Murine Colonic Organoid and Monolayer Culture

To obtain 3D murine colonoids, intestinal crypts were isolated from the colon of male and female Cldn23^ERΔIEC and Cldn23^f/f mice (mice were age and sex matched between genotypes for each experiment), embedded in Matrigel (Corning, 365237, Lot 9112015), and maintained in LWRN-conditioned media supplemented with 50 ng/ml of recombinant human EGF (R&D Systems, 236-EG) and antibiotics/antimycotic (Corning, 30-003-Cl) as described previously^{94 (link)}. To acutely deplete CLDN23, colonoid cultures were treated for 72 h with 1 μM (Ζ)−4-hydroxytamoxifen (Sigma-Aldrich, H7904) in complete media followed by passage and maintenance in Ζ−4-hydroxytamoxifen-free complete media. Direct 2D colonoid monolayers were generated directly from intestinal crypts as described in⁹⁵ . Isolated crypts were seeded onto collagen and laminin coated plates, Transwells, and/or cover slips. Murine 2D cultures were maintained in LWRN complete media for 24–48 h, until monolayers attained confluency, and then media was changed to differentiation media for at least 24 h to allow for epithelial differentiation. For colonoid cocultures, equal numbers of isolated crypts from Cldn23^ERΔIEC and Cldn23^f/f mice were mixed and seeded onto collagen- and laminin-coated cover slips.