In vitro chemosensitivity testing of multilayered microcultures.

A potential limitation of in vitro microtiter cytotoxicity assays as compared to in vivo antitumor studies is that the complex three-dimensional structure of the solid tumor is lost in monolayer cultures in vitro. We investigated whether more in vivo like cell-cell interactions could be easily and reproducibly obtained in an in vitro cytotoxicity assay. HT29 human colon adenocarcinoma cells were seeded in 96-well microtiter plates with "V"-shaped wells and allowed to form postconfluent multilayered cultures. Cross-sections of microcultures fixed after 2 and 3 weeks following plating revealed approximately 7 and 35 cell layers, respectively. Using a tetrazolium assay to assess cytotoxicity, the EC50 (drug concentration which gives absorbance readings 50% lower than those of non-treated wells) of multilayered cultures exposed to doxorubicin for 24 h was 12 times higher (p less than 0.05) than that determined for subconfluent monolayered cells simultaneously exposed to the drug. This system offers an alternative to study the chemosensitivity of three-dimensionally organized cells using semiautomated microtiter plate technology.