Derivation and Maintenance of Intestinal Organoids

Derivation and maintenance of HIOs followed published protocols^{1 (link), 25 (link)}. Briefly, HIOs were embedded in Matrigel (BD Biosciences) and overlaid with Advanced DMEM-F12 medium (Invitrogen, Carlsbad, CA) containing 1X B27 supplement (Invitrogen), 1X GlutaMAX (Life Technologies, Carlsbad, CA), 10 µM Hepes, 10% pen/strep, 100 ng/mL rhNoggin (R&D Systems), 100 ng/mL epidermal growth factor (R&D Systems), and approximately 500 ng/mL R-Spondin1 (RSPO1). RSPO1 was obtained from conditioned media collected from a HEK293 cell line that was stably transfected and zeocin-selected for the RSPO1 expression vector. Media was changed every two to four days, and HIOs were transferred to fresh Matrigel once a week until they reached approximately 2 to 3 mm in diameter for experiments. This size was reached on average 48 days after initial spheroid formation.