Serum-Free Cryopreservation of Primary Rat Hepatocytes in a Modified Cold Storage Solution: Improvement of Cell Attachment and Function

Aims: The use of primary hepatocytes in pharmacological and toxicological research as well as for clinical and biotechnological applications requires adequate storage options for these cells. However, hepatocytes are very susceptible to cryopreservation injury. Based on experience in hypothermic storage of hepatocytes, we, in this study, aimed to optimize hepatocyte cryopreservation. Materials and Methods: Rat hepatocytes were cryopreserved in serum-containing cell culture medium or in serum-free solutions optimized for hypothermic storage, all with 10% dimethyl sulfoxide, using a standard protocol (-1 degrees C/min in a controlled-rate freezer). After rapid thawing, cells were seeded in supplemented Leibovitz-15 cell culture medium without further purification steps. Cell attachment and metabolic activity were assessed. Results: Cell attachment (37%15% vs. 9%+/- 7% of noncryopreserved control cells) and metabolic activity (resazurin reduction: 47%+/- 23% vs. 25%+/- 8%; glucose release: 44%+/- 6% vs. 15%+/- 7%; and urea production: 31%+/- 16% vs. 5%+/- 8%) were significantly higher after cryopreservation in the new solution compared to cryopreservation in cell culture medium. Experiments with modified solutions suggested that the protective effect of the new solution is multifactorial. Conclusions: In summary, significant improvement of cell attachment and function compared to cell culture medium was achieved after cryopreservation in serum-free hepatocyte cold storage solution.