WAVE生物反应器应用于CIK细胞培养

WAVE bioreactors for cytokine induced killer cell culure

目的:探讨利用波浪(WAVE)生物反应器培养扩增肿瘤患者CIK细胞的效能及其对肿瘤细胞的杀伤活性。方法:分离8例肿瘤患者外周血单个核细胞,分别采用CIK细胞传统扩增技术(CIK组)和WAVE反应器培养方案(WAVE组),应用锥虫蓝染色并进行细胞计数,利用流式细胞术比较两组CIK细胞的扩增效率、活化情况及其亚群的组成变化,并以K562细胞为靶细胞检测扩增后CIK细胞的杀伤活性。结果:WAVE组细胞增殖数较CIK组明显提高(P<0.05或P<0.01);在第14天,WAVE组CD3^+CD8^+、CD3^+CD56^+细胞亚群比例明显高于CIK组[(78.56±2.99)%vs(74.54±3.02)%,(48.33±7.01)%vs(40.69±6.43)%,均P<0.05],而Treg细胞比例显著降低(P<0.05);当效靶比为10∶1、20∶1时WAVE组扩增的CIK细胞对K562的杀伤率明显高于CIK组(P<0.05或P<0.01),CIK细胞中CD3^+CD8^+CD107a^+的比例明显升高[(29.43±4.97)%vs(25.19±4.91)%,P<0.05]。结论:WAVE生物反应器扩增培养体系获得的CIK细胞数量多、纯度高,其中CD3^+CD8^+和CD3^+CD56^+NKT细胞比例较高,其对K562肿瘤细胞的杀伤效果明显高于传统培养技术获得的CIK细胞。

Objective：To investigate the efficacy of WAVE bioreactorin the culture and amplification of cytokine induced killer （CIK） cells and their killing activity on tumor cells. Methods： Peripheral blood mononuclear cells （PBMCs） from 8 cancer patients were isolated and cultured by using traditional CIK amplification method （CIK group） and WAVE bioreactors （WAVE group）respectively. Trypan blue staining was used for cell counting, and Flow cytomery was used to comparethe amplification efficiency, functionality and subtype compositionbetween two groups; K562 cell, as the target cell, was used to detect the killing activity of amplified CIK cells of two methods. Results： The proliferated number of CIK cells in WAVE groupwassignificantly higher than that of CIK group（P〈0.05 or P〈0.01）; the proportions of CD3＋CD8＋ cells and CD3＋CD56＋ cells in WAVE group were significantly higher than those in CIK group on Day 14 （[78.56±2.99]% vs [74.54±3.02]%, [48.33±7.01]% vs [40.69±6.43]%, all P〈0.05）; However, the proportion of Tregs cell was significantly decreased （P〈0.05） in WAVE group. Moreover, it was observed that CIK cells culturedin the WAVE bioreactor group displayed a significantly higher cytotoxic capacitythan that in CIK group at the E/T ratio of 10∶1 and 20∶1, and the proportion of CD3＋CD8＋CD107a＋ in CIK cells of WAVE group was significantly higher （[29.43±4.97]% vs [25.19±4.91]%, P〈0.05）. Conclusion： WAVE bioreactor system could produce more CIK cells with high purity, andCD3＋CD8＋ cells and CD3＋CD56＋NKT cells account higher proportion; CIK cells amplified by WAVE bioreactor exhibited higher killing effect on tumor K562 cells compared to the CIK cells amplified by traditional culture technique.