Cryopreservation of Primary Hepatocytes for Repeated Investigational Uses

Dong, Seok Ho; Kim, Byung Ho; Sung, Se Ra; Kim, Young Il; Park, Jai Kyung; Kim, Kyeong Jin; Kim, Hyo Jong; Chang, Young Woon; Lee, Joung Il; Chang, Rin

Abstract

BACKGROUND/AIMS
The cryopreservation of primary hepatocytes could avoid unnecessary isolation of hepatocytes and meet repeated investigational demands. We tried to find out an optimal cryopreservation method of rat hepatocytes.
METHODS
Primary hepatocytes with more than 90% viability were cryopreserved with the manual or computer-programmable freezing method. We analyzed the effects of the composition (basal medium or fetal bovine serum, FBS) of cryopreservation media, cell concentration, and freezing method on cell viability.
RESULTS
Two-step addition of cryopreservation medium (4%--<16% DMSO) improved cell viability, compared to its one-step addition (82.7+/-2.5 vs 73.3+/-2.1%, p=0.008). In the manual method, the cell viability was about 60% and the culture attachment rate was less than 1%. They were not related with the composition of media used. These results showed that the manual method was not efficacious for cryopreservation. However, about 80% of the cell viability and 50% of the culture attachment rate could be obtained with an optimal computer-programmable method (-2C degrees slow cooling rate with a shock cooling, 2x106/mL cell concentration, 10-20% FBS, 10% DMSO). Moreover, the culture attachment rate was increased up to 75% when Percoll density purification was applied.
CONCLUSIONS
Primary hepatocytes can be effectively cryopreserved by an optimal computer-programmable freezing method and Percoll density purification, but not by the manual method.