Phase I and II metabolism and carbohydrate metabolism in cultured cryopreserved porcine hepatocytes.
Primary porcine hepatocytes were cryopreserved using freezing boxes or a programmable freezer (PF). Upon thawing and culturing in 12-well plates cryopreserved hepatocytes were compared with their fresh controls on days 1 and 2 after plating. Cryopreserved hepatocytes attached approximately as well as fresh hepatocytes and useful cultures were obtained. In cryopreserved hepatocytes, coumarin 7-hydroxylation, 6beta-testosterone hydroxylation and p-nitrophenol glucuronidation were reduced to about 10-40, 35 and 40%, respectively, compared to their fresh counterparts. Glycogen synthesis in cryopreserved hepatocytes was reduced to about 30% on day 1 of culture and about 47% on day 2 of culture compared to the synthesis in fresh hepatocytes. Both fresh and cryopreserved hepatocytes increased the synthesis by twofold in response to stimulation with insulin. Reduced basal levels of glycogen and of glycogen synthesis could be explained by an increased energy demand in cryopreserved hepatocytes needing to repair damages caused by cryopreservation. Glycogenolysis was reduced to about 50% in cryopreserved hepatocytes and gluconeogenesis to about 40% of the glucose production in fresh hepatocytes. In both fresh and cryopreserved hepatocytes the glucose production from glycogenolysis and gluconeogenesis, respectively, was increased fourfold in response to stimulation with glucagon. Overall, the hepatocytes cryopreserved in boxes had a tendency to perform better than hepatocytes cryopreserved in a programmable freezer. In conclusion, the cryopreserved hepatocytes were metabolic active; however, to a lower extent than the fresh hepatocytes, although, the cryopreserved hepatocytes responded as well as the fresh hepatocytes to insulin and glucagon.
Loven
Anne-Dorthe Bonde
AD
Department of Veterinary Pathobiology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark. adhl@mail.tele.dk
Olsen
Anette Kristensen
AK
Friis
Christian
C
Andersen
Birgitte
B
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D051544:Cytochrome P-450 CYP3A; D005260:Female; D005947:Glucose / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism*; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013552:Swine
D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C029009:steroid hormone 7-alpha-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D014453:Glucuronosyltransferase; D005947:Glucose