Development of Culture system for LAK cells and its application to adoptive immunotheraphy for lung cancer.

用于癌症的手术辅助治疗。我们设计了一种独特的采用淋巴因子激活的杀伤细胞（LAK）和重组白细胞介素-2（IL-2）的过继免疫治疗方案。该方案的特点是长时间（连续6天）皮下施用低剂量IL-2和从手术时获得的区域淋巴结淋巴细胞转移离体产生的LAK细胞。根据这个养生法。19例病理Ⅰ期肺癌患者于肺叶切除术后2周左右接受免疫治疗。临床毒性包括发热。疲劳体重增加<5%。所有毒副反应均在治疗后4天内逆转。13例患者。治疗后24小时取外周血淋巴细胞，体外培养显示LAK活性。因此，从患者术后情况来看，该方案耐受性好，具有免疫活性。我们开发了一种新的培养系统（LIFROC液体填充旋转培养：正式命名为JCC装置）。LIFROC系统基本上基于透析培养。透析膜将培养容器与基础培养基的外部循环分开。旋转培养容器以使细胞适当分散，并通过硅管提供氧气。使用该LIFROC系统。细胞密度达到最大值2.7 × 10^7个细胞/ml，具有大于90%的存活率和相当水平的LAK活性。与传统的组织培养皿分批培养系统进行了比较。LIFROC系统能够减少基础培养基的消耗。白细胞介素2。培养容器、贮液器和从培养液中回收LAK细胞的工具均设计成一次性使用。该培养体系可安全地用于过继免疫治疗的LAK细胞培养。

For application with surgical adjuvant therapy of cancer. we have designed a unique regimen of adopuve immunotherapy with lymphokineacuvated killer (LAK) cells and recombinant interleukin-2 (IL-2). The regimen features the prolonged (6 consecutive days) subcutaneous administration of a low-dose IL-2 and the transfer of ex vivo generated LAK cells from regional lymph node lymphocytes, obtained at the time of operations. According to this regimen. 19 patients with pathological stage-1 lung cancer received immunotheraph about 2 weeks after pulmonary lobectomy. Clinical toxicities included fever. fatigue. <5% weight gain. and edema.All toxicities reversed within 4 days after the complection of therapy. In 13 patients. peripheral blood lymphecytes obtained 24h after therapy exhibited in vitro LAK activity. Thus the regimen is considered to be well-tolerable and immunologically active, in regard to the postoperative state of the patients.To improve the routine performance of the generation and expansion of LAK cells. we have developed a new culture system (LIFROC Liquid-Filled Rotary Culture : formaly designated JCC device). The LIFROC system is essentially based on dialysis culture. A dialysis membrane divides the culture vessel from the outside circulation of basal medium. The culture vessel is rotated for the proper dispersion of cells and supplied with oxygen through a silicon tube.With this LIFROC system. the cell density reached a maximum 2.7x10^7 cells/ml with greater than 90% viability and a substantial level of LAK activity. In comparison with a convenstonal batchwise culture system suing tissue culture dishes. the LIFROC system was able to reduce the consumption of basal medium. IL-2. and serum by 20%, 84%, and96%, respectively.Culture vessel, reservoir, and tools for the recovery of LAK cells from culture medium were devised to be disposable. This culture system is apphed with safe to culture LAK cells for adoptive immunotheraphy.