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NamJu Kim,Ji-Hyun Bang,Hee Yi,Moon Her,Hyun-Ok Ku,Byung-Suk Jeon 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7
In vitro cytotoxicity testing has become an integral aspect of drug-induced toxicity study because it is a convenient, cost-effective and predictive means of characterizing the toxic potential of drugs. The present study aimed to investigate the drug-induced cytotoxicity and effects of the liver enzymes such as ALT, AST and LDH enzyme activity in human cell. However, there are no reports of a comparison of ALT, AST, and LDH enzyme activity after hepatotoxicants treatment in 2D and 3D cell cultures. Therefore, the purpose of this study is to investigate the difference in expression of liver injury factors after treatment with hepatotoxicants in 2D and 3D culture models. Cell cytotoxicity and liver injury factors were confirmed by human induced-pluripotent stem cells. The expression of the liver injury factors were increasd, as results of the WST-1 and CellTiter decreased on concentration-dependently. In conclusion, the expression of liver injury factors were found to be higher in 2D cultures. However, it was confirmed that the reactivity was different depending on the cell death mechanism of the drugs.
NamJu Kim,Ji-Hyun Bang,Hee Yi,Moon Her,Hyun-Ok Ku,Byung-Suk Jeon 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7
In vitro Cell viability assay is one of the most fundamental tests in different form of cell culture, which tests the number of healthy cells in a sample. Also, cell viability assay has become an integral aspect of drug-induced toxicity study because it is a convenient, cost-effective, and predictive means of characterizing the toxic potential of drugs. Recently, utilization of induced-pluripotent stem cells (iPSc) and multistep hepatic differentiation recapitulating in vivo hepatic development opens the new era in medicine and pharmaceutical industry. The aim of this study was to compare the in vitro cytotoxicity assay and the ability to detect early cytotoxic events in 2D and 3D cultures of induced pluripotent stem cells. Human iPSC (QIA7) was established from adipose tissue-derived stromal cells from our previous study. These cells were mechanically passaged using Dispase (STEMCELL Technologies, Canada) and maintained on a Matrigel (hESC-qualified matrix, Corning) coated plate with mTeSR1 medium (STEMCELL Technologies). The 2D culture cells were seeded at the density of 1.5 x 104 cells/well on 96-well matrigel-coated plates and the cells were incubated at 37°C in a 5% CO2 humidified incubator. The 3D cells were cultured under the same conditions as 2D cells, and the cells were seeded on 96-well U-bottom matrigel non-coated plate. After 24 hours post-seeding, cells were treated with Acetaminophen (AAP, 1, 3, 10, 30 mM) for 24 hours. Following to the drugs treated, cytotoxicity was determined with the 4-[3(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (Tetrazolium salts, WST-1), 7-Hydroxy-10- oxidophenoxazin-10-ium-3-one (Resazurin, Alamarblue) and Cell Titer-Glo<SUP>®</SUP> luminescence assay (Promega, Fitchburg, WI). Overall, it was concluded that the best way to evaluate the potential cytotoxicity of hepatotoxicants is CellTiter and WST-1 assays. CellTiter and WST-1 assay showed effect of reducing cell viability in 2D and 3D cultures depending on the concentration of the drug. Alamarblue assays showed significant results in 2D culture, but the reagent did not respond well in 3D cultures.