Cryopreservation of primary hepatocyte monolayers may provide a means of long-term storage of the cells for in vitro studies of xenobiotic metabolism and toxicity. Rat hepatocytes can be stored at −70 °C as simple monolayers attached to collagen-coated dishes, and post-thaw cultures can be continued for up to 72 h. Throughout this post-thaw period viability of the cells was demonstrated by retention of intracellular fluorescence after exposure to carboxyfluorescein diacetate (CFDA) and examination by confocal laser scanning microscopy (CLSM). CLSM images revealed an uneven distribution of CFDA-derived fluorescence within hepatocytes post-thaw, particularly in Williams' E medium, indicating generation and retention of carboxyfluorescein within the intracellular organelles. The membranes of the intracellular organelles appear to be less sensitive to freeze/thaw damage than the cell membrane. Viability was not compromised with storage for up to 28 days at −70 °C. Cytochrome P450 content was retained in post-thaw culture to a similar extent as in non-frozen cultures. Cryopreserved rat hepatocyte monolayers may provide a useful in vitro model for studying xenobiotic metabolism and toxicity.
- confocal microscopy