Establishment and Validation of Caco-2 Cell in Vitro Absorption Model
LYU Xiaojun;CAO Yidan;HE Kaiyong;Hubei Institute for Drug Control;Hubei University of Chinese Medicine;
Objective To establish and validate the Caco-2 cell in vitro absorption model,so as to play a foundation for next study of drug absorption and transport. Methods Caco-2 cells were cultured on the polycarbonate membrane inserts fixed in the 24-well transwell transport chamber with three types cell density of low,medium and high concentration( 5×10~4,1×10~5,2×10~5·mL~(-1)) respectively.After being cultured for 3,6,9,12,15,18,21 d,the integrity of monolayer were assessed and compared by the cell morphology,growth characters and transepithelial electrical resistance( TEER),aiming to determine the inoculum density and culture time.Then the permeability and polarity were validated by the apical-to-basolateral amount of Lucifer Yellow across cell monolayer,the alkaline phosphatase activity in the apical side( AP),the basolateral side( BL) and intracellular activity. Results The cells of low,medium and high concentration group had fusion into a integrate cell monolayer and the maximum absorbance after being cultured for 15,12,9 d respectively. However,conglobated and dead cells were observed at the later growth stage in the medium and high concentration group and the TEER of cell monolayer were smaller than the low concentration group,which could reach 300 Ω · cm~2 after cultured 15 d and keep a relatively stable value,then cells were cultured with 5×10~4·mL~(-1) cell density for 21 d. The Lucifer Yellow apparent permeability coefficient( Papp) was 3. 57 × 10~(-7) cm·s~(-1) which was lower than 5. 0 × 10~(-7) cm · s~(-1) according to provision. And the intracellular alkaline phosphates' activity increased,AP/BL increased by 5 times in day 21. Conclusion The integrity,permeability and polarity of the established Caco-2 cell model in our laboratory was validated,and it can be used as an in vitro model similar with small intestinal epithelium for absorption and transport studies.
【CateGory Index】： R96