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目的采用普通48孔平底培养板结合悬滴培养法建立人结肠癌HT29细胞的三维培养模型,并通过比较选择适合三维培养的细胞活力检测方法。方法以237.5、316.4、421.8、562.5、750.0、1 000.0/μL、每孔10μL的接种量接种HT29细胞于48孔平底培养板底面形成液滴,倒置培养2 d形成细胞球,补充培养液后常规培养3 d,通过倒置显微镜测量细胞球体积;采用酸性磷酸酶法(APH)、MTT法及CCK-8法(直接测定及消化后测定)测定细胞活力,比较不同检测方法的优劣。结果 HT29细胞以237.5~1 000.0/μL、每孔10μL悬滴培养能形成较为规则的细胞球,237.5~750.0/μL细胞球体积与接种量呈良好的线性关系;APH法A值随细胞接种量增加而增大;MTT及CCK-8未经消化直接测定组A值随细胞接种量增大增加缓慢,消化后测定的A值-细胞接种量曲线与APH法相似,但细胞球消化操作复杂,且对细胞活力造成损伤。结论采用48孔培养板悬滴法建立三维细胞培养模型,并结合APH法进行细胞活力检测,经济、准确、便于操作。
OBJECTIVE: To establish a three-dimensional culture model of human colon cancer HT29 cells by using ordinary 48-well flat-bottom culture plate and hanging-drop culture method and to select the cell viability test method suitable for three-dimensional culture by comparison. Methods HT29 cells were inoculated on the bottom of 48-well flat-bottomed plates to form droplets with an inoculum size of 237.5, 316.4, 421.8, 562.5, 750.0, and 1000 μL, and each well was inoculated with 10 μL. After cultured for 3 days, the cell volume was measured by inverted microscope. The cell viability was measured by acid phosphatase (APH), MTT assay and CCK-8 assay (direct assay and post-digestion assay), and the advantages and disadvantages of different assays were compared. Results HT29 cells with a volume of 237.5-1 000.0 / μL and 10μL per well could form more regular cytoplasm. The volume of 237.5-750.0 / μL cell sphere showed a good linear relationship with the inoculum size. The value of APH with the inoculum size Increase and increase; MTT and CCK-8 non-digestible direct measurement group A value increased slowly with the cell inoculum increased after digestion measured value of A-cell inoculum curve similar to the APH method, but the cell ball digestion operation is complicated, And damage to cell viability. Conclusion The cell culture model was established by the hanging drop method of 48-well culture plate and the cell viability assay combined with APH method was economical, accurate and easy to operate.