This study aimed to create a more efficient Caco-2 model for in vitro drug permeability assessment. The conventional Caco-2 cell monolayer method, requiring 21 days, prompted us to develop a 3-day model that maintains monolayer quality and transporter functions better than other short-duration models. To mitigate monolayer leakage, we tested media additives for improved tight junction formation and identified safe compounds replicating the 21-day model\'s functions within this shorter duration.

Our 3-day model exhibited enhanced monolayer integrity evident from higher trans-epithelial electrical resistance (TEER) and the presence of mRNA levels of tight junction proteins. Transporter mRNA levels were aligned with the 21-day model, except for P-glycoprotein (P-gp). We also confirmed the roles of P-gp and peptide uptake transporter 1 (PEPT-1) through inhibition assays, verifying their functionality in our model.

In summary, our 3-day screening model addresses limitations in shorter Caco-2 cell monolayer models, improving monolayer integrity and transporter functionality. This advancement enhances compound permeability screening, considerably reducing the testing time.