An experimental study on a chemosensitivity test with alginate microcapsule. Feasibility of in vivo succinic dehydrogenase inhibition test

A new chemosensitivity test was evaluated by the MTT colorimetric assay with human tumor cell lines encapsulated in alginate microcapsules with semipermeable membranes. The proliferation of KATOIII in the microcapsules rapidly increased on the 4th day affer the encapsulation. The change expressed on the proliferation curve of the encapsulated KATOIII was approximately 2 days behind the proliferation of the suspension culture. The encapsulated cell number reversed and further proliferation was recognized after the 12th day. After the incubation for 5 hours of encapsulated KATOIII with the medium supplemented with 0.5% MTT, a blue formazan crystal formation was observed radiating around the cells in the capsules. MTT assay depends on the cellular reduction of the absorbance spectra at 540 nm (OD(540nm)), for complete solubilization of the formazan by DMSO. The formazan formation was observed more significantly in serum medium culture than in serum free medium. In MIT assay when 0.1 mol succinic acid was added, OD(540nm) of encapsulated KATOIII increased by approximately 50% and its sensitivity also increased greatly. In comparison the results of MTT assay for encapsulated KATOIII and MKN28 with suspended cells under the same conditions (0.1, 1, 10 μg/ml of MMC and ADR, 0.5, 5, 50 μg/ml of 5FU, 10, 30, 50 μg/ml of CDDP), the calculated inhibition index (%) with encapsulated cells were similar to the percentages obtained in the former MTT assay. In this study with microcapsules, the formazan formation in the capsules and the absorbance were macroscopically inhibited when the drug concentration was increased. The encapsulated KATOIII, which was implanted intraperitoneally into rat with a 16-gauge needle, was recovered at a rate of 70.8% on the 8th day and at a rate of 54.5% on the 16th day. The recovered encapsulated KATOIII proliferated remarkably forming cell clots on the 8th day after implantation. Incubation with MTT promoted formazan formation and sufficient cell viability was recognized. The Tegafur concentration in the intraperitoneal microcapsules and the microcapsules containing KATOIII after the intravenous administration of Tegafur was similar to the intrahepatic level. The 5FU level in the microcapsules containing KATOIII was higher than that in the capsules alone. In an attempt to conduct an in vivo chemosensitivity test, encapsulated KATOIII and MKN28 were intraperitoneally implanted, 4 mg/kg of MMC, ADR and CDDP, and 75 mg/kg of 5FU were intravenously administered on the 2nd and 4th days after the implantation. On the 6th day, MTT assay was performed on the recovered microcapsules containing cells and the inhibition index was calculated. The respective inhibition index for encapsulated KATOIII was as follows; MMC (56.2%), ADR (69.5%), 5FU (38.6%), CDDP (67.2%), for encapsulated MKN28; MMC (69.7%), ADR (52.4%), 5FU (69.7%), CDDP (59.0%). It was easy to heteroimplant encapsulated human tumor cells by needle and syringe into rats and there was no immunological rejection. If was possible to perform MTT assay on recovered encapsulated cells from the intraperitoneum without rupture of the capsules. Therefore it is suggested that a new in vivo succinic dehydrogenase inhibition test has been developed using alginate microencapsulated target cells.