitro. Additionally, we also assessed and compared the in vivo efficacy of human hepatocytes cryopreserved in different media in an animal model of acute liver failure. Human hepatocytes cryopreserved in the new GMP solution offered better in vitro and in vivo functionality compared with those cryopreserved in the standard medium. Overall, the results indicate that the new tested GMP solution maintains better hepatic functions and, most importantly, shows better results in vivo, which could imply an increase in long-term efficacy when used in patients.
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Sharma A, Bischof JC, Finger EB. Liver Cryopreservation for Regenerative Medicine Applications. REGENERATIVE ENGINEERING AND TRANSLATIONAL MEDICINE 2019. [DOI: 10.1007/s40883-019-00131-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
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22
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de Vries RJ, Banik PD, Nagpal S, Weng L, Ozer S, van Gulik TM, Toner M, Tessier SN, Uygun K. Bulk Droplet Vitrification: An Approach to Improve Large-Scale Hepatocyte Cryopreservation Outcome. LANGMUIR : THE ACS JOURNAL OF SURFACES AND COLLOIDS 2019; 35:7354-7363. [PMID: 30514081 PMCID: PMC6548701 DOI: 10.1021/acs.langmuir.8b02831] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/27/2023]
Abstract
Loss of hepatocyte viability and metabolic function after cryopreservation is still a major issue. Although vitrification is a promising alternative, it has generally been proven to be unsuitable for vitrification of large cell volumes which is required for clinical applications. Here, we propose a novel bulk droplet (3-5 mm diameter) vitrification method which allows high throughput volumes (4 mL/min), while using a low preincubated CPA concentration (15% v/v) to minimize toxicity and loss of cell viability and function. We used rapid (1.25 s) osmotic dehydration to concentrate a low preincubated intracellular CPA concentration ahead of vitrification, without the need of fully equilibrating toxic CPA concentrations. We compared direct postpreservation viability, long-term viability, and metabolic function of bulk droplet vitrified, cryopreserved, and fresh hepatocytes. Simulations and cooling rate measurements confirmed an adequate concentration of the intracellular CPA concentration (up to 8.53 M) after dehydration in combination with high cooling rates (960-1320 °C/min) for successful vitrification. In comparison to cryopreserved hepatocytes, bulk droplet vitrified hepatocytes had a significantly higher viability, directly after preservation and after 1 day in culture. Moreover, bulk droplet vitrified hepatocytes had evidently better morphology and showed significantly higher metabolic activity than cryopreserved hepatocytes in long-term collagen sandwich cultures. In conclusion, we developed a novel bulk droplet vitrification method of which we validated the theoretical background and demonstrated the feasibility to use this method to vitrify large cell volumes. Moreover, we showed that this method results in improved hepatocyte viability and metabolic function as compared to cryopreservation.
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Affiliation(s)
- Reinier J. de Vries
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
- Department of Surgery, University of Amsterdam, Amsterdam, the Netherlands
| | - Peony D. Banik
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | - Sonal Nagpal
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | - Lindong Weng
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | - Sinan Ozer
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | | | - Mehmet Toner
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | - Shannon N. Tessier
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
| | - Korkut Uygun
- Department of Surgery, Massachusetts General Hospital, Boston, MA, USA
- Center for Engineering in Medicine, Harvard Medical School, Boston MA, USA
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23
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Acun A, Oganesyan R, Uygun BE. Liver Bioengineering: Promise, Pitfalls, and Hurdles to Overcome. CURRENT TRANSPLANTATION REPORTS 2019; 6:119-126. [PMID: 31289714 PMCID: PMC6615568 DOI: 10.1007/s40472-019-00236-3] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
PURPOSE OF REVIEW In this review, we discuss the recent advancements in liver bioengineering and cell therapy and future advancements to improve the field towards clinical applications. RECENT FINDINGS 3D printing, hydrogel-based tissue fabrication, and the use of native decellularized liver extracellular matrix as a scaffold are used to develop whole or partial liver substitutes. The current focus is on developing a functional liver graft through achieving a non-leaky endothelium and a fully constructed bile duct. Use of cell therapy as a treatment is less invasive and less costly compared to transplantation, however, lack of readily available cell sources with low or no immunogenicity and contradicting outcomes of clinical trials are yet to be overcome. SUMMARY Liver bioengineering is advancing rapidly through the development of in vitro and in vivo tissue and organ models. Although there are major challenges to overcome, through optimization of the current methods and successful integration of induced pluripotent stem cells, the development of readily available, patient-specific liver substitutes can be achieved.
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Affiliation(s)
- Aylin Acun
- Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, USA
| | - Ruben Oganesyan
- Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, USA
| | - Basak E. Uygun
- Center for Engineering in Medicine, Massachusetts General Hospital, Shriners Hospitals for Children, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, USA
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24
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Clemens MM, Kennon-McGill S, Apte U, James LP, Finck BN, McGill MR. The inhibitor of glycerol 3-phosphate acyltransferase FSG67 blunts liver regeneration after acetaminophen overdose by altering GSK3β and Wnt/β-catenin signaling. Food Chem Toxicol 2019; 125:279-288. [PMID: 30654094 PMCID: PMC6443093 DOI: 10.1016/j.fct.2019.01.014] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2018] [Revised: 12/23/2018] [Accepted: 01/13/2019] [Indexed: 12/15/2022]
Abstract
Repair mechanisms after acetaminophen (APAP) hepatotoxicity are poorly understood. We recently discovered that phosphatidic acid (PA) increases in mice and humans after APAP overdose, and is critical for liver regeneration. Here, we hypothesized that PA inhibits glycogen synthase kinase-3β (GSK3β), a component of canonical Wnt/β-catenin signaling, after APAP overdose. To test that, we treated mice with 300 mg/kg APAP at 0 h followed by vehicle or 20 mg/kg of the glycerol 3-phosphate acyltransferase inhibitor FSG67 at 3, 24 and 48 h. Some mice also received the GSK3 inhibitor L803-mts. Blood and liver were collected at multiple time points. Consistent with our earlier results, FSG67 did not affect toxicity (ALT, histology), APAP bioactivation (total glutathione), or oxidative stress (oxidized glutathione), but did reduce expression of proliferating cell nuclear antigen (PCNA) at 52 h. We then measured GSK3β phosphorylation and found it was dramatically decreased by FSG67 at 24 h, before PCNA dropped. Expression of cyclin D1, downstream of Wnt/β-catenin, was also reduced. To determine if the effect of FSG67 on GSK3β is important, we treated mice with FSG67 and L803-mts after APAP. Importantly, L803-mts rescued hepatocyte proliferation and survival. Our data indicate PA and lysoPA may support recovery after APAP overdose by inhibiting GSK3β.
