Caco-2细胞单层模型的建立和评估

Establishment and evaluation of Caco-2 cell monolayer model

目的建立Caco-2细胞单层的培养和模型评价的方法,为后续研究普萘洛尔(propranolo)对映异构体转运动力学特征奠定基础。方法常规培养条件下,将Caco-2细胞分别以2.5×105、5.5×105cells/ml分别接种于Transwell的聚碳酸脂膜上(0.4ml/well),在培养10d、15d和21d后,以细胞单层跨膜电阻(Transepithelial electrical resistance,TEER)、标志物的表观通透系数(The apparent permeability coefficients,Papp)初步评价Caco-2细胞单层模型的完整性,以扫描电镜和透射电镜验证模型的完整性,以期确定建模时的细胞接种密度和培养时间;检测细胞单层模型在HBSS液和不同浓度的普萘洛尔溶液中培养不同时间时的TEER值,以评价单层模型的稳定性。结果以5.5×105cells/ml接种21d后,TEER>200Ω*cm2、Papp<1.0×10-6cm/s,形态学观察可见细胞融合形成连续、完整的单细胞层,细胞表面覆盖一层刷状缘微绒毛;细胞单层模型在HBSS液和盐酸普萘洛尔药液(≤640mmol/L)中孵育8h后TEER>200Ω*cm2。结论以5.5×105cells/ml为细胞接种密度且培养至21d后,Caco-2细胞单层形态与小肠上皮细胞类似,跨膜电阻、标志物透过量均达到要求,具有良好的完整性,而且细胞单层的完整性在HBSS液和盐酸普萘洛尔药液(≤640mmol/L)中可稳定8h,表明该细胞模型建立成功,可用于后续普萘洛尔对映异构体药物吸收动力学研究。

Objective To establish the Caco-2 cell monolayer model and evaluate the model＇ integrity and stability, so as to play a foundation for next study of propranolol of drug absorption and transport. Methods In conventional culture condition, the Caco-2 cells were inoculated on the Transwell polycarbonate （0. 4ml/well）with inoculum density of 2. 5 × 105 cells/ml and 5.5 ×105 cells/ml respectively; after cultured 10d,15d,21d, the Caco-2 cell monolayer model＇integrity was preliminary estimated with transepithelial electrical resistance（TEER） and the apparent permeability coefficients（Papp） ,and verified by scanning elec- tron microscope（SEM） and transmission electron micrescope（TEM） ,aimed to determine the inoculum density and culture time; the suibility of the cell monolayer in HBSS and different concentration propranolol solutions（20,40,80,160,320,640,1280 mmol/ L） was evaluated with TEER after cultured 0h,2h,4h,6h,Sh. Results After been cultured for 21 days with inoculum density of 2. 5 × 105cells/ml, the Caco-2 cell monolayer had not form, howerer, as inoculum density of 5.5 × 105cells/ml,the cells had fu- sion into a continuous and integrate cell monolayer with brush border microvillus, as well, with Papp 〈 1.0 × 106cm/s and TEER 〉 200 Ω ＊ cm2. The TEER of cell monolayer in HBSS and propranolol solutions（≤640mmol/L） after gh were higher than 200 Ω ＊ cm2. Conclusion The inoculum density of 2. 5 × I05 ceUs/ml was not avail to Caco-2 cell monolayer. With inoculum density of 5.5 × 105cells/ml and cultured for 21d, the appearance of Caco-2 cell monolayer were same as intestinal mucosa, as well, the TEER and Papp fitted to the requirement which hinted that the Caco-2 cell monolayer model had good integrity. Moreo- ver, the integrity could retain 8h in HBSS and propranolol solutions（≤640mmol/L）. All of thht above showed that the cell mono- layer was established successfully and appreciate to next study of propranolol enantiomers absorption and transport.