| 培养板悬滴法三维细胞培养模型建立及细胞活力检测方法比较 |
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| 引用本文： | 林鹤,王婉秋,焦佳媛,杨昊珅,任利翔,邢立国. 培养板悬滴法三维细胞培养模型建立及细胞活力检测方法比较[J]. 现代药物与临床, 2017, 40(8): 1103-1106 |
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| 作者姓名： | 林鹤 王婉秋 焦佳媛 杨昊珅 任利翔 邢立国 |
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| 作者单位： | 沈阳化工研究院有限公司安评中心, 辽宁 沈阳 110021;沈阳化工研究院有限公司医药研究室, 辽宁 沈阳 110021;沈阳化工研究院有限公司医药研究室, 辽宁 沈阳 110021;沈阳化工大学, 辽宁 沈阳 110142;沈阳化工研究院有限公司安评中心, 辽宁 沈阳 110021;沈阳化工研究院有限公司安评中心, 辽宁 沈阳 110021 |
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| 基金项目： | 沈阳化工研究院资助项目（2016-YTR02-12） |
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| 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10 μL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10 μL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。
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| 关 键 词： | 悬滴三维培养法 HT29细胞 细胞活力检测 酸性磷酸酶法(APH) MTT CCK-8 |
| 收稿时间： | 2017-03-09 |
Establishment of hanging drop 3D cell culture model in cell culture plate and comparison of cell viability detection methods |
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| LIN He,WANG Wan-qiu,JIAO Jia-yuan,YANG Hao-shen,REN Li-xiang and XING Li-guo. Establishment of hanging drop 3D cell culture model in cell culture plate and comparison of cell viability detection methods[J]. Drugs & Clinic, 2017, 40(8): 1103-1106 |
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| Authors: | LIN He WANG Wan-qiu JIAO Jia-yuan YANG Hao-shen REN Li-xiang XING Li-guo |
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| Affiliation: | Safety Evaluation Center, Shenyang Research Institute of Chemical Industry Co., Ltd, Shenyang 110021, China;Pharmaceutical Research Laboratory, Shenyang Research Institute of Chemical Industry Co., Ltd, Shenyang 110021, China;Pharmaceutical Research Laboratory, Shenyang Research Institute of Chemical Industry Co., Ltd, Shenyang 110021, China;Shenyang University of Chemical Technology, Shenyang 110142, China;Safety Evaluation Center, Shenyang Research Institute of Chemical Industry Co., Ltd, Shenyang 110021, China;Safety Evaluation Center, Shenyang Research Institute of Chemical Industry Co., Ltd, Shenyang 110021, China |
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| Abstract: | 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 after 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. |
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| Keywords: | Hanging drop cell culture HT29 cells Cell viability detection Acid phosphatase assay (APH) MTT CCK-8 |
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