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Affiliation(s)
- Melissa M Clemens
- Dept. of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Interdisciplinary Biomedical Sciences Graduate Program, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Stefanie Kennon-McGill
- Dept. of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Udayan Apte
- Dept. of Pharmacology, Toxicology, and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS, USA
| | - Laura P James
- Dept. of Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA
| | - Brian N Finck
- Div. of Geriatrics and Nutritional Sciences, Dept. of Internal Medicine, Washington University School of Medicine, St. Louis, MO, USA
| | - Mitchell R McGill
- Dept. of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Dept. of Environmental and Occupational Health, Fay W. Boozman College of Public Health, University of Arkansas for Medical Sciences, Little Rock, AR, USA; Center for Dietary Supplement Research, University of Arkansas for Medical Sciences, Little Rock, AR, USA.
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25
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Kanamori T, Togawa Iwata Y, Segawa H, Yamamuro T, Kuwayama K, Tsujikawa K, Inoue H. Metabolism of Fentanyl and Acetylfentanyl in Human-Induced Pluripotent Stem Cell-Derived Hepatocytes. Biol Pharm Bull 2018; 41:106-114. [PMID: 29311471 DOI: 10.1248/bpb.b17-00709] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
To evaluate the capability of human-induced pluripotent stem cell-derived hepatocytes (h-iPS-HEP) in drug metabolism, the profiles of the metabolites of fentanyl, a powerful synthetic opioid, and acetylfentanyl, an N-acetyl analog of fentanyl, in the cells were determined and analyzed. Commercially available h-iPS-HEP were incubated with fentanyl or acetylfentanyl for 24 or 48 h. After enzymatic hydrolysis, the medium was deproteinized with acetonitrile, then analyzed by LC/MS. Desphenethylated metabolites and some hydroxylated metabolites, including 4'-hydroxy-fentanyl and β-hydroxy-fentanyl, were detected as metabolites of fentanyl and acetylfentanyl in the medium. The main metabolite of fentanyl with h-iPS-HEP was the desphenethylated metabolite, which was in agreement with in vivo results. These results suggest that h-iPS-HEP may be useful as a tool for investigating drug metabolism.
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26
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Iansante V, Dhawan A, Masmoudi F, Lee CA, Fernandez-Dacosta R, Walker S, Fitzpatrick E, Mitry RR, Filippi C. A New High Throughput Screening Platform for Cell Encapsulation in Alginate Hydrogel Shows Improved Hepatocyte Functions by Mesenchymal Stromal Cells Co-encapsulation. Front Med (Lausanne) 2018; 5:216. [PMID: 30140676 PMCID: PMC6095031 DOI: 10.3389/fmed.2018.00216] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2018] [Accepted: 07/16/2018] [Indexed: 12/31/2022] Open
Abstract
Hepatocyte transplantation has emerged as an alternative to liver transplant for liver disease. Hepatocytes encapsulated in alginate microbeads have been proposed for the treatment of acute liver failure, as they are able to provide hepatic functions while the liver regenerates. Furthermore, they do not require immunosuppression, as the alginate protects the hepatocytes from the recipient's immune cells. Mesenchymal stromal cells are very attractive candidates for regenerative medicine, being able to differentiate into cells of the mesenchymal lineages and having extensive proliferative ability. When co-cultured with hepatocytes in two-dimensional cultures, they exert a trophic role, drastically improving hepatocytes survival and functions. In this study we aimed to (i) devise a high throughput system (HTS) to allow testing of a variety of different parameters for cell encapsulation and (ii) using this HTS, investigate whether mesenchymal stromal cells could have beneficial effects on the hepatocytes when co-encapsulated in alginate microbeads. Using our HTS platform, we observed some improvement of hepatocyte behavior with MSCs, subsequently confirmed in the low throughput analysis of cell function in alginate microbeads. Therefore, our study shows that mesenchymal stromal cells may be a good option to improve the function of hepatocytes microbeads. Furthermore, the platform developed may be used for HTS studies on cell encapsulation, in which several conditions (e.g., number of cells, combinations of cells, alginate modifications) could be easily compared at the same time.
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Affiliation(s)
- Valeria Iansante
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Anil Dhawan
- Paediatric Liver, GI and Nutrition Centre, King's College London, King's College Hospital, London, United Kingdom
| | - Fatma Masmoudi
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Charlotte A Lee
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Raquel Fernandez-Dacosta
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Simon Walker
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Emer Fitzpatrick
- Paediatric Liver, GI and Nutrition Centre, King's College London, King's College Hospital, London, United Kingdom
| | - Ragai R Mitry
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
| | - Céline Filippi
- Dhawan Lab at Mowat Labs, Institute of Liver Studies, King's College London, King's College Hospital, London, United Kingdom
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27
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Pless-Petig G, Rauen U. Serum-Free Cryopreservation of Primary Rat Hepatocytes in a Modified Cold Storage Solution: Improvement of Cell Attachment and Function. Biopreserv Biobank 2018; 16:285-295. [DOI: 10.1089/bio.2018.0002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/29/2023] Open
Affiliation(s)
- Gesine Pless-Petig
- Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany
| | - Ursula Rauen
- Institut für Physiologische Chemie, Universitätsklinikum Essen, Essen, Germany
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28
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Jitraruch S, Dhawan A, Hughes RD, Filippi C, Lehec SC, Glover L, Mitry RR. Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation. Cell Transplant 2018; 26:1341-1354. [PMID: 28901189 PMCID: PMC5680969 DOI: 10.1177/0963689717720050] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
Intraperitoneal transplantation of hepatocyte microbeads is an attractive option for the management of acute liver failure. Encapsulation of hepatocytes in alginate microbeads supports their function and prevents immune attack of the cells. Establishment of banked cryopreserved hepatocyte microbeads is important for emergency use. The aim of this study was to develop an optimized protocol for cryopreservation of hepatocyte microbeads for clinical transplantation using modified freezing solutions. Four freezing solutions with potential for clinical application were investigated. Human and rat hepatocytes cryopreserved with University of Wisconsin (UW)/10% dimethyl sulfoxide (DMSO)/5% (300 mM) glucose and CryoStor CS10 showed better postthawing cell viability, attachment, and hepatocyte functions than with histidine-tryptophan-ketoglutarate/10% DMSO/5% glucose and Bambanker. The 2 freezing solutions that gave better results were studied with human and rat hepatocytes microbeads. Similar effects on cryopreserved microbead morphology (external and ultrastructural), viability, and hepatocyte-functions post thawing were observed over 7 d in culture. UW/DMSO/glucose, as a basal freezing medium, was used to investigate the additional effects of cytoprotectants: a pan-caspase inhibitor (benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone [ZVAD]), an antioxidant (desferoxamine [DFO]), and a buffering and mechanical protectant (human serum albumin [HSA]) on RMBs. ZVAD (60 µM) had a beneficial effect on cell viability that was greater than with DFO (1 mM), HSA (2%), and basal freezing medium alone. Improvements in the ultrastructure of encapsulated hepatocytes and a lower degree of cell apoptosis were observed with all 3 cytoprotectants, with ZVAD tending to provide the greatest effect. Cytochrome P450 activity was significantly higher in the 3 cytoprotectant groups than with fresh microbeads. In conclusion, developing an optimized cryopreservation protocol by adding cytoprotectants such as ZVAD could improve the outcome of cryopreserved hepatocyte microbeads for future clinical use.
