目的 采用普通48孔平底培養(yǎng)板結(jié)合懸滴培養(yǎng)法建立人結(jié)腸癌HT29細(xì)胞的三維培養(yǎng)模型，并通過比較選擇適合三維培養(yǎng)的細(xì)胞活力檢測方法。方法 以237.5、316.4、421.8、562.5、750.0、1 000.0/μL、每孔10 μL的接種量接種HT29細(xì)胞于48孔平底培養(yǎng)板底面形成液滴，倒置培養(yǎng)2 d形成細(xì)胞球，補(bǔ)充培養(yǎng)液后常規(guī)培養(yǎng)3 d，通過倒置顯微鏡測量細(xì)胞球體積；采用酸性磷酸酶法（APH）、MTT法及CCK-8法（直接測定及消化后測定）測定細(xì)胞活力，比較不同檢測方法的優(yōu)劣。結(jié)果 HT29細(xì)胞以237.5~1 000.0/μL、每孔10 μL懸滴培養(yǎng)能形成較為規(guī)則的細(xì)胞球，237.5~750.0/μL細(xì)胞球體積與接種量呈良好的線性關(guān)系；APH法A值隨細(xì)胞接種量增加而增大；MTT及CCK-8未經(jīng)消化直接測定組A值隨細(xì)胞接種量增大增加緩慢，消化后測定的A值-細(xì)胞接種量曲線與APH法相似，但細(xì)胞球消化操作復(fù)雜，且對細(xì)胞活力造成損傷。結(jié)論 采用48孔培養(yǎng)板懸滴法建立三維細(xì)胞培養(yǎng)模型，并結(jié)合APH法進(jìn)行細(xì)胞活力檢測，經(jīng)濟(jì)、準(zhǔn)確、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 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.

沈陽化工研究院資助項(xiàng)目（2016-YTR02-12）