Viability and function in primary culture of adult hepatocytes from various animal species and human beings after cryopreservation.
Cryopreserved hepatocytes from various animal species and human beings were tested for their ability to survive and function in primary culture. The freeze/thaw protocol primarily designed for rat hepatocytes was used with slight modifications for the cells of all other species; it consisted of suspending parenchymal cells in the Leibovitz L15 medium containing 10% fetal calf serum and 10% to 16% dimethyl sulfoxide. After transient storage at 4 degrees C cell suspensions were transferred to -20 degrees C and then to -70 degrees C before being plunged in liquid nitrogen. Hepatocytes were stored for a few weeks to 4 yr. Prolonged storage did not augment loss of cell viability and function. Cell viability after thawing was estimated by the trypan blue exclusion test, and attachment efficiency to plastic was estimated by measuring intracellular lactate dehydrogenase content. Similar values were obtained for most species tested; after cryopreservation cell viability and attachment were decreased by 10% to 25% and by 40% to 50%, respectively. A lower attachment rate was found with dog hepatocytes. Total cytochrome P-450 and protein synthesis were compared in fresh and cryopreserved cells from four species after 4, 24, 48 or 72 hr of culture. Similar values were found in both cells after 24 or 48 hr of culture. In addition, drug-metabolizing activities were measured in human hepatocytes from five donors. In most cases phenacetin deethylation activity was decreased whereas procainamide N-acetylation and paracetamol sulfoconjugation and glucuronidation were increased in cryopreserved cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Chesné
C
C
Unité de Recherches Hépatologiques, INSERM U 49, Hôpital de Pontchaillou, Rennes, France.
Guyomard
C
C
Fautrel
A
A
Poullain
M G
MG
Frémond
B
B
De Jong
H
H
Guillouzo
A
A
D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D015925:Cryopreservation; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004285:Dogs; D006801:Humans; D008099:Liver / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D008252:Macaca fascicularis; D051379:Mice; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D014343:Trypan Blue
D004364:Pharmaceutical Preparations; D011506:Proteins; D003577:Cytochrome P-450 Enzyme System; D014343:Trypan Blue; D004121:Dimethyl Sulfoxide