Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing

Abstract

Several cryopreservation protocols for hepatocytes have been proposed over the past few years, but their effectiveness varies greatly as a function of the characteristics of the method used. One factor in the success of cryopreservation is the quality of cells before freezing. The results suggest that the cryopreservation of hepatocytes in a medium containing polyvinylpyrrolidone (PVP), in addition to DMSO, constitutes a convenient means of long-term storage of hepatocytes for preparing primary cultures to be used in drug metabolism studies. The combined use of the two cryoprotectants is particularly critical for low-viability cell suspensions. An interesting alternative to increase cell viability is the preculture of hepatocytes before cryopreservation. By the use of this procedure, high-quality cells, estimated in terms of post-thaw recovery, viability, adaptation of hepatocytes to culture, drug-metabolizing capability and cytochrome P450 induction, are obtained. Therefore, cryopreserved hepatocytes can provide a regular source of metabolically competent cells for in vitro investigations of the metabolic profile of new drugs and drug–drug interactions in pharmaco-toxicological research.

Key words::

• Cryopreservation
• cytochrome P450
• hepatocytes
• cytochrome P450 induction
• polyvinylpyrrolidone
• preculture

Acknowledgements

The authors would like to express their thanks to E. Belenchón for expert technical assistance. The present research was supported by grants from the ALIVE Foundation, Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III of Spain (FIS 03/0339), and the European Union (LSHB-CT-2004 512051).