Porcine Intestinal Epithelial Cell Culture

Crypt cells were extracted from the small intestine (ileum) as described previously [24 (link)], with slight modifications detailed in Guo et al. [7 (link)]. The culture conditions for porcine IEs’ (PIEs’) maintenance and differentiation were formulated as follows: complete medium without growth factors (CMGF(-) medium) consisted of advanced DMEM/F12 medium (Invitrogen, US) supplemented with 100 U/mL penicillin–streptomycin (PS, Invitrogen), 10 mM HEPES buffer (Invitrogen), and 1% of GlutaMAX-100x (Invitrogen) [24 (link),28 (link)]. The complete cell culture medium contained IntestiCult Organoid Growth Medium (Component A: Component B ratio = 1:1) (STEMCELL), 100 U/mL of PS (Invitrogen), and 2.5 µM of CHIR99021 (Stemgent). The differentiation medium (DM) contained IntestiCult Organoid Growth Medium Components A and B added at a ratio of 1:0.8, supplemented with 100 U/mL of PS (Invitrogen).