Cryopreservation of suckling pig hepatocytes

To determine the best and simplest method for cryopreservation of pig hepatocytes, we compared immediate cryopreservation with cryopreservation after short-term culture. Suckling pig hepatocytes were isolated by a modified 2-step in situ collagenase perfusion method, suspended in serum-free medium, and preserved for 10 da by two cryopreservation methods. Serial measurements were made of cell viability, LDH release, synthesis of protein, urea and glucose, glucose-6-phosphatase (G-6-Pase) activity, and diazepam transformation after thawing. These measurements were performed on both groups of cultured hepatocytes, and on freshly isolated hepatocytes, which served as a control. High viability (>95%)of thawed hepatocytes was obtained and maintained in both cryopreservation groups. There were no significant differences in cell viability, protein synthesis, glucose synthesis, G-6-Pase activity, or diazepam transformation between the two cryopreservation groups. In the immediate cryopreservation group, urea synthesis was less than in the group with cryopreservation after short-term culture. Protein synthesis, glucose synthesis, and diazepam transformation were lower in both cryopreserved groups than in the controls. The results showed that a protocol of immediate cryopreservation of hepatocytes in RPMI-1640 medium containing 10% DMSO, hormones, growth factors, and 10% newborn bovine serum, together with rate-controlled freezing and rapid thawing, provides indices of cell viability and function during subsequent serum-free culture that are comparable to hepatocytes cryopreserved after short-term culture, except for lower urea production. This simple procedure can be used in studies of bioartificial liver and hepatocyte transplantation.