FUNCTIONAL RECOVERY OF PORCINE HEPATOCYTES AFTER HYPOTHERMIC OR CRYOGENIC PRESERVATION FOR LIVER SUPPORT SYSTEMS

The provision of an immediate supply of isolated porcine hepatocytes f or artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions, By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatm ent facilities, Additionally, cryopreservation provides an adequate pe riod for quality control testing to be completed prior to use of any s pecific cell lot, We are reporting a dual approach, namely the preserv ation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents wi th Chee's modified Eagle's culture media (CEM), collagenase isolated h epatocytes stored at 4 degrees C for 24 h maintained 80% of the initia l diazepam metabolism measured in freshly isolated cells and nearly 10 0% of initial function a as preserved in hepatocytes stored up to 6 mo at -196 degrees C, University of Wisconsin solution (UW) was also tes ted and while adequate for 4 degrees C storage, it certainly did not m atch the performance of the CEM formulations for preservation of metab olic function of cells stored in liquid nitrogen, Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN 2 frozen cells, Other findings in these studies underlined the importa nce of the gradual introduction of DMSO in the prefreezing process, th e period of osmotic equilibration, and the rapid postthaw withdrawal o f this agent to minimize cytotoxic effects at these critical stages. O ur freezing methodology provides the foundation for further technologi cal developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices . (C) 1997 Elsevier Science Inc.