Improvement of human tumor cloning assay by suspension of fibroblasts into the bottom layer of agarose

Abstract

Twenty-nine human tumors were cultured on a soft agarose cloning assay under three conditions: (1) standard (control); (2) standard with varying numbers of mitomycin C-treated 3T3 Swiss mouse embryonic fibroblasts suspended into the bottom layer of agarose; and (3) standard with varying concentrations of conditioned medium derived from those same fibroblasts. Suspension of 1 X 10(5) fibroblasts into the bottom layer of agarose was found to significantly increase the number of colonies formed over control specimens, as did cultures with 30% conditioned medium. In addition, compared with control, both of these techniques increased the number of specimens which would allow optimal vitro chemotherapy sensitivity testing. Specifically, growth of at least 30 colonies per plate increased from 7% of specimens treated under control conditions to 36% and 52% of specimens treated with 30% conditioned medium and 1 X 10(5) fibroblast-supplemented agar, respectively. This data indicate that 3T3 Swiss mouse fibroblasts improve cloning efficiency when suspended in the bottom layer of agarose or when used to produce conditioned medium. As a consequence, these techniques may permit a better opportunity to define the role of the cloning assay for cancer chemotherapy.