Functional assessment of the quality of human hepatocyte preparations for cell transplantation.
Hepatocyte transplantation is an alternative therapy to orthotopic liver transplantation for the treatment of liver diseases. Good quality freshly isolated or cryopreserved human hepatocytes are needed for clinical transplantation. However, isolation, cryopreservation, and thawing processes can seriously impair hepatocyte viability and functionality. The aim of the present study was to develop a fast and sensitive procedure to estimate the quality of hepatocyte preparations prior to clinical cell infusion. To this end, cell viability, attachment efficiency, and metabolic competence (urea synthesis and drug-metabolizing P450 activities) were selected as objective criteria. Viability of hepatocyte suspension was estimated by trypan blue staining. DNA content of attached cells 50 min after hepatocyte platting to fibronectin/collagen-coated dishes was quantified to estimate adherence capacity. Urea production was determined after incubating hepatocyte suspensions with 2 mM C1NH4 for 30 min. The cytochrome P450 function was assayed by a 30-min incubation of hepatocyte suspension with a cocktail mixture containing selective substrates for seven individual P450 activities (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1, and 3A4). The assay can be applied to both freshly isolated and cryopreserved hepatocyte suspensions, and the results are available within 1 h, which could help to make short-term decisions: 1) to assess the suitability for cell transplantation of a preparation of freshly isolated hepatocytes or a particular batch of thawed cells, or 2) to estimate the convenience of banking a particular cell preparation.
Donato
María Teresa
MT
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Valencia, Spain.
Lahoz
Agustín
A
Montero
Sandra
S
Bonora
Ana
A
Pareja
Eugenia
E
Mir
José
J
Castell
José V
JV
Gómez-Lechón
María José
MJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D002470:Cell Survival; D017690:Cell Transplantation / Q000379:methods; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism / Q000502:physiology* / Q000637:transplantation*; D006801:Humans; D008111:Liver Function Tests; D016031:Liver Transplantation / Q000379:methods; D008297:Male; D008875:Middle Aged; D011786:Quality Control; D013048:Specimen Handling / Q000379:methods; D014019:Tissue Donors; D014508:Urea / Q000378:metabolism; D055815:Young Adult
D014508:Urea; D003577:Cytochrome P-450 Enzyme System