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Simplified isolation and spheroidal aggregate culture of rat hepatocytes.

ABSTRACT: AIM:To explore a simplified method for isolation of hepatocytes and establish a method of primary hepatocyte culture with more aggregates and longer persisence.METHODS: Wistar rat hepatocytes were isolated by a single extracorporeal two-step perfusion method, and the cells were seeded on poly-HEMA coated flasks and cultured with hormonally defined medium and gentle shaking at regular intervals.RESULTS: The total yield of isolating hepatocytes amounted to 10(8) cells for each rat liver with the viability of more than 90% in all isolations. Under the nonadherent environments, the cells were found to attach to each other and form multicellular aggregates rapidly, and the aggregates became spheroidal shape after two days in culture. The morphologic characteristics and albumin synthetic unction of the multicellular spheroidal aggregates can be maintained for one month.CONCLUSION: The simple and reliable isolation as well as large scale and longer time culture of hepatocytes can be used for experiments in liver cell transplantation and bioartificial liver support system.

SUBMITTER: Wang YJ

PROVIDER: S-EPMC4767772 | biostudies-literature | 1998 Feb

REPOSITORIES: biostudies-literature

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Simplified isolation and spheroidal aggregate culture of rat hepatocytes.

Wang Ying-Jie YJ Li Meng-Dong MD Wang Yu-Min YM Ding Jian J Nie Qing-He QH

World journal of gastroenterology 19980201 1

AIM:To explore a simplified method for isolation of hepatocytes and establish a method of primary hepatocyte culture with more aggregates and longer persisence.METHODS: Wistar rat hepatocytes were isolated by a single extracorporeal two-step perfusion method, and the cells were seeded on poly-HEMA coated flasks and cultured with hormonally defined medium and gentle shaking at regular intervals.RESULTS: The total yield of isolating hepatocytes amounted to 10(8) cells for each rat liver with the v ...[more]

PMID: 11819238

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Project description:Sandwich-cultured rat hepatocytes are used in drug discovery for pharmacological and toxicological assessment of drug candidates, yet their utility as a functional model for drug transporters has not been fully characterized. To evaluate the system as an in vitro model for drug transport, expression changes of hepatic transporters relative to whole liver and freshly isolated hepatocytes (day 0) were examined by real-time quantitative reverse transcription-polymerase chain reaction for 4 consecutive days of culture. No significant differences in transporter expression levels were observed between freshly isolated hepatocytes and whole liver. Two distinct mRNA profiles were detected over time showing 1) a more than 5-fold decline in levels of uptake transporters such as Na(+)-taurocholate cotransporting polypeptide (Ntcp), organic anion transporter (Oat) 2, organic anion-transporting polypeptide (Oatp) 1a1, Oatp1a4, and Oatp1b2 and 2) a greater than 5-fold increase of efflux transporters P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-related proteins (Mrp) 1, 2, 3, and 4. In addition, protein levels and functional activities for selected transporters were also determined. Protein levels for Mrp2, Bcrp, P-gp, Ntcp, and Oatp1a4 corresponded to changes in mRNA. Functional activities of Oatps and Oct1 exhibited a 3- and 4-fold decrease on day 2 and day 4, respectively, relative to that on day 0, whereas a more than 10-fold reduction in Oat2 activity was observed. These results indicate that the cell culture conditions used herein did not provide an optimal environment for expression of all hepatic transporters. Significant time-dependent alterations in basal gene expression patterns of transporters were detected compared with those in liver or freshly isolated hepatocytes. Further work and new strategies are required to improve the validity of this model as an in vitro tool for in vivo drug transport or biliary clearance prediction.

Dataset Information

Simplified isolation and spheroidal aggregate culture of rat hepatocytes.

Publications

Simplified isolation and spheroidal aggregate culture of rat hepatocytes.

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