Chin K, Shimizu K, Shoji T
Second Department of Surgery, Nippon Medical School First Hospital, Tokyo, Japan.
Nihon Ika Daigaku Zasshi. 1994 Oct;61(5):422-34. doi: 10.1272/jnms1923.61.422.
A new chemosensitivity test was evaluated by the MTT colorimetric asay 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 after 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 (OD540nm), 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, OD540nm 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 micrograms/ml of MMC and ADR, 0.5, 5, 50 micrograms/ml of 5FU, 10, 30, 50 micrograms/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 75mg/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.(ABSTRACT TRUNCATED AT 400 WORDS)
采用MTT比色法对一种新的化学敏感性试验进行评估，该试验使用包裹在具有半透膜的海藻酸盐微胶囊中的人肿瘤细胞系。封装后第4天，微胶囊中KATOIII的增殖迅速增加。封装的KATOIII增殖曲线上的变化比悬浮培养的增殖滞后约2天。封装细胞数在第12天后出现逆转并进一步增殖。用补充有0.5%MTT的培养基孵育封装的KATOIII 5小时后，观察到在胶囊中的细胞周围有蓝色甲臜晶体形成。MTT试验取决于细胞在540nm（OD540nm）处对吸收光谱的还原，以便用二甲基亚砜完全溶解甲臜。在血清培养基培养中比在无血清培养基中观察到更明显的甲臜形成。在MTT试验中，当加入0.1mol琥珀酸时，封装的KATOIII的OD540nm增加约50%，其敏感性也大大提高。在相同条件下（0.1、1、10微克/毫升的丝裂霉素C和阿霉素、0.5、5、50微克/毫升的5-氟尿嘧啶、10、30、50微克/毫升的顺铂）对封装的KATOIII和MKN28与悬浮细胞进行MTT试验比较，封装细胞的计算抑制指数（%）与前一次MTT试验获得的百分比相似。在这项关于微胶囊的研究中，当药物浓度增加时，胶囊中的甲臜形成和吸光度在宏观上受到抑制。用16号针头将封装的KATOIII腹腔内植入大鼠体内，第8天回收率为70.8%，第16天回收率为54.5%。回收的封装KATOIII在植入后第8天显著增殖形成细胞凝块。用MTT孵育促进了甲臜形成，并确认有足够的细胞活力。静脉注射替加氟后，腹腔内微胶囊和含有KATOIII的微胶囊中的替加氟浓度与肝内水平相似。含有KATOIII的微胶囊中的5-氟尿嘧啶水平高于单独的胶囊。为了进行体内化学敏感性试验，腹腔内植入封装的KATOIII和MKN28，在植入后第2天和第4天静脉注射4毫克/千克的丝裂霉素C、阿霉素和顺铂，以及75毫克/千克的5-氟尿嘧啶。第6天，对回收的含细胞微胶囊进行MTT试验并计算抑制指数。（摘要截断于400字）
