J Korean Surg Soc.  2013 Apr;84(4):202-208. 10.4174/jkss.2013.84.4.202.

Successful mouse hepatocyte culture with sandwich collagen gel formation

• Choi HJ
• Choi D

• KMID: 2212464
• DOI: http://doi.org/10.4174/jkss.2013.84.4.202

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.

Keyword

Collagen; Culture; Hepatocyte

MeSH Terms

Adult
Animals
Bile Canaliculi
Collagen
Gels
Gene Expression
Glucose-6-Phosphatase
Hepatocyte Nuclear Factor 4
Hepatocytes
Humans
Longevity
Mice
Oxidoreductases
Tissue Donors
Tryptophan Oxygenase
Tyrosine Transaminase
Collagen
Gels
Glucose-6-Phosphatase
Hepatocyte Nuclear Factor 4
Oxidoreductases
Tryptophan Oxygenase
Tyrosine Transaminase

Figure

• 

  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.

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