Strategies to improve cryopreservation of hepatocytes for use in drug metabolism and toxicity studies
Cryopreservation of hepatocytes would provide a readily available supply of cells for investigating drug metabolism and toxicity in vitro providing that the functional capacities of the cells are maintained at comparable levels to those found in freshly isolated cells. The aim of this thesis was to develop strategies to improve cryopreservation of hepatocytes in an attempt to maintain membrane integrity during incubation at 37oC, and to improve post thaw attachment. Initial optimisation studies showed no difference in post thaw viability when cells were cryopreserved in 10% (v/v) DMSO in FCS and L15VS25 (25% v/v) vitrification solution prepared in L15 medium. In addition, viability was also independent of the storage temperature used, the temperature of cryoprotectant addition/removal and the volume of cryoprotectant used. However, high speed centrifugation steps (10,000g) during the cryoprotectant removal processes, were detrimental to the percentage viability and recovery of cryopreserved cells. Reduction of the speed to 50g resulted in approximately 70% of the total cells cryopreserved being recovered, with an approximate viability of 35%. Attempts to increase initial viability by using Euro-Collins solution (electrolyte composition similar to intracellular fluid), as the aqueous component of the cryoprotectant (ECVS25) was successful in increasing the number of cells excluding Trypan Blue, compared with the use of tissue culture media (52% c.f. 38%). However such increases were not reflected in the ability of the cells to maintain membrane integrity during incubation at 37oC or to attach post thawing. In addition, no improvement was observed when the cells underwent Percoll centrifugation prior to incubation or culture. It was thought that the loss of viability observed following incubation of cryopreserved cells at 37oC may be the result of damage to the Na+K+ ATPase.