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 <0.05) than that determined for subconfluent monolayered cells simultaneouslv exposed to the drug. This system offers an alternative to study the chemosensitivity of three-dimensionally organized cells using semiautomated microtiter plate technology.