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Affiliation(s)
- Suttiruk Jitraruch
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom.,2 Department of Pediatrics, King Chulalongkorn Memorial Hospital, Bangkok, Thailand
| | - Anil Dhawan
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
| | - Robin D Hughes
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
| | - Celine Filippi
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
| | - Sharon C Lehec
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
| | - Leanne Glover
- 3 Centre for Ultrastructural Imaging, King's College London, London, United Kingdom
| | - Ragai R Mitry
- 1 Dhawan Lab at Institute of Liver Studies, King's College London School of Medicine at King's College Hospital, London, United Kingdom
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29
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Wu H, Chang Q. The cryoprotectant trehalose could inhibit ERS-induced apoptosis by activating autophagy in cryoprotected rat valves. PLoS One 2018. [PMID: 29522567 PMCID: PMC5844695 DOI: 10.1371/journal.pone.0194078] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Valvular diseases are common health problems that are strongly related to high morbidity and mortality; aortic valve allograft transplantation may be a promising way to improve survival and relieve symptoms. However, ideal tissue viability has not been observed with common valve cryopreservation methods, which could lead to apoptosis and necrosis in cryopreserved tissue. It has been observed that trehalose plays a positive role by acting to maintain cell structures and protect cells from stress responses. In this study, we studied the effects of trehalose in protecting rat valve tissue from the cooling process. We found improved higher cell function in rat valves treated with trehalose during cryopreservation than in those treated with dimethyl sulphoxide (DMSO). To further explore the mechanisms, we found that trehalose could down-regulate the expression of cleaved caspase-3, an important molecule involved in cell apoptosis. In addition, treatment with trehalose also decreased Glucose-regulated protein 78 (GRP78) and CCAAT/enhancer-binding protein homologous protein (CHOP), the key proteins in the endoplasmic reticulum stress (ERS) process. Intriguingly, we observed that trehalose promotes cryoprotected rat valve cell autophagy via an mTOR-independent but p38 MAPK-dependent signaling pathway. Additionally, miR-221 and miR-32 have been implicated in such cell activities. In summary, our study offers a new and meaningful cryopreservation approach for valve allograft storage.
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Affiliation(s)
- Hongyan Wu
- Department of Cardiovascular Surgery, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
| | - Qing Chang
- Department of Cardiovascular Surgery, The Affiliated Hospital of Qingdao University, Qingdao University, Qingdao, China
- * E-mail:
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30
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Lee CA, Dhawan A, Iansante V, Lehec S, Khorsandi SE, Filippi C, Walker S, Fernandez-Dacosta R, Heaton N, Bansal S, Mitry RR, Fitzpatrick E. Cryopreserved neonatal hepatocytes may be a source for transplantation: Evaluation of functionality toward clinical use. Liver Transpl 2018; 24:394-406. [PMID: 29356341 DOI: 10.1002/lt.25015] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/31/2017] [Revised: 12/13/2017] [Accepted: 12/15/2017] [Indexed: 12/27/2022]
Abstract
Neonatal livers are a potential source of good-quality hepatocytes for clinical transplantation. We compared viability and function of neonatal hepatocytes (NHs) and adult hepatocytes (AHs) and report their clinical use both intraportally and in alginate microbeads. Following isolation from donor livers, hepatocyte function was assessed using albumin, alpha-1-antitrypsin, and factor VII. Metabolic function was investigated by measuring resorufin conjugation, ammonia metabolism, uridine diphosphate glucuronosyltransferase enzyme activity, and cytochrome P450 (CYP) function following induction. Activation of the instant blood-mediated inflammatory reaction by NHs and AHs was investigated using an in vitro blood perfusion model, and tissue factor expression was analyzed using real-time polymerase chain reaction (RT-PCR). Clinical hepatocyte transplantation (HT) was undertaken using standard protocols. Hepatocytes were isolated from 14 neonatal livers, with an average viability of 89.4% ± 1.8% (mean ± standard error of the mean) and average yield of 9.3 × 106 ± 2.0 × 106 cells/g. Hepatocytes were isolated from 14 adult livers with an average viability of 78.6% ± 2.4% and yield 2.2 × 106 ± 0.5 × 105 cells/g. NHs had significantly higher viability after cryopreservation than AHs, with better attachment efficiency and less plasma membrane leakage. There were no differences in albumin, alpha-1-antitrypsin, and factor VII synthesis between NHs and AHs (P > 0.05). Neonatal cells had inducible phase 1 enzymes as assessed by CYP function and functional phase 2 enzymes, in which activity was comparable to AHs. In an in vitro blood perfusion model, AHs elicited increased thrombus formation with a greater consumption of platelets and white cells compared with NHs (28.3 × 109 versus 118.7 × 109 and 3.3 × 109 versus 6.6 × 109 ; P < 0.01). Intraportal transplantation and intraperitoneal transplantation of alginate encapsulated hepatocytes was safe, and preliminary data suggest the cells may activate the immune response to a lesser degree than adult cells. In conclusion, we have shown NHs have excellent cell viability, function, and drug metabolism making them a suitable alternative source for clinical HT. Liver Transplantation 24 394-406 2018 AASLD.
