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KMID : 0371320130840040202
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Journal of the Korean Surgical Society
2013 Volume.84 No. 4 p.202 ~ p.208
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Successful mouse hepatocyte culture with sandwich collagen gel formation
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Choi Hyun-Jung
Choi Dong-Ho
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Abstract
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Purpose : Primary mammalian hepatocytes largely retain their liver-specific functions when they are freshly derived from donors. However, long-term cultures of functional hepatocytes are difficult to establish. To increase the longevity and maintain the differentiated functions of hepatocytes in primary culture, cells can be cultured in a sandwich configuration of collagen. In such a configuration, hepatocytes can be cultured for longer periods compared with cultures on single layers of collagen. However, research regarding mouse hepatocytes in sandwich culture is lacking. Methods : Primary mouse hepatocytes were sandwiched between two layers of collagen to maintain the stability of their liver-specific functions. After gelation, 2 mL of hepatocyte culture medium was applied. Results : After 24 hours, 5, 10 days of culture, the collagen gel sandwich maintained the cellular border and numbers of bile canaliculi more efficiently than a single collagen coating in both high and low density culture dishes. Reverse transcription-polymerase chain reaction analysis of alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), the tyrosine aminotransferase gene, glucose-6-phosphatase, glyceraldehyde-3-phosphate dehydrogenase for mouse primary hepatocytes cultured on collagen coated dishes and collagen gels showed superior hepatocyte-related gene expression in cells grown using the collagen gel sandwich culture system. AAT, HNF4A, albumin, TO were found to be expressed in mouse hepatocytes cultured on collagen gels for 5 and 10 days. In contrast, mouse hepatocytes grown on collagen-coated dishes did not express these genes after 5 and 10 days of culture. Conclusion : The collagen gel sandwich method is suitable for primary culture system of adult mouse hepatocytes.
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KEYWORD
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Collagen, Culture, Hepatocyte
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