Quantifying Paracellular Permeability in Cocultures

To measure apparent paracellular permeability (P_app), 50 g/mL Cascade Blue (3 kDa, Thermo Fisher Scientific) was added to the antibiotic-free expansion media in the top epithelial channel with a flow rate of 30 μL/hr. Every 24 hours, the effluents from the epithelial and endothelial channels were collected. The concentration of Cascade Blue that diffused across the membrane into the endothelial channel effluent was quantified with a Varioskan LUX multimode microplate reader (Thermo Fisher Scientific) with excitation at 400 nm and emission at 425 nm. The following formula was used to calculate P_app (cm/s) as in Kasendra et al. (16 (link)): P_app = (Endothelial_Output × Flow Rate)/(Epithelial_Input × Surface Area), where Endothelial_Output is the concentration (mg/mL) of Cascade Blue from the endothelial channel effluent, Flow Rate is the fluid flow rate (mL/s) of the media through both channels, Epithelial_Input is the concentration (mg/mL) of Cascade Blue originally added to the epithelial channel media, and Surface Area is the area (cm²) of the channel where coculture occurs.