基于人肝癌细胞系SMMC-7721的三维培养条件研究

目的 基于人肝癌细胞系SMMC-7721考察合适的海藻酸钠、氯化钙浓度，形成良好的海藻酸钙凝胶，优选三维条件下肿瘤细胞的培养条件。方法 在96孔板中培养SMMC-7721细胞，基于L₉（3⁴）正交表设计实验，以MTT法检测并计算氯化钙溶液、柠檬酸钠溶液不同浓度与作用时间下的细胞存活率；结合正交优选结果，考察适宜的海藻酸钠浓度，筛选适合的成胶、溶胶条件，并在三维培养模式下进行细胞活力确证。结果 最佳凝胶应用条件为1%氯化钙溶液与1%海藻酸钠溶液作用15 min内用于成胶，10%柠檬酸钠溶液作用凝胶10 min内用于溶胶，MTT法检测细胞存活率为94.97%。该三维培养条件下的肝癌细胞72 h内生长状态良好，存活率可达92.10%，细胞堆积紧密，出现肿瘤细胞球样聚集体。结论 本实验筛选出适合肿瘤细胞三维培养的成胶、溶胶条件。在该条件下培养肝癌细胞SMMC-7721，细胞生长状态良好，且细胞生长与聚集形态与传统二维培养存在差异，更接近细胞体内生存环境。基于该方法进行肿瘤细胞三维培养，可为深入研究肿瘤细胞生长状态，为抗肿瘤药物筛选提供更有利的方式。

Research of the 3D Cell Culture Conditions Based on Human Liver Cancer Cells SMMC-7721

OBJECTIVE To optimize 3D culturing conditions for the tumor cells based on human liver cancer cells SMMC-7721, find out the proper concentrations of sodium alginate and calcium chloride to form calcium alginate gel. METHODS SMMC-7721 cells were cultured in 96-well plate. The cell survival rate under different concentrations and treatment durations of calcium chloride and sodium citrate solutions were detected by MTT method based on L₉(3⁴) orthogonal design. According to the result of orthogonal optimization, the suitable concentration of sodium alginate was investigated, and optimal conditions of gelatin forming and dissolving were screened. Then cell viability was confirmed under the 3D culture mode. RESULTS The optimal conditions were as follows:1% calcium chloride act on the sodium alginate within 15 min to form gel, 10% sodium citrate act on the calcium alginate gel within 10 min to dissolve gel, which achieved a 94.97% cell survival rate by MTT method. The SMMC-7721 cells cultured in the calcium alginate gel showed a fine state within 72 h, the survival rate was 92.10%, and showed a compact agminated status which was called multicellular spheroid. CONCLUSION The conditions for gel forming and dissolving used for tumor cells 3D culture is compatible for cell culturing, SMMC-7721 cells show a fine growth state, and the agminated status shows some difference compared with the cell cultured in 2D condition. Tumor cells cultured by this method could provide a better way to study the living status for tumor cells and to screen more anti-tumor drugs.