Examination of in vitro chemosensitivity test using collagen gel droplet culture method with colorimetric endpoint quantification

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of co lorimetric quantitative determination of images for evaluation of anticance r effect against cancer cells alone in the presence of fibroblasts, based o n differences in proliferative morphology and stainability with neutral red of cells within collagen gel drops determined using a video-microscope and NIH Image software. In examinations using a human cancer cell line and a f ibroblast cell line, a high degree of linearity between number of cancer ce lls and image-optical density was found within the range of 10(2)-10(6) cel ls/droplet (r(2)=0.933). Using NIH Image, fibroblast cells could be elimina ted at a cut-off value of 128, and an immonocytochemical method demonstrate d that the cells eliminated from the image were indeed fibroblasts, and tho se remaining were cancer cells. CD-DST was carried out with mixtures of can cer cells with fibroblasts at various ratios, and the feasibility of evalua ting anticancer activity in cancer cells alone with no effect of fibroblast s at any mixing ratio was confirmed, In addition, for CD-DST of primary cel l cultures of human lung cancers collected at the time of surgery, a high c orrelation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimet ric quantification method was obtained (r=0.933). These results indicate th at introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for cli nical chemosensitivity testing using primary cell cultures of human cancers .