Preparation of equine isolated hepatocytes

Bakala, A.; Karlik, Wojciech; Wiechetek, M.

doi:10.1016/s0887-2333(03)00112-7

Cited by 14 publications

(21 citation statements)

References 20 publications

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“…Hepatocyte viability was on average 84.1 ± 2.62% (Table ) and is comparable to that reported by Bakala et al. () of 82.7 ± 10.2% and Stefanski et al. () of 84.6 ± 6.4%.…”

Section: Resultssupporting

confidence: 88%

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Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Shibany

Tötemeyer

Pratt

et al. 2016

Pharmacology Res & Perspec

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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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“…Hepatocyte viability was on average 84.1 ± 2.62% (Table ) and is comparable to that reported by Bakala et al. () of 82.7 ± 10.2% and Stefanski et al. () of 84.6 ± 6.4%.…”

Section: Resultssupporting

confidence: 88%

“…Fresh equine primary hepatocytes have successfully been isolated and cultured (Bakala et al. ; Stefanski et al. ).…”

Section: Introductionmentioning

confidence: 99%

Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Shibany

Tötemeyer

Pratt

et al. 2016

Pharmacology Res & Perspec

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“…This is possibly due to the complex cytoskeletal reorganizations required during this process; cell attachment has not yet been reported after cold storage periods exceeding 72 h [1], [2], [30]. Here, we showed that crude cell suspensions stored in the new hepatocyte storage solutions (solutions 2 and 6) for even one week were still able to form normal adherent cell cultures – without further processing of the cell suspensions and with a cell function close to cultures from non-stored control cells, a functionality not yet described for stored hepatocyte suspensions.…”

Section: Discussionmentioning

confidence: 99%

Cold Storage of Rat Hepatocyte Suspensions for One Week in a Customized Cold Storage Solution – Preservation of Cell Attachment and Metabolism

2012

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Background & AimsPrimary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells.MethodsRat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed.ResultsCold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage.ConclusionIn the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

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“…Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.CT-1248 Cell Transplantation Early Epub; provisional acceptance 01/12/2015 6 This approach has been widely explored in experimental settings using different organ preservative solutions or defined culture media and isolated (2,34,37,40,41) or cultured hepatocytes (8,26,39), mainly of animal origin. Most of these studies focused on the recovery of the cell metabolic capacities after rewarming, with the aim of providing a convenient cell source for in vitro drug discovery platforms.…”

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confidence: 99%

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy

et al. 2015

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Hepatocyte transplantation is a promising alternative therapy for the treatment of hepatic failure, hepatocellular deficiency, and genetic metabolic disorders. Hypothermic preservation of isolated human hepatocytes is potentially a simple and convenient strategy to provide on-demand hepatocytes in sufficient quantity and of the quality required for biotherapy. In this study, first we assessed how cold storage in three clinically safe preservative solutions (UW, HTS-FRS, and IGL-1) affects the viability and in vitro functionality of human hepatocytes. Then we evaluated whether such cold-preserved human hepatocytes could engraft and repopulate damaged livers in a mouse model of liver failure. Human hepatocytes showed comparable viabilities after cold preservation in the three solutions. The ability of fresh and cold-stored hepatocytes to attach to a collagen substratum and to synthesize and secrete albumin, coagulation factor VII, and urea in the medium after 3 days in culture was also equally preserved. Coldstored hepatocytes were then transplanted in the spleen of immunodeficient mice previously infected with adenoviruses containing a thymidine kinase construct and treated with a single dose of ganciclovir to induce liver injury. Engraftment and liver repopulation were monitored over time by measuring the blood level of human albumin and by assessing the expression of specific human hepatic mRNAs and proteins in the recipient livers by RT-PCR and immunohistochemistry, respectively. Our findings show that cold-stored human hepatocytes in IGL-1 and HTS-FRS preservative solutions can survive, engraft, and proliferate in a damaged mouse liver. These results demonstrate the usefulness of human hepatocyte hypothermic preservation for cell transplantation.

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Preparation of equine isolated hepatocytes

Cited by 14 publications

References 20 publications

Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Cold Storage of Rat Hepatocyte Suspensions for One Week in a Customized Cold Storage Solution – Preservation of Cell Attachment and Metabolism

Cold Preservation of Human Adult Hepatocytes for Liver Cell Therapy

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