title: Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices.
abstract: New assays were compared with tritiated thymidine (3HTdR) and trypan blue dye exclusion for evaluating hepatocyte viability on cytodex 3 beads and in microcapsules, the matrices used in bioartificial liver support systems. They were the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) 5 min qualitative assay and the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 -sulfophenyl)-2H -tetrazolium, inner salt (MTS) 1 h quantitative assay. Both tetrazolium salts are cleaved in active mitochondria, the reaction occurring, therefore, in living cells only. After bathing at 39 degrees C with MTT for 5 min, porcine or rat hepatocytes on cytodex 3 beads were detached by collagenase while those in microcapsules were released by citrate treatment or passage through a fine needle. Cell viability was determined directly by microscope. The MTT 5 min metabolic inclusion test and MTS 1 h quantitative assay results correlated closely with those obtained by the 3HTdR, trypan blue dye exclusion, and fluorescein diacetate (FDA) methods. Both the new assays are sensitive, accurate, simple, and time-saving.
authors.affiliation: Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital, Sydney, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: M A
authors.initials: MA
authors.lastname: Waring
authors.forename: C
authors.initials: C
authors.lastname: Wang
authors.forename: K K
authors.initials: KK
authors.lastname: Woodman
authors.forename: A G
authors.initials: AG
authors.lastname: Sheil
mesh_terms: D000818:Animals; D001672:Biocompatible Materials / Q000378:metabolism; D002469:Cell Separation; D002470:Cell Survival / Q000502:physiology; D017690:Cell Transplantation; D002478:Cells, Cultured; D003911:Dextrans / Q000378:metabolism; D005562:Formazans / Q000737:chemistry* / Q000378:metabolism; D008099:Liver / Q000166:cytology*; D019164:Liver, Artificial / Q000592:standards*; D008863:Microspheres; D008930:Mitochondria, Liver / Q000378:metabolism; D051381:Rats; D012984:Software; D013552:Swine; D013778:Tetrazolium Salts / Q000737:chemistry* / Q000378:metabolism; D013844:Thiazoles / Q000737:chemistry* / Q000378:metabolism; D013936:Thymidine; D014343:Trypan Blue / Q000378:metabolism
chemical_list: D001672:Biocompatible Materials; D003911:Dextrans; D005562:Formazans; D013778:Tetrazolium Salts; D013844:Thiazoles; C079782:MTT formazan; C052839:Cytodex; C022616:thiazolyl blue; D014343:Trypan Blue; D013936:Thymidine