Successful mouse hepatocyte culture with sandwich collagen gel formation

Abstract

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.

Keywords: Collagen; Culture; Hepatocyte.

Fig. 1

Comparison of mouse hepatocyte morphology between cells cultured in a collagen gel sandwich and those grown in a collagen-coated dish at low cellular concentration (5 × 10⁴ cells/35 mm² well). Cellular borders and the numbers of bile canaliculi of the primary hepatocytes are well preserved in the collagen gel sandwich (A-C) compared to the collagen-coated dish (D-F) at 1 day, 5 days, and 10 days (H&E, ×100).

Fig. 2

Comparison of mouse hepatocyte morphology between cells cultured in a collagen gel sandwich and those grown in a collagen-coated dish at high cellular concentration (1 × 10⁶ cells/35 mm² well). Cellular borders and the numbers of bile canaliculi of the primary hepatocytes are well preserved in the collagen gel sandwich (A-C) compared to collagen-coated dish (D-F) at 1 day, 5 days, and 10 days (H&E, ×100).

Fig. 3

Schematic figure of the hepatic sinusoid compared to the intestinal epithelium (A) and magnified morphology of mouse primary hepatocytes cultured in a collagen gel sandwich (B). Mouse hepatocytes cultured between collagen gels showed prominent bile canaliculi and dense cell-to-cell contact (B). Arrows show apical and basal surfaces and the space of Disse in the hepatocytes (×200).

Fig. 4

Representative reverse transcription-polymerase chain reaction data of mouse hepatocytes grown in a collagen gel sandwich and a collagencoated dish. Alpha-1-antitrypsin (AAT), hepatocyte nuclear factor 4 alpha (HNF4A), alphafetoprotein, albumin, tryptophan oxygenase (TO), tyrosine aminotransferase (TAT) gene, glucose-6-phosphatase (G6P), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in mouse primary hepatocytes cultured in collagen-coated and collagen gel dishes. 1st row corresponds to mouse hepatocytes and 2nd and 3rd row correspond to mouse hepatocytes grown on collagen gel for 5 days and 10 days, respectively. 4th and 5th row represent mouse hepatocytes cultured in a collagen-coated dish for 5 and 10 days, respectively. AFP, alphafetoprotein.