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Affiliation(s)
- Charlotte A Lee
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Anil Dhawan
- Pediatric Liver, GI and Nutrition Centre, King's College London at King's College Hospital, London, UK
| | - Valeria Iansante
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Sharon Lehec
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Shirin E Khorsandi
- Institute of Liver Studies, King's College Hospital National Health Service Foundation Trust, London, UK
| | - Celine Filippi
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Simon Walker
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Raquel Fernandez-Dacosta
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Nigel Heaton
- Institute of Liver Studies, King's College Hospital National Health Service Foundation Trust, London, UK
| | - Sanjay Bansal
- Pediatric Liver, GI and Nutrition Centre, King's College London at King's College Hospital, London, UK
| | - Ragai R Mitry
- Dhawan Lab, Institute of Liver Studies and, King's College London at King's College Hospital, London, UK
| | - Emer Fitzpatrick
- Pediatric Liver, GI and Nutrition Centre, King's College London at King's College Hospital, London, UK
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31
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Thacker SE, Nautiyal M, Otieno MA, Watkins PB, Mosedale M. Optimized Methods to Explore the Mechanistic and Biomarker Potential of Hepatocyte-Derived Exosomes in Drug-Induced Liver Injury. Toxicol Sci 2018; 163:92-100. [DOI: 10.1093/toxsci/kfy015] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/19/2022] Open
Affiliation(s)
- Sarah E Thacker
- Division of Pharmacotherapy and Experimental Therapeutics and Institute for Drug Safety Sciences, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Manisha Nautiyal
- Division of Pharmacotherapy and Experimental Therapeutics and Institute for Drug Safety Sciences, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Monicah A Otieno
- Preclinical Development and Safety, Janssen Research and Development, LLC, Spring House, Pennsylvania 19477
| | - Paul B Watkins
- Division of Pharmacotherapy and Experimental Therapeutics and Institute for Drug Safety Sciences, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Merrie Mosedale
- Division of Pharmacotherapy and Experimental Therapeutics and Institute for Drug Safety Sciences, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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32
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Iansante V, Mitry RR, Filippi C, Fitzpatrick E, Dhawan A. Human hepatocyte transplantation for liver disease: current status and future perspectives. Pediatr Res 2018; 83:232-240. [PMID: 29149103 DOI: 10.1038/pr.2017.284] [Citation(s) in RCA: 139] [Impact Index Per Article: 19.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/15/2017] [Accepted: 10/02/2017] [Indexed: 12/16/2022]
Abstract
Liver transplantation is the accepted treatment for patients with acute liver failure and liver-based metabolic disorders. However, donor organ shortage and lifelong need for immunosuppression are the main limitations to liver transplantation. In addition, loss of the native liver as a target organ for future gene therapy for metabolic disorders limits the futuristic treatment options, resulting in the need for alternative therapeutic strategies. A potential alternative to liver transplantation is allogeneic hepatocyte transplantation. Over the last two decades, hepatocyte transplantation has made the transition from bench to bedside. Standardized techniques have been established for isolation, culture, and cryopreservation of human hepatocytes. Clinical hepatocyte transplantation safety and short-term efficacy have been proven; however, some major hurdles-mainly concerning shortage of donor organs, low cell engraftment, and lack of a long-lasting effect-need to be overcome to widen its clinical applications. Current research is aimed at addressing these problems, with the ultimate goal of increasing hepatocyte transplantation efficacy in clinical applications.
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Affiliation(s)
- V Iansante
- DhawanLab, Paediatric Liver GI and Nutrition Center and MowatLabs, Institute of Liver Studies, King's College London, Faculty of Life Sciences and Medicine, King's College London, King's College Hospital, London, UK
| | - R R Mitry
- DhawanLab, Paediatric Liver GI and Nutrition Center and MowatLabs, Institute of Liver Studies, King's College London, Faculty of Life Sciences and Medicine, King's College London, King's College Hospital, London, UK
| | - C Filippi
- DhawanLab, Paediatric Liver GI and Nutrition Center and MowatLabs, Institute of Liver Studies, King's College London, Faculty of Life Sciences and Medicine, King's College London, King's College Hospital, London, UK
| | - E Fitzpatrick
- DhawanLab, Paediatric Liver GI and Nutrition Center and MowatLabs, Institute of Liver Studies, King's College London, Faculty of Life Sciences and Medicine, King's College London, King's College Hospital, London, UK
| | - A Dhawan
- DhawanLab, Paediatric Liver GI and Nutrition Center and MowatLabs, Institute of Liver Studies, King's College London, Faculty of Life Sciences and Medicine, King's College London, King's College Hospital, London, UK
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33
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Human embryoid bodies to hepatocyte-like clusters: Preparing for translation. LIVER RESEARCH 2017. [DOI: 10.1016/j.livres.2017.08.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
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Kilbride P, Lamb S, Gibbons S, Bundy J, Erro E, Selden C, Fuller B, Morris J. Cryopreservation and re-culture of a 2.3 litre biomass for use in a bioartificial liver device. PLoS One 2017; 12:e0183385. [PMID: 28841674 PMCID: PMC5572048 DOI: 10.1371/journal.pone.0183385] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/31/2016] [Accepted: 07/28/2017] [Indexed: 12/29/2022] Open
Abstract
For large and complex tissue engineered constructs to be available on demand, long term storage using methods, such as cryopreservation, are essential. This study optimised parameters such as excess media concentration and warming rates and used the findings to enable the successful cryopreservation of 2.3 litres of alginate encapsulated liver cell spheroids. This volume of biomass is typical of those required for successful treatment of Acute Liver Failure using our Bioartificial Liver Device. Adding a buffer of medium above the biomass, as well as slow (0.6°C/min) warming rates was found to give the best results, so long as the warming through the equilibrium melting temperature was rapid. After 72 h post thaw-culture, viable cell number, glucose consumption, lactate production, and alpha-fetoprotein production had recovered to pre-freeze values in the 2.3 litre biomass (1.00 ± 0.05, 1.19 ± 0.10, 1.23 ± 0.18, 2.03 ± 0.04 per ml biomass of the pre-cryopreservation values respectively). It was also shown that further improvements in warming rates of the biomass could reduce recovery time to < 48 h. This is the first example of a biomass of this volume being successfully cryopreserved in a single cassette and re-cultured. It demonstrates that a bioartificial liver device can be cryopreserved, and has wider applications to scale-up large volume cryopreservation.
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Affiliation(s)
- Peter Kilbride
- Asymptote, General Electric Healthcare, Cambridge, United Kingdom
- Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom
- * E-mail:
| | - Stephen Lamb
- Asymptote, General Electric Healthcare, Cambridge, United Kingdom
| | - Stephanie Gibbons
- Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom
| | - James Bundy
- Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom
| | - Eloy Erro
- Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom
| | - Clare Selden
- Institute for Liver and Digestive Health, Royal Free Hospital Campus, University College London, London, United Kingdom
| | - Barry Fuller
- Department of Surgery, Royal Free Hospital Campus, University College London, London, United Kingdom
| | - John Morris
- Asymptote, General Electric Healthcare, Cambridge, United Kingdom
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Chabot D, Tremblay T, Paré I, Bazin R, Loubaki L. Transient warming events occurring after freezing impairs umbilical cord-derived mesenchymal stromal cells functionality. Cytotherapy 2017; 19:978-989. [PMID: 28606762 DOI: 10.1016/j.jcyt.2017.04.005] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Revised: 03/28/2017] [Accepted: 04/27/2017] [Indexed: 12/29/2022]
Abstract
BACKGROUND Mesenchymal stromal cells (MSCs) have shown promising results for the treatment of refractory acute graft-versus-host disease. While safety of MSC infusion has been demonstrated, the use of cryopreserved MSCs in clinical trials has raised concerns regarding the retention of their functional activity. This has led to the recommendation by experts in the field to use freshly harvested MSCs, even though this approach is much less practical from a logistic point of view. In the present study, we revisited the impact of cryopreservation on MSC functionality and addressed the possibility that warming events on frozen cells rather than cryopreservation per se could impact MSC functionality. METHODS Following controlled-rate freezing to -130°C, umbilical cord-derived MSCs were left at room temperature (RT) for 2-10 min or on dry ice for 10 min, before being transferred into liquid nitrogen (LqN2). MSCs of each group were subsequently tested (viability, functionality and cellular damage) and compared with their freshly harvested counterparts. RESULTS We demonstrated that freshly harvested MSCs as well as cryopreserved MSCs that were left on dry ice following step-down freezing have comparable viability, functionality and integrity. In contrast, cryopreserved MSCs that were left at RT before being transferred into LqN2 were functionally impaired and showed cellular damage upon thawing even though they exhibited high viability. DISCUSSION Warming events after freezing and not cryopreservation per se significantly impair MSC functionality, indicating that cryopreserved MSCs can be an advantageous alternative to freshly harvested cells for therapeutic purposes.
