Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests

We investigated the feasibility of hypothermic- or cryogenically-preserved human hepatoma Hep G2 cell precultured in 96-well plates in cytotoxicity te stings. First, we observed that microplates precoated with both collagen (C N) and pronectin (PN) showed significantly improved living cell adhesion (7 1.0 +/- 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing cult ure medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 +/- 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at -20 degrees C allowed cells to be us ed within a week of the storage period. Only cryopreservation in a deep fre ezer (-85 degrees C) gave satisfactory results in much longer period of sto rage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr o f exposure using hypothermically - (4 degrees C for 2 days) or cryogenicall y - (-85 degrees C for 7 days) preserved cells cultured in CN/PN-precoated microplates in comparison with results from freshly inoculated cells. Altho ugh almost the same LD50 values were obtained, LD10 values of relatively hy drophilic chemicals obtained with cryopreserved cell were significantly low ered. These results shown that CN/PN-precoating is effective in keeping cel ls attached even in recultivation of preserved cells and that the toxicitie s of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.