Cryopreservation of Rat Hepatocytes for the Use of Primary Culture

Jeong, Yong Hee; Kim, Byung Ho; Chang, Rin; Lee, Byoung Wook; Han, Yo Seb; Dong, Seok Ho; Kim, Hyo Jong; Chang, Young Woon; Lee, Joung Il

Abstract

BACKGROUND/AIMS: The cryopreservation of primary hepatocytes could avoid unnecessary isolation of hepatocytes and supply them on demand. We compared two programs of computer-controlled freezing methods on the efficacy of hepatocyte cryopreservation. METHODS: Rat hepatocytes were cryopreserved by computer-controlled freezing methods. In program I, the overall cooling rate was 1.33degrees C/min and single-step shock cooling was used to reduce the latent heat of fusion. In program II, the cooling rate of 2degrees C/min and two-step shock cooling were used. Two programs were compared in regard to hepatocyte viability and long-term cryopreservation and thawing temperature were also evaluated. RESULTS: The hepatocyte viability showed the highest value of 74.5+/-2.5% when cryopreserved using the program II and at 2 X 106/mL cell concentration in cryopreservation medium. The hepatocyte attachment rate on culture was similar in every occasion, more or less 50%. The hepatocyte viability was improved by 10% when thawed at 40degrees C, compared to the value at 37degrees C in the program I. The hepatocyte viability was decreased to 38.2+/-5.5% in the program I and 45.4+/-1.6% in the program II after long-term cryopreservation. CONCLUSIONS: The program II showed better survival of hepatocytes at 2 X 10(6)/mL cell concentration. However, overall efficacy of hepatocyte cryopreservation was less than 35% and decreased more after long-term cryopreservation. Further studies are needed to develop a more effective program for hepatocyte cryopreservation.