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Affiliation(s)
- Dominique Chabot
- Department of Research and Development, Héma-Québec, Québec City, QC, Canada; Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec City, QC, Canada
| | - Tony Tremblay
- Department of Research and Development, Héma-Québec, Québec City, QC, Canada
| | - Isabelle Paré
- Department of Research and Development, Héma-Québec, Québec City, QC, Canada
| | - Renée Bazin
- Department of Research and Development, Héma-Québec, Québec City, QC, Canada; Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec City, QC, Canada
| | - Lionel Loubaki
- Department of Research and Development, Héma-Québec, Québec City, QC, Canada; Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec City, QC, Canada.
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36
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Clinical Application of Pluripotent Stem Cells: An Alternative Cell-Based Therapy for Treating Liver Diseases? Transplantation 2017; 100:2548-2557. [PMID: 27495745 DOI: 10.1097/tp.0000000000001426] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
The worldwide shortage of donor livers for organ and hepatocyte transplantation has prompted the search for alternative therapies for intractable liver diseases. Cell-based therapy is envisaged as a useful therapeutic option to recover and stabilize the lost metabolic function for acute liver failure, end-stage and congenital liver diseases, or for those patients who are not considered eligible for organ transplantation. In recent years, research to identify alternative and reliable cell sources for transplantation that can be derived by reproducible methods has been encouraged. Human pluripotent stem cells (PSCs), which comprise both embryonic and induced PSCs, may offer many advantages as an alternative to hepatocytes for liver cell therapy. Their capacity for expansion, hepatic differentiation and self-renewal make them a promising source of unlimited numbers of hepatocyte-like cells for treating and repairing damaged livers. Immunogenicity and tumorigenicity of human PSCs remain the bottleneck for successful clinical application. However, recent advances made to develop disease-corrected hepatocyte-like cells from patients' human-induced PSCs by gene editing have opened up many potential gateways for the autologous treatment of hereditary liver diseases, which may likely reduce the risk of rejection and the need for lifelong immunosuppression. Well-defined methods to reduce the expression of oncogenic genes in induced PSCs, including protocols for their complete and safe hepatic differentiation, should be established to minimize the tumorigenicity of transplanted cells. On top of this, such new strategies are currently being rigorously tested and validated in preclinical studies before they can be safely transferred to clinical practice with patients.
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37
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Alternative Cell Sources to Adult Hepatocytes for Hepatic Cell Therapy. Methods Mol Biol 2016; 1506:17-42. [PMID: 27830543 DOI: 10.1007/978-1-4939-6506-9_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/17/2023]
Abstract
Adult hepatocyte transplantation is limited by scarce availability of suitable donor liver tissue for hepatocyte isolation. New cell-based therapies are being developed to supplement whole-organ liver transplantation, to reduce the waiting-list mortality rate, and to obtain more sustained and significant metabolic correction. Fetal livers and unsuitable neonatal livers for organ transplantation have been proposed as potential useful sources of hepatic cells for cell therapy. However, the major challenge is to use alternative cell sources for transplantation that can be derived from reproducible methods. Different types of stem cells with hepatic differentiation potential are eligible for generating large numbers of functional hepatocytes for liver cell therapy to treat degenerative disorders, inborn hepatic metabolic diseases, and organ failure. Clinical trials are designed to fully establish the safety profile of such therapies and to define target patient groups and standardized protocols.
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38
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Shibany KA, Tötemeyer S, Pratt SL, Paine SW. Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Pharmacol Res Perspect 2016; 4:e00268. [PMID: 27713829 PMCID: PMC5045944 DOI: 10.1002/prp2.268] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2016] [Accepted: 08/27/2016] [Indexed: 11/15/2022] Open
Abstract
Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two‐step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse‐specific drug metabolites.
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Affiliation(s)
- Khaled A Shibany
- School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom
| | - Sabine Tötemeyer
- School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom
| | - Stefanie L Pratt
- School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom
| | - Stuart W Paine
- School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom
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39
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Omidi M, Niknahad H, Mohammadi-Bardbori A. Dithiothreitol (DTT) rescues mitochondria from nitrofurantoin-induced mitotoxicity in rat. J Biochem Mol Toxicol 2016; 30:588-592. [PMID: 27373690 DOI: 10.1002/jbt.21825] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2016] [Revised: 05/24/2016] [Accepted: 06/14/2016] [Indexed: 11/09/2022]
Abstract
Nitrofurantoin (N-(5-nitro-2-furfurylidine) 1-amino-hydantoine; NIT) is mainly used for the treatment of acute urinary tract infections. However, its administration can be associated with liver failure or cirrhosis. The aim of this study was to determine whether NIT is a mitochondrial toxicant, if so, what mechanism(s) is involved. The rat liver mitochondria were isolated and treated with different doses of NIT alone or in combination with a reagent of choice for protecting thiol groups, dithiothreitol (DTT). Several mitochondrial parameters, including succinate dehydrogenase activity (also called 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide assay), lipid peroxidation, superoxide dismutase activity, Reduced glutathione (GSH), and oxidized glutathione (GSSG), and GSSG (oxidized glutathione) levels were determined. The results from this study showed that simultaneous treatment of mitochondria with NIT and DTT significantly reduces the toxicity. Here, we provide evidence that mitochondrial dysfunction followed by depletion of reduced glutathione can be reversed by DTT administration.
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Affiliation(s)
- Mahmoud Omidi
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, 71345-1583, Iran
| | - Hossein Niknahad
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, 71345-1583, Iran.,Pharmaceutical Science Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Afshin Mohammadi-Bardbori
- Department of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Fars, 71345-1583, Iran
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40
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Gómez-Lechón MJ, Tolosa L. Human hepatocytes derived from pluripotent stem cells: a promising cell model for drug hepatotoxicity screening. Arch Toxicol 2016; 90:2049-2061. [PMID: 27325232 DOI: 10.1007/s00204-016-1756-1] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2015] [Accepted: 06/09/2016] [Indexed: 01/09/2023]
Abstract
Drug-induced liver injury (DILI) is a frequent cause of failure in both clinical and post-approval stages of drug development, and poses a key challenge to the pharmaceutical industry. Current animal models offer poor prediction of human DILI. Although several human cell-based models have been proposed for the detection of human DILI, human primary hepatocytes remain the gold standard for preclinical toxicological screening. However, their use is hindered by their limited availability, variability and phenotypic instability. In contrast, pluripotent stem cells, which include embryonic and induced pluripotent stem cells (iPSCs), proliferate extensively in vitro and can be differentiated into hepatocytes by the addition of soluble factors. This provides a stable source of hepatocytes for multiple applications, including early preclinical hepatotoxicity screening. In addition, iPSCs also have the potential to establish genotype-specific cells from different individuals, which would increase the predictivity of toxicity assays allowing more successful clinical trials. Therefore, the generation of human hepatocyte-like cells derived from pluripotent stem cells seems to be promising for overcoming limitations of hepatocyte preparations, and it is expected to have a substantial repercussion in preclinical hepatotoxicity risk assessment in early drug development stages.
