<i>In vitro</i>analysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

Hang, Hualian; Shi, Xiaolei; Gu, Guang xiang; Wu, Yuzhong; Gu, Jin-yang; Ding, Yi-Tao

doi:10.1111/j.1478-3231.2009.02197.x

Cited by 24 publications

(15 citation statements)

References 38 publications

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“…Others have employed encapsulation methods to improve the vitality of human hepatocytes after cryopreservation. For example, Hang et al (2010) prepared human hepatocytes by first pre-incubating for 12–24 h and then microencapsulating them in alginate-poly- l -lysine-alginate. Compared to cells which had been immediately cryopreserved, encapsulated hepatocytes exhibited higher mRNA and protein levels in attached cells, and higher secretion of albumin and urea levels after thawing.…”

Section: Cryopreservation Of Hepatocytes and Recent Developmentsmentioning

confidence: 99%

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Hewitt²,

et al. 2013

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This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell–derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-013-1078-5) contains supplementary material, which is available to authorized users.

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Section: Cryopreservation Of Hepatocytes and Recent Developmentsmentioning

confidence: 99%

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Hewitt²,

et al. 2013

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“…The strategies currently taken include ͑1͒ refinement of the isolation technology, and incubation of the isolated cells with medium containing antioxidants, at low temperature, before freezing. [42][43][44] This can effectively reduce the isolation damage to the cells and improve their quality prefreezing. ͑2͒ Modification of the formula of the cryopreserving medium and the protective agent.…”

Section: Hepatocyte Cryopreservation For Ebalmentioning

confidence: 99%

Current development of bioreactors for extracorporeal bioartificial liver (Review)

et al. 2010

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The research and development of extracorporeal bioartificial liver is gaining pace in recent years with the introduction of a myriad of optimally designed bioreactors with the ability to maintain long-term viability and liver-specific functions of hepatocytes. The design considerations for bioartificial liver are not trivial; it needs to consider factors such as the types of cell to be cultured in the bioreactor, the bioreactor configuration, the magnitude of fluid-induced shear stress, nutrients' supply, and wastes' removal, and other relevant issues before the bioreactor is ready for testing. This review discusses the exciting development of bioartificial liver devices, particularly the various types of cell used in current reactor designs, the state-of-the-art culturing and cryopreservation techniques, and the comparison among many today's bioreactor configurations. This review will also discuss in depth the importance of maintaining optimal mass transfer of nutrients and oxygen partial pressure in the bioreactor system. Finally, this review will discuss the commercially available bioreactors that are currently undergoing preclinical and clinical trials.

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“…[8][9][10][11] The challenges are the high water content (>90%) together with the relatively large size of the microbeads and the number of cells compared to single cells. This makes microbeads susceptible to cryodamage by icecrystal formation, leading to cell death.…”

Section: Introductionmentioning

confidence: 99%

“…Most of the previous studies of cryopreservation of microencapsulated hepatocytes used a cryopreservation medium consisting of culture medium, 10% fetal calf serum (FCS), and 10% dimethyl sulfoxide (DMSO) placed into cryovials and stored in liquid nitrogen. [8][9][10]13 Although some of these protocols provided relatively good viability and function after thawing, they are not acceptable for clinical transplantation.…”

Section: Introductionmentioning

confidence: 99%

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

et al. 2017

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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 mM) 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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In vitroanalysis of cryopreserved alginate-poly-l-lysine-alginate-microencapsulated human hepatocytes

Cited by 24 publications

References 38 publications

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME

Current development of bioreactors for extracorporeal bioartificial liver (Review)

Cryopreservation of Hepatocyte Microbeads for Clinical Transplantation

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