基于中心组合设计和响应面分析的血清替代物浓度优化

Optimization of Serum Substitute Concentration in Serum-Free Media for CIK Cells Using Central Composite Design and Response Surface Analysis

细胞因子诱导杀伤细胞(Cytokine-induced killer cells,CIK cells)是一类广泛应用的肿瘤过继免疫疗法的效应细胞,其体外扩增过程中常需要使用无血清培养基。今以胰岛素、亚麻酸、胆固醇和乙醇胺4种可替代血清促进细胞扩增的组份为研究对象,采用中心组合设计和响应面分析相结合的方法,考察上述组分的浓度以及相互作用对单个核细胞扩增的影响,得到了可支持单个核细胞扩增的最优浓度,分别为10、5.66、19.86和1.22 mg?L?1。将上述4种组分以优化的浓度添加到DMEM/F12和IMDM以1:1体积比混合而成的基础培养基中,制成无血清培养基Optimizer。以含10%FBS的RPMI 1640为对照,采用半量稀释法培养脐血单个核细胞,以培养14天的总细胞扩增倍数、细胞组成、CD3+CD56+细胞扩增倍数以及对K562细胞的杀伤活性为指标,评价了制成的无血清培养基Optimizer支持CIK细胞扩增的效果。结果显示,采用所制备的无血清培养基Optimizer,总细胞扩增倍数为56.54±18.87,明显高于SFM1的5.14±1.03(p<0.05)和SFM2的3.59±0.56(p<0.05),与含10%FBS的RPMI 1640的35.24±20.92(p>0.05)接近;CD3+细胞为97.98%±1.41%,与含10%FBS的RPM I1640的94.34%±1.29%相当(p>0.05);尽管CD3+CD56+细胞比例18.17%±7.38%低于含10%FBS的RPMI 1640中的数值25.49%±3.35%(p<0.05),但两种培养基的CD3+CD56+细胞扩增倍数分别为440.86±222.89和429.27±249.16,没有显著性差异(p>0.05);当效靶分别为9:1时分别采用两种培养基扩增的细胞对K562细胞的杀伤活性均能达到50%以上。该研究结果为用于CIK细胞体外扩增的无血清培养基的开发提供了依据。

CIK cells （cytokine-induced killer cells） are one type of effector cells in adoptive cell immunotherapy for tumors. Serum-free media are often necessary when CIK cells are expanded in vitro. This study investigated the applicability of insulin, linolenic acid, cholesterol and ethanolamine as substitutes of serum in promoting cell proliferation. Composite combination design and response surface analysis were combined to optimize the concentration of these chemicals to obtain the maximum expansion fold of total mononuclear cells. Their final optimized concentrations are 10, 5.66 19.86, 1.22 mg·L^-1, respectively. These four components were added at their optimized concentrations to the basic medium consists of DMEM/F 12 and IMDM at volume ratio of 1：1 to prepare serum-free media named Optimizer. Umbilical cord blood mononuclear cells were cultured for 14 days in Optimizer via half amount of dilution, and RPMI 1640 with 10% FBS was used as a control. Cells composition, expansion fold of CD3^＋CD56^＋cells and killing activity on K562 cells were assessed. The results indicate that the expansion fold of total cells in Optimizer is 56.54±18.87, which is significantly higher than 5.14±1.03（p〈0.05） in SFM1 and 3.59±0.56（p〈0.05） in SFM2, and it is similar to 35.24±20.92（p〉0.05） in RPMI1640 with 10% FBS. CD3^＋ cell proportion is 97.98%±1.41% in Optimizer, which is similar to 94.34%±1.29% iri RPMI 1640 with 10% FBS（p〉0.05）. CD3^＋CD56^＋ cell proportion is 18.17±7.38% and it is lower than 25,49%±3.35%（p〈0.05） in RPMI 1640 with 10% FBS. However, the expansion fold of CD3＋CD56＋ cells is 440.86±222.89 and 429.27±249.16 respectively in these two media, which shows no significant difference（p〉0.05）, When the ratio of effector cell and target cell is 9：1, the killing activities on K562 cells in these two media are bOtfi higher than 50%. These results provide fundamental data fo r the development of serum-free media for the expansion of CIK cells in vitro.