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Affiliation(s)
- María José Gómez-Lechón
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Hospital Universitario y Politécnico La Fe de Valencia, Torre A, 6ª Planta, Avenida Fernando Abril Martorell 106, 46026, Valencia, Spain.,CIBERehd, FIS, 08036, Barcelona, Spain
| | - Laia Tolosa
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe, Hospital Universitario y Politécnico La Fe de Valencia, Torre A, 6ª Planta, Avenida Fernando Abril Martorell 106, 46026, Valencia, Spain.
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41
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Kilbride P, Gonzalez-Molina J, Maurmann N, Mendonça da Silva J, Gibbons S, Selden C, Fuller B, Morris J. Impact of Storage at -80°C on Encapsulated Liver Spheroids After Liquid Nitrogen Storage. Biores Open Access 2016; 5:146-54. [PMID: 27298755 PMCID: PMC4900228 DOI: 10.1089/biores.2016.0017] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
For many bioengineered tissues to have practical clinical application, cryopreservation for use on demand is essential. This study examined different thermal histories on warming and short holding periods at different subzero temperatures on subsequent functional recoveries of alginate encapsulated liver spheroids (ELS) for use in a bioartificial liver device. This mimicked transport at liquid nitrogen (−196°C) or dry ice (∼−80°C) temperatures. Holding at −80°C on warming after −196°C storage resulted in ELS expressing significant (p < 0.001) damage compared with direct thaw from liquid nitrogen, with viable cell number falling from 74.0 ± 8.4 million viable cells/mL without −80°C storage to 1.9 ± 0.6 million viable cells/mL 72 h post-thaw after 8 days storage at −80°C. Even 1 day at −80°C after −196°C storage resulted in lower viability (down 21% 24 h post-thaw), viable cell count (down 29% 24 h post-thaw), glucose, and alpha-1-fetoprotein production (reduced by 59% and 95% 24 h from 1 day post-thaw, respectively). Storage at −80°C was determined to be harmful only during the warming cycle. Chemical measurements of the alginate component of ELS were unchanged by cryogenic exposure in either condition.
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Affiliation(s)
- Peter Kilbride
- Asymptote Ltd., St. John's Innovation Centre, Cambridge, United Kingdom.; UCL Institute for Liver and Digestive Health, Royal Free Hospital Campus, London, United Kingdom
| | - Jordi Gonzalez-Molina
- UCL Institute for Liver and Digestive Health , Royal Free Hospital Campus, London, United Kingdom
| | - Natasha Maurmann
- UCL Institute for Liver and Digestive Health, Royal Free Hospital Campus, London, United Kingdom.; UCL Department of Surgery, Royal Free Hospital Campus, London, United Kingdom
| | - Joana Mendonça da Silva
- UCL Institute for Liver and Digestive Health , Royal Free Hospital Campus, London, United Kingdom
| | - Stephanie Gibbons
- UCL Institute for Liver and Digestive Health , Royal Free Hospital Campus, London, United Kingdom
| | - Clare Selden
- UCL Institute for Liver and Digestive Health , Royal Free Hospital Campus, London, United Kingdom
| | - Barry Fuller
- UCL Department of Surgery , Royal Free Hospital Campus, London, United Kingdom
| | - John Morris
- Asymptote Ltd. , St. John's Innovation Centre, Cambridge, United Kingdom
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Ibars EP, Cortes M, Tolosa L, Gómez-Lechón MJ, López S, Castell JV, Mir J. Hepatocyte transplantation program: Lessons learned and future strategies. World J Gastroenterol 2016; 22:874-886. [PMID: 26811633 PMCID: PMC4716085 DOI: 10.3748/wjg.v22.i2.874] [Citation(s) in RCA: 43] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Accepted: 12/01/2015] [Indexed: 02/06/2023] Open
Abstract
This review aims to share the lessons we learned over time during the setting of the hepatocyte transplantation (HT) program at the Hepatic Cell Therapy Unit at Hospital La Fe in Valencia. New sources of liver tissue for hepatocyte isolation have been explored. The hepatocyte isolation and cryopreservation procedures have been optimized and quality criteria for assessment of functionality of hepatocyte preparations and suitability for HT have been established. The results indicate that: (1) Only highly viable and functional hepatocytes allow to recover those functions lacking in the native liver; (2) Organs with steatosis (≥ 40%) and from elderly donors are declined since low hepatocyte yields, viability and cell survival after cryopreservation, are obtained; (3) Neonatal hepatocytes are cryopreserved without significant loss of viability or function representing high-quality cells to improve human HT; (4) Cryopreservation has the advantage of providing hepatocytes constantly available and of allowing the quality evaluation and suitability for transplantation; and (5) Our results from 5 adults with acute liver failure and 4 from children with inborn metabolic diseases, indicate that HT could be a very useful and safe cell therapy, as long as viable and metabolically functional human hepatocytes are used.
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43
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Tolosa L, Caron J, Hannoun Z, Antoni M, López S, Burks D, Castell JV, Weber A, Gomez-Lechon MJ, Dubart-Kupperschmitt A. Transplantation of hESC-derived hepatocytes protects mice from liver injury. Stem Cell Res Ther 2015; 6:246. [PMID: 26652177 PMCID: PMC4676869 DOI: 10.1186/s13287-015-0227-6] [Citation(s) in RCA: 57] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2015] [Revised: 10/25/2015] [Accepted: 11/06/2015] [Indexed: 12/25/2022] Open
Abstract
Background Hepatic cell therapy has become a viable alternative to liver transplantation for life-threatening liver diseases. However, the supply of human hepatocytes is limited due to the shortage of suitable donor organs required to isolate high-quality cells. Human pluripotent stem cells reflect a potential renewable source for generating functional hepatocytes. However, most differentiation protocols use undefined matrices or factors of animal origin; as such, the resulting hepatocytes are not Good Manufacturing Practice compliant. Moreover, the preclinical studies employed to assess safety and function of human embryonic stem cell (hESC)-derived hepatocytes are generally limited to immunodeficient mice. In the present study, we evaluate the generation of hepatocytes under defined conditions using a European hESC line (VAL9) which was derived under animal-free conditions. The function capacity of VAL9-derived hepatocytes was assessed by transplantation into mice with acetaminophen-induced acute liver failure, a clinically relevant model. Methods We developed a protocol that successfully differentiates hESCs into bipotent hepatic progenitors under defined conditions, without the use of chromatin modifiers such as dimethyl sulphoxide. These progenitors can be cryopreserved and are able to generate both committed precursors of cholangiocytes and neonate-like hepatocytes. Results Thirty days post-differentiation, hESCs expressed hepatocyte-specific markers such as asialoglycoprotein receptor and hepatic nuclear factors including HNF4α. The cells exhibited properties of mature hepatocytes such as urea secretion and UGT1A1 and cytochrome P450 activities. When transplanted into mice with acetaminophen-induced acute liver failure, a model of liver damage, the VAL9-derived hepatocytes efficiently engrafted and proliferated, repopulating up to 10 % of the liver. In these transplanted livers, we observed a significant decrease of liver transaminases and found no evidence of tumourigenicity. Thus, VAL9-derived hepatocytes were able to rescue hepatic function in acetaminophen-treated animals. Conclusions Our study reveals an efficient protocol for differentiating VAL9 hESCs to neonatal hepatocytes which are then able to repopulate livers in vivo without tumour induction. The human hepatocytes are able to rescue liver function in mice with acetaminophen-induced acute toxicity. These results provide proof-of-concept that replacement therapies using hESC-derived hepatocytes are effective for treating liver diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13287-015-0227-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Laia Tolosa
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Unidad de Hepatología Experimental, IIS LA Fe, Valencia, S-46026, Spain. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
| | - Jérôme Caron
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
| | - Zara Hannoun
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
| | - Marc Antoni
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
| | - Silvia López
- Unidad de Hepatología Experimental, IIS LA Fe, Valencia, S-46026, Spain.
