摘要
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 hanging drop 3D cell culture model of human colon cancer cell （HT29） in 48-well cell culture plate, at the same time, through the comparison of several cell viability detection methods to determine the appropriate one for this cell culture way. Methods HT29 cells of 2 375, 3 164, 4 218, 5 625, 7 500 and 10 000/well were seeded in the bottom of the 48-well culture plate to form droplets. After 2 d of inversion culture, the cell spheroids were formed and incubated in medium for another 3 d. The volume of cell spheroids were measured, and the absorbance （A） values were detected through APH assay, MTT assay, MTT assay after digestion, CCK-8 assay and CCK-8 assay after digestion. The results were compared among different methods. Results After 5 d of culture, the cell spheroids were formed perfectly at the density of 2 375--10 000/well, and the volumes were in good linear with the original cell inoculation number at the density of 2 375--7 500/well. The A values of APH assay, MTT assay al%r digestion and CCK-8 assay after digestion increased with the increase of cell inoculation amount; But the cell ball digestion process was complex, and the cell viability was damaged. However, the A values of MTT and CCK-8 assay increased slowly. Conclusion The method of a hanging drop 3D cell culture model in 48-well culture plate combining with APH assay to detect cell viability is economical, accurate and easy to operate.
出处
《药物评价研究》
CAS
2017年第8期1103-1106,共4页
Drug Evaluation Research
基金
沈阳化工研究院资助项目(2016-YTR02-12)
