Cryopreserved porcine hepatocyte cultures.
Cryopreservation of freshly isolated hepatocytes is regarded the standard technique for long term storage of liver cells. Frankly, we were not successful in reproducing viability rates of about 70% of that which have been reported by most authors as results of various freezing protocols for hepatocyte suspensions. In fact, we saw mostly devastating results. We assume that intracellular ice crystal formation as well as osmotic changes during freezing and thawing of liver cells cause hazardous effects, especially on membranes of cells after enzymatic isolation, and, thus, generally result in a severe loss in number and impaired specific hepatocyte functions in subsequent culture. We tried to improve results by freezing cell cultures instead. We allowed hepatocytes to regain a more stable condition prior to storage and placed them in tissue flasks in a uniform configuration, rather than to pack cell suspensions in vials or bags under rather indefinable conditions. Porcine hepatocytes (viability 92+/-2%) were isolated from slaughterhouse organs and cultured in a double gel (sandwich) configuration. At day 3, cultures were rate controlled frozen (RCF) and stored in a cell bank for three hours (Group A) or 14 days at -80 degrees C (Group B), respectively. Non-frozen cells (NF) and cultures subjected to a linear freezing rate of -10 degrees C/min (LFR, Group C) with 3 h of storage served as controls from identical cell batches. Upon thawing, at day 2 of subsequent culture, fluorescence microscopy studies revealed a survival rate of 75+/-10% (mean+/-S.D.) in the RCF groups. Time of storage (3 h, 14 d) did not influence results. Survival in Group C was 10+/-5%. Cell integrity was measured by LDH-release, which indicated a larger damage of cells in the LFR group, and thereby resembled the morphological findings. Functional parameters, such as albumin synthesis and CYT P 450-activity were comparable to non-frozen liver cells at 48 h after thawing in the RCF groups (A + B), and showed significantly higher levels in these groups as compared to the LFR Group (C). We recommend to freeze hepatocytes in culture in a rate controlled fashion rather than cell suspensions. This way a cell bank of cryopreserved hepatocyte cultures for batch controlled investigations can be easily obtained. This could ameliorate the availability of rare (human) cell material and might also improve the quality of data generated from in vitro experiments in hepatology or pharmacology/toxicology with liver cells from identical sources. It remains to be seen whether this technique might also be of value in hybrid bioartificial liver devices to make these systems become readily available upon clinical demand.
Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University of Munich, Germany. koebe_hg@compuserve.com
H G
HG
Koebe
B
B
Mühling
C J
CJ
Deglmann
F W
FW
Schildberg
D000418:Albumins / Q000378:metabolism; D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D009926:Organ Preservation / Q000379:methods*; D013552:Swine
D000418:Albumins; D003577:Cytochrome P-450 Enzyme System; D007770:L-Lactate Dehydrogenase