A modified method of isolation and primary culture of rat hepatocytes

Objective To explore and modify the method of isolation and primary culture of rat hepatocytes. Methods Six-well plates were pre-coated with collagen m about 24 h before use. Rat hepa- tocytes were isolated by a modified two-step collagenase perfusion technique in situ, seeded in eligible den-sity,and cultured in RPMI 1640 medium containing 10% fetal bovine serum. The yield,viability,attach-ment and growth of hepatocytes were observed to estimate the feasibility. Results The isolation time of rat hepatocytes was shortened obviously. One-third of collagenase IV was saved. About(1.81±0.65)x 108 hepatytes were obtained from every rat,and the viability was (87.46±6.90 )%. The cells attached to the plates about 4 h after seeded ,grew well and had a two-to three-week survival. Conclusion The modi-fied method is economical,and less labor and time for isolation and culture of primary rat hepatocytes. The yield, viability and attachment rate are high enough to be used for various experiments. Key words: Hepatocytes;  Collagenases ;  Cell culture