Morphology, viability and functions of suckling pig hepatocytes cultured in serum-free medium at high density.
BACKGROUND
In bioartificial liver preparation, serum-contained medium is ordinarily replaced by serum-free medium and hepatocytes are generally cultured at high density. This study was to undertaken to evaluated the dynamic changes in morphology, viability and functions of porcine hepatocytes in serum-free medium at high density.


METHODS
Hepatocytes were isolated from suckling pigs by modified two-step in situ collagenase perfusion method and cultured in serum-free medium at high density. Morphology, viability, protein and glucose syntheses, G-6-Pase activity, diazepam transformation of hepatocytes and release of LDH in supernatant during 7 days of culture were evaluated. These measurements were also determined on both groups of hepatocytes cultured at low-density in serum-free medium and serum-contained medium, which served as control groups.


RESULTS
Morphology and protein synthesis of hepatocytes cultured in serum-free medium at high density were stable over the course of 7 days. High viability (>90%) was obtained though it declined with time. Diazepam transformation of cells was higher on days 2 and 3. Glucose synthesis of cells declined from day 3 to day 7. G-6-Pase activity of the hepatocytes declined apparently after 1 day of culture and it was maintained at a low level from day 1 to day 7. Release of LDH in supernatant was higher on days 1, 2 and 3. There were no significant differences in viability and functions of hepatocytes except for G-6-Pase activity at low-density culture between the serum-free medium group and the serum-contained medium group. The functions of hepatocytes cultured at high density were lower than at low-density culture.


CONCLUSIONS
The results showed that the morphology, viability, protein synthesis and diazepam transformation of hepatocytes cultured in serum-free medium at high density were maintained during 7 days of culture. The serum-free medium provided indices of cell viability and functions that were comparable to serum-contained medium. The functions of hepatocyte cultured at high density (1 x 10(7) cells/ml) were lower than at low density (5 x 10(5) cells/ml).
Chen
Zhong
Z
Department of Hepatobiliary Surgery, Affiliated Drum Tower Hospital, Medical School, Nanjing, PR China. zchen9999@sina.com
Ding
Yitao
Y
Zhang
Heyun
H
D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003470:Culture Media; D003975:Diazepam / Q000494:pharmacology; D005947:Glucose / Q000096:biosynthesis; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D014176:Protein Biosynthesis; D013552:Swine
D003470:Culture Media; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D005947:Glucose; D003975:Diazepam