Optimisation of porcine hepatocyte cryopreservation by comparison of viability and enzymatic activity of fresh and cryopreserved cells

Abstract

Cryopreservation of porcine hepatocytes would ensure the accessibility of cells for laboratory use, permit the standardisation of experiments and save lives of animals. Therefore, in this study, we sought the optimal procedure for cryopreservation of porcine hepatocytes for both laboratory and clinical purposes. Hepatocytes were isolated from the liver lobe of a mini-pig by two-step collagenase perfusion. The cells were frozen with 20% foetal calf serum and 15% DMSO in two different media in four different concentrations ranging from 1 × 106 cells/ml to 5 × 106 cells/ml. For this purpose, 1.8 ml cryotubes and 120 ml Baxter bags were used. Cells were cryopreserved either in a controlled freezer Sylab or step by step in a styro-foam box and stored at -196°C. The quality of fresh and cryopreserved hepatocytes was assessed by trypan blue exclusion test and by the evaluation of cytochrome P450 isoenzymes and glutathione-S-transferase activities; primary cultures were evaluated morphologically and by MTT test. Cryopreserved hepatocytes did not form the typical monolayer of polygonal cells in primary cultures and remained round, unlike fresh hepatocytes. Lifetime of viable culture was shortened from 7-8 days to 4 days in cryopreserved cells. Viability of fresh cells was 88 ± 2% and decreased to 36-63% in cryopreserved hepatocytes. Enzyme activities of cryopreserved cells were reduced to 60% when compared with fresh hepatocytes. Concentrations of 3 × 106 cells/ml and 5 × 106 cells/ml and controlled freezing gave the best results. The use of Baxter bags was more convenient due to easier manipulation. Freezing media appeared to have no influence.