Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices.
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.
Sun
J
J
Australian National Liver Transplantation Unit, Royal Prince Alfred Hospital, Sydney, Australia.
Wang
L
L
Waring
M A
MA
Wang
C
C
Woodman
K K
KK
Sheil
A G
AG
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
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