| | - Deborah Burks
- CIBERDEM, Centro de Investigacion Prıncipe Felipe, Valencia, S-46012, Spain.
| | - Jose Vicente Castell
- Unidad de Hepatología Experimental, IIS LA Fe, Valencia, S-46026, Spain. .,CIBERehd, FIS, Barcelona, S-08036, Spain.
| | - Anne Weber
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
| | - Maria-Jose Gomez-Lechon
- Unidad de Hepatología Experimental, IIS LA Fe, Valencia, S-46026, Spain. .,CIBERehd, FIS, Barcelona, S-08036, Spain.
| | - Anne Dubart-Kupperschmitt
- INSERM, U 1193, Hôpital Paul Brousse, Villejuif, F-94807, France. .,Univ Paris-Sud, UMR-S 1193, Villejuif, F-94800, France. .,DHU Hepatinov, Villejuif, F-94800, France.
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44
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Dhawan A. Clinical human hepatocyte transplantation: Current status and challenges. Liver Transpl 2015; 21 Suppl 1:S39-44. [PMID: 26249755 DOI: 10.1002/lt.24226] [Citation(s) in RCA: 41] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 07/27/2015] [Accepted: 07/29/2015] [Indexed: 02/07/2023]
Affiliation(s)
- Anil Dhawan
- Department of Pediatric Hepatology, Cell Therapy Unit, National Institute for Health Research/Wellcome Trust King's Clinical Research Facility, King's College Hospital, London, UK
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45
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Guo X, Wang S, Dou YL, Guo XF, Chen ZL, Wang XW, Shen ZQ, Qiu ZG, Jin M, Li JW. A Convenient and Efficient Method to Enrich and Maintain Highly Proliferative Human Fetal Liver Stem Cells. Rejuvenation Res 2015; 18:211-24. [PMID: 25556695 DOI: 10.1089/rej.2014.1619] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Pluripotent human hepatic stem cells have broad research and clinical applications, which are, however, restricted by both limited resources and technical difficulties with respect to isolation of stem cells from the adult or fetal liver. In this study, we developed a convenient and efficient method involving a two-step in situ collagenase perfusion, gravity sedimentation, and Percoll density gradient centrifugation to enrich and maintain highly proliferative human fetal liver stem cells (hFLSCs). Using this method, the isolated hFLSCs entered into the exponential growth phase within 10 days and maintained sufficient proliferative activity to permit subculture for at least 20 passages without differentiation. Immunocytochemistry, immunofluorescence, and flow cytometry results showed that these cells expressed stem cell markers, such as c-kit, CD44, epithelial cell adhesion molecule (EpCAM), oval cell marker-6 (OV-6), epithelial marker cytokeratin 18 (CK18), biliary ductal marker CK19, and alpha-fetoprotein (AFP). Gene expression analysis showed that these cells had stable mRNA expression of c-Kit, EpCAM, neural cell adhesion molecule (NCAM), CK19, CK18, AFP, and claudin 3 (CLDN-3) throughout each passage while maintaining low levels of ALB, but with complete absence of cytochrome P450 3A4 (C3A4), phosphoenolpyruvate carboxykinase (PEPCK), telomeric repeat binding factor (TRF), and connexin 26 (CX26) expression. When grown in appropriate medium, these isolated liver stem cells could differentiate into hepatocytes, cholangiocytes, osteoblasts, adipocytes, or endothelial cells. Thus, we have demonstrated a more economical and efficient method to isolate hFLSCs than magnetic-activated cell sorting (MACS). This novel approach may provide an excellent tool to isolate highly proliferative hFLSCs for tissue engineering and regenerative therapies.
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Affiliation(s)
- Xuan Guo
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Shu Wang
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Ya-ling Dou
- 3 Peking Union Medical College Hospital , Chinese Medical Academy, Beijing, China
| | - Xiang-fei Guo
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Zhao-li Chen
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Xin-wei Wang
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Zhi-qiang Shen
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Zhi-gang Qiu
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Min Jin
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
| | - Jun-wen Li
- 1 Institute of Health and Environmental Medicine , Tianjin, China .,2 Key Laboratory of Risk Assessment and Control for Environment & Food Safety , Tianjin, China
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Forbes SJ, Gupta S, Dhawan A. Cell therapy for liver disease: From liver transplantation to cell factory. J Hepatol 2015; 62:S157-69. [PMID: 25920085 DOI: 10.1016/j.jhep.2015.02.040] [Citation(s) in RCA: 218] [Impact Index Per Article: 21.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/29/2014] [Revised: 02/20/2015] [Accepted: 02/27/2015] [Indexed: 02/08/2023]
Abstract
Work over several decades has laid solid foundations for the advancement of liver cell therapy. To date liver cell therapy in people has taken the form of hepatocyte transplantation for metabolic disorders with a hepatic basis, and for acute or chronic liver failure. Although clinical trials using various types of autologous cells have been implemented to promote liver regeneration or reduce liver fibrosis, clear evidence of therapeutic benefits have so far been lacking. Cell types that have shown efficacy in preclinical models include hepatocytes, liver sinusoidal endothelial cells, mesenchymal stem cells, endothelial progenitor cells, and macrophages. However, positive results in animal models have not always translated through to successful clinical therapies and more realistic preclinical models need to be developed. Studies defining the optimal repopulation by transplanted cells, including routes of cell transplantation, superior engraftment and proliferation of transplanted cells, as well as optimal immunosuppression regimens are required. Tissue engineering approaches to transplant cells in extrahepatic locations have also been proposed. The derivation of hepatocytes from pluripotent or reprogrammed cells raises hope that donor organ and cell shortages could be overcome in the future. Critical hurdles to be overcome include the production of hepatocytes from pluripotent cells with equal functional capacity to primary hepatocytes and long-term phenotypic stability in vivo.
