Kedar E, Ikejiri B L, Bonnard G D, Herberman R B
Eur J Cancer Clin Oncol. 1982 Oct;18(10):991-1000. doi: 10.1016/0277-5379(82)90248-6.
A rapid and simple technique for the isolation of viable tumor cells from human and mouse solid neoplasms is described. It consists of a 5 to 10-min treatment with trypsin-collagenase-DNase mixture, followed by mechanical disaggregation of the tumor tissue and subsequently by a brief centrifugation on a discontinuous Percoll gradient. With the tumors employed, this procedure usually requires less than 1 hr and results in preparations comprising greater than 80% tumor cells with viability of 80-90%. Cell-mediated cytotoxic response was measured with: (a) unsensitized lymphocytes freshly obtained from tumor-bearing hosts; (b) lymphocytes propagated in culture with T cell growth factor; and (c) lymphocytes stimulated in cocultures with autologous or syngeneic tumor cells. The cytotoxic activity was assessed in a modified [51Cr]-release assay adapted for solid tumor cells, allowing a long incubation period (24 hr) and the use of a low number (200-1000) of highly labeled target cells (2-10 counts/min/cell).
本文描述了一种从人和小鼠实体瘤中分离活肿瘤细胞的快速简便技术。该技术包括用胰蛋白酶 - 胶原酶 - DNA酶混合物处理5至10分钟，随后对肿瘤组织进行机械解离，然后在不连续的Percoll梯度上进行短暂离心。对于所使用的肿瘤，该过程通常需要不到1小时，得到的制剂中肿瘤细胞含量超过80%，活力为80 - 90%。细胞介导的细胞毒性反应通过以下方法测量：(a) 从荷瘤宿主新鲜获取的未致敏淋巴细胞；(b) 用T细胞生长因子在培养中增殖的淋巴细胞；(c) 与自体或同基因肿瘤细胞共培养刺激的淋巴细胞。细胞毒性活性在一种改良的适用于实体瘤细胞的[51Cr]释放试验中进行评估，该试验允许长时间孵育（24小时）并使用少量（200 - 1000）高标记的靶细胞（2 - 10计数/分钟/细胞）。
