Influence of the isolation method on the stability of differentiated phenotype in cultured rat hepatocytes.
Primary cultures of adult rat hepatocytes were established using two different isolation procedures: a two-step collagenase perfusion and a method using ethylenediaminetetraacetate (EDTA) as the dissociating agent. Both techniques provided good yields of hepatocytes with comparable viability. The evolution of hepato-specific protein levels and several drug-metabolizing enzyme activities were followed for 8 days in cultured hepatocytes obtained by both methods. EDTA-isolated hepatocytes maintained a low gamma glutamyltransferase (GGT) activity, whereas collagenase-treated cells acquired a high GGT level. Transferrin secretion and tyrosine aminotransferase (TAT), alanine aminotransferase (ALT), and microsomal epoxide hydrolase (mEH) activities were stable in both EDTA- and collagenase-isolated hepatocytes, whereas albumin secretion, aspartate amino transferase (AST) activity, total cytochromes P-450 content, IA1 and IIB1 P-450 isoenzymes, NADPH-cytochrome P-450 reductase (EC 1.6.2.4) levels, and bilirubin glucuronidation decreased faster in collagenase-treated cells. The most important difference observed was the maintainance of the mixed-function oxidase system in EDTA-isolated hepatocytes. These results emphasize the critical role of isolation technique in stabilization of differentiated hepatocytes in primary culture.
Bayad
J
J
Centre du Médicament, URA CNRS 597, Faculté des Sciences Pharmaceutiques et Biologiques, Nancy, France.
Sabolovic
N
N
Bagrel
D
D
Magdalou
J
J
Siest
G
G
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D004492:Edetic Acid / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D003012:Microbial Collagenase / Q000494:pharmacology; D010641:Phenotype; D011519:Proto-Oncogenes; D051381:Rats; D011919:Rats, Inbred Strains
D004492:Edetic Acid; D003012:Microbial Collagenase