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Affiliation(s)
- Stuart J Forbes
- MRC Centre for Regenerative Medicine, Scottish Centre for Regenerative Medicine, 5 Little France Drive, Edinburgh EH16 4UU, United Kingdom.
| | - Sanjeev Gupta
- Departments of Medicine and Pathology, Albert Einstein College of Medicine, Jack and Pearl Resnick Campus, 1300 Morris Park Avenue, Ullmann Building, Room 625, Bronx, NY 10461, United States
| | - Anil Dhawan
- Paediatric Liver GI and Nutrition Center and NIHR/Wellcome Cell Therapy Unit, King's College Hospital at King's College, London SE59RS, United Kingdom
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Park S, Seawright A, Park S, Craig Dutton J, Grinnell F, Han B. Preservation of tissue microstructure and functionality during freezing by modulation of cytoskeletal structure. J Mech Behav Biomed Mater 2015; 45:32-44. [PMID: 25679482 DOI: 10.1016/j.jmbbm.2015.01.014] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Revised: 01/13/2015] [Accepted: 01/16/2015] [Indexed: 02/06/2023]
Abstract
Cryopreservation is one of the key enabling technologies for tissue engineering and regenerative medicine, which can provide reliable long-term storage of engineered tissues (ETs) without losing their functionality. However, it is still extremely difficult to design and develop cryopreservation protocols guaranteeing the post-thaw tissue functionality. One of the major challenges in cryopreservation is associated with the difficulty of identifying effective and less toxic cryoprotective agents (CPAs) to guarantee the post-thaw tissue functionality. In this study, thus, a hypothesis was tested that the modulation of the cytoskeletal structure of cells embedded in the extracellular matrix (ECM) can mitigate the freezing-induced changes of the functionality and can reduce the amount of CPA necessary to preserve the functionality of ETs during cryopreservation. In order to test this hypothesis, we prepared dermal equivalents by seeding fibroblasts in type I collagen matrices resulting in three different cytoskeletal structures. These ETs were exposed to various freeze/thaw (F/T) conditions with and without CPAs. The freezing-induced cell-fluid-matrix interactions and subsequent functional properties of the ETs were assessed. The results showed that the cytoskeletal structure and the use of CPA were strongly correlated to the preservation of the post-thaw functional properties. As the cytoskeletal structure became stronger via stress fiber formation, the ET's functionality was preserved better. It also reduced the necessary CPA concentration to preserve the post-thaw functionality. However, if the extent of the freezing-induced cell-fluid-matrix interaction was too excessive, the cytoskeletal structure was completely destroyed and the beneficial effects became minimal.
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Affiliation(s)
- Seungman Park
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
| | - Angela Seawright
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
| | - Sinwook Park
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA
| | - J Craig Dutton
- Department of Aerospace Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, USA
| | - Frederick Grinnell
- Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA
| | - Bumsoo Han
- School of Mechanical Engineering, Purdue University, West Lafayette, IN, USA; Weldon School of Biomedical Engineering, Purdue University, West Lafayette, IN, USA; Birck Nanotechnology Center, Purdue University, West Lafayette, IN, USA.
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Abstract
The use of cryopreserved hepatocytes has increased in the last decade due to the improvement of the freezing and thawing methods, and has even achieved acceptance by the US Food and Drug Administration for use in drug metabolizing enzyme induction studies. This chapter provides an overview of the theories behind the process of cryopreservation as well as practical advice on methods to cryopreserve hepatocytes, which retain functions similar to fresh cells after thawing. Parameters, such as cell density, cryoprotectants, freezing media, storage conditions, and thawing techniques, should be critically considered. Special emphasis is put on human hepatocytes, but information for the cryopreservation of animal hepatocytes is also described.
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Jitraruch S, Dhawan A, Hughes RD, Filippi C, Soong D, Philippeos C, Lehec SC, Heaton ND, Longhi MS, Mitry RR. Alginate microencapsulated hepatocytes optimised for transplantation in acute liver failure. PLoS One 2014; 9:e113609. [PMID: 25438038 PMCID: PMC4249959 DOI: 10.1371/journal.pone.0113609] [Citation(s) in RCA: 88] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/13/2014] [Accepted: 10/27/2014] [Indexed: 11/24/2022] Open
Abstract
Background and Aim Intraperitoneal transplantation of alginate-microencapsulated human hepatocytes is an attractive option for the management of acute liver failure (ALF) providing short-term support to allow native liver regeneration. The main aim of this study was to establish an optimised protocol for production of alginate-encapsulated human hepatocytes and evaluate their suitability for clinical use. Methods Human hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality. Results The optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×106 cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function. Conclusion An optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.
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Affiliation(s)
- Suttiruk Jitraruch
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Anil Dhawan
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
- * E-mail: (AD); (RRM)
| | - Robin D. Hughes
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Celine Filippi
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Daniel Soong
- British Heart Foundation Centre of Excellence Cardiovascular Division, King's College London School of Medicine, London, United Kingdom
| | - Christina Philippeos
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Sharon C. Lehec
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Nigel D. Heaton
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Maria S. Longhi
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
| | - Ragai R. Mitry
- Institute of Liver Studies, King's College London School of Medicine, London, United Kingdom
- * E-mail: (AD); (RRM)
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Bissoyi A, Nayak B, Pramanik K, Sarangi SK. Targeting cryopreservation-induced cell death: a review. Biopreserv Biobank 2014; 12:23-34. [PMID: 24620767 DOI: 10.1089/bio.2013.0032] [Citation(s) in RCA: 75] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
Despite marked developments in the field of cryopreservation of cells and tissues for research and therapeutic applications, post-thaw cell death remains a significant drawback faced by cryobiologists. Post cryopreservation apoptosis and necrosis are normally observed within 6 to 24 h after post-thaw culture. As a result, massive loss of cell viability and cellular function occur due to cryopreservation. However, in this new generation of cryopreservation science, scientists in this field are focusing on incorporation of apoptosis and necrosis inhibitors (zVAD-fmk, p38 MAPK inhibitor, ROCK inhibitor, etc.) to cryopreservation and post-thaw culture media. These inhibitors target and inhibit various proteins such as caspases, proteases, and kinases, involved in the cell death cascade, resulting in reduced intensity of apoptosis and necrosis in the cryopreserved cells and tissues, increased cell viability, and maintenance of cellular function; thus improved overall cryopreservation efficiency is achieved. The present article provides an overview of various cell death pathways, molecules mediating cryopreservation-induced apoptosis and the potential of certain molecules in targeting cryopreservation-induced delayed-onset cell death.
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Affiliation(s)
- A Bissoyi
- 1 Department of Biotechnology and Medical Engineering, National Institute of Technology , Rourkela, India
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