肠上皮细胞模型不同培养条件的优化及适应性研究

目的:建立肠上皮(Caco-2)细胞单层模型,探讨不同培养条件对细胞单层模型的影响,优化适宜的条件,提供稳定、重复性好的Caco-2细胞模型。方法:将细胞接种Transwell培养板上进行体外培养,并比较3种Transwell培养板及包被鼠尾胶原蛋白对细胞单层的影响,通过透射电镜和倒置显微镜观察细胞单层的形态学特征,通过测定细胞单层的电阻值、细胞两侧碱性磷酸酶含量、荧光素钠的透过量验证细胞单层的完整性、极性、通透性。结果:经过21 d培养,24孔Transwell培养的细胞单层的完整性、极性高于12孔Transwell培养的细胞单层,而透过性低于12孔Transwell培养的细胞单层;包被鼠尾胶原蛋白的Transwell于细胞单层生长前期跨上皮细胞电阻值(transepithelium electrical resistance,TEER)迅速升高,对细胞单层生长的中后期无显著性影响。结论:3种Transwell均可成功培养Caco-2细胞单层,24孔站立式Transwell适用于样品需求量少、检测灵敏度高的物质,可应用于大量样品的高通量筛选;12孔悬挂式Transwell提供检测的样品量和透过率较大,适于实验室的常规操作和常用仪器的样品需求。

Comparative Study on Different Culture Conditions for Caco-2 Cell Model

Objective: To study the impacts of culture conditions on the Caco-2 cell model,to optimize the appropriate conditions for a stable,reproducible Caco-2 model. Mehtod: The environment of small intestine was in vitro simulated,and rat tail collagen was coated on three types of Transwell monolayerly and compared.Morphological features were observed by microscope,the integrity,polarity and permeability of monolayer were measured by the resistance,alkaline phosphatase,the amount of sodium fluorescein. Result: After culture of 21 days,the integrity,polarity of Caco-2 monolayer cultured on 24-well Transwell were higher than that on 12-well Transwell,but the permeability were the opposite.The TEER of the monolayer coated with rat tail collagen was increased rapidly in early stages,and was no significant effect at the middle and late stages. Conclusion: Three types of Transwell can be successfully used to culture Caco-2 cell monolayer.24-well Transwell is suitable for small samples with high sensitivity and high-throughput screening of a large number of samples.12-well Transwell has higher volume of sample and permeability,is suitable for routine laboratory operations and equipment.