Permeability

Since our previous work [22] has demonstrated that dextran is suitable for determining intestinal permeability to macromolecules, we used 4 to 10 kDa fluorescein isothiocyanate (FITC)-labeled dextrans that have a Stokes radius of 1.3 to 2 nm [23] to characterize the steric properties of the leak pathway of organoids enriched in different cell types. It seems that 40 kDa dextrans having ~4.5 nm radius is impermeable to the TJs of typical organoids [24]. The paracellular permeability of organoids was tested using FITC-dextran (4 kDa and 10 kDa) [24]. Briefly, directed organoids were generated in individual Matrigel wells each housing TYP, ISC, ENT, GOB, and PAN organoids. Wells were washed twice with phosphate buffered saline (PBS; pH 7.4) and incubated for 30 min in 4 and 10 kDa FITC-dextran at a final concentration of 1.25 μM for 30 min at room temperature, to impose a chemical serosal-to-lumen gradient. We determined by fluorescence intensity the amount that would accumulate in the organoid lumen. After incubation, the FITC-dextran was removed and the organoids were gently washed five times with PBS to remove the background. Fluorescence within an organoid was estimated (ImageJ) by focusing on the entire luminal area inside the organoid. The representative background fluorescence was estimated from four different areas surrounding the organoid. The net fluorescence in an organoid is the integrated luminal fluorescence density less the mean fluorescence of background readings and the mean autofluorescence. The net fluorescence was then averaged from several representative organoids from different wells, and this mean fluorescence was normalized to that of a TYP organoid.