Multiparametric characterization by flow cytometry of flow-sorted subpopulations of a human hepatoma cell line useful for drug research.
BACKGROUND
Primary cultured hepatocytes are the closest model to the liver for drug research. However, to overcome its limited availability, the search for hepatic cell lines as an alternative to primary cultures is a matter of current interest. In particular, highly differentiated hepatocellular carcinomas have been proposed as in vitro tools for routine experiments in hepatotoxicity and drug metabolism.


METHODS
Cell populations were selected by fluorescence-activated cell sorting based on low and high relative expressions of P-glycoprotein. These cell lines were characterized after 21 days in culture by multiparametric analysis with flow cytometry providing direct information on key cellular functions (stability in culture, intracellular ionic homeostasis, plasmatic and mitochondrial membrane-related parameters, red-ox status, drug transport, and metabolism).


RESULTS
Two subpopulations (ADV-1 and ADV-2) from the differentiated and well-characterized human hepatoma BC2 cell line showed increased activity of drug transport and drug biotransformation capability (cytochrome P450 [CYP] 1A2, CYP2B6, CYP3A4, and CYP2Cs). These subpopulations were characterized extensively by multiparametric flow cytometric analysis.


CONCLUSIONS
ADV-1 subpopulation showed greater stability in culture, better efficiency regarding intracellular pH maintenance through the operation of Na+/H+ exchange antiporter, and significantly greater CYP-dependent biotransformation activity than the BC2 parental cells and ADV-2 cells.
O'Connor
José-Enrique
JE
Centro de Citometría y Citómica, Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Martínez
Alicia
A
Castell
José V
JV
Gómez-Lechón
María-José
MJ
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism; D000975:Antioxidants / Q000378:metabolism; D001692:Biological Transport; D001711:Biotransformation; D002118:Calcium / Q000378:metabolism; D006528:Carcinoma, Hepatocellular; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D008113:Liver Neoplasms; D010084:Oxidation-Reduction; D017382:Reactive Oxygen Species / Q000378:metabolism; D020112:Rhodamine 123 / Q000493:pharmacokinetics; D012964:Sodium / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000975:Antioxidants; D002412:Cations; D005456:Fluorescent Dyes; D007527:Isoenzymes; D017382:Reactive Oxygen Species; D020112:Rhodamine 123; D003577:Cytochrome P-450 Enzyme System; D012964:Sodium; D002118:Calcium