Examination of in vitro Chemosensitivity Test Using Collagen Gel Droplet Culture Method with Colorimetric Endpoint Quantification

Abstract

To develop a simpler method of performing the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), we examined the introduction of colorimetric quantitative determination of images for evaluation of anticancer effect against cancer cells alone in the presence of fibroblasts, based on 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 fibroblast cell line, a high degree of linearity between number of cancer cells and image-optical density was found within the range of 10 2 -10 6 cells/droplet (r 2 = = = =0.933). Using NIH Image, fibroblast cells could be eliminated at a cut-off value of 128, and an immunocytochemical method demonstrated that the cells eliminated from the image were indeed fibroblasts, and those remaining were cancer cells. CD-DST was carried out with mixtures of cancer cells with fibroblasts at various ratios, and the feasibility of evaluating anticancer activity in cancer cells alone with no effect of fibroblasts at any mixing ratio was confirmed. In addition, for CD-DST of primary cell cultures of human lung cancers collected at the time of surgery, a high correlation between results obtained with the volume supplementation method, a current cell quantification method, and those with the imaging colorimetric quantification method was obtained (r = = = =0.933). These results indicate that introduction of imaging colorimetric quantification utilizing NIH Image makes CD-DST a quick and simple method that should be highly useful for clinical chemosensitivity testing using primary cell cultures of human cancers.

Three-dimensional cellular microarray for high-throughput toxicology assays

“…with more conventional plate assays, the accuracy of cytotoxicity data is not adversely affected. Finally, the stamping procedure is performed for 6 h; however, for more conventional growthinhibition studies, the drug or drug candidate remains in contact with the cells for anywhere from 1 to 7 days (23,(26)(27)(28)(29). Thus, the DataChip enables rapid cellular uptake that results in sufficient exposure to deliver a representative cytotoxic dose of drug.…”

Impact of the individualization of the first‑line chemotherapy for advanced colorectal cancer based on collagen gel droplet‑embedded drug sensitivity test

“…Tumor tissue was excised from primary surgical specimens and subjected to the CD-DST. The CD-DST allows for the evaluation of drug sensitivity using isolated 3-dimensionally cultured tumor cells in a small collagen gel droplet, and was used to evaluate the sensitivity of the tumors to 5-FU, which was performed according to a previous description by Kobayashi et al (17,18). Each specimen was washed 5 times with 50 ml saline, followed by additional washing 5 times with 50 ml antibiotic fluid containing 1.0 mg/ml piperacillin and 0.5 mg/ml kanamycin.…”

Parkin regulates paclitaxel sensitivity in breast cancer via a microtubule‐dependent mechanism

“…Paclitaxel was added at a concentration of 10 µg/ml and incubated for 24 h. After removal of the medium containing paclitaxel, each well was overlaid with PCM-2 medium (Nitta Gelatin), and the cells were incubated for 7 days. At the end of the incubation, neutral red was added to each well and colonies in the collagen gel droplets were stained for 2 h. Each collagen droplet was fixed with 10% neutral formalin buffer, air-dried and quantified by image analysis, as described previously [17]. Paclitaxel sensitivity was expressed as the T : C ratio, where T was the reading of the treated group and C was the reading of the control group; a T : C ratio of 50% or less was regarded as being sensitive.…”