Gibco™ DMEM/F-12, GlutaMAX™ supplement

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Change media every 3-4 days and after 12 days organoids harvest organoids for further processing

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Change media every 3-4 days and after 12 days organoids harvest organoids for further processing

Publication protocol

Uterine epithelial organoids: The uteri collected from adult female mice were washed with Mg2+ and Ca2+ free Hank’s Balanced Salt Solution (HBSS). Uterine epithelium was isolated as described previously (Syed et al., 2020). Briefly, uterine arm was slit open and cut longitudinally into 3-4 mm pieces. The tissue fragments were then incubated in Pronase/DNase enzyme solution [10mg/ml Pronase (Sigma-Aldrich) from Streptomyces griseus and 0.5 mg/ml DNase I (Sigma-Aldrich)] at 4°C on a shaker for 13-14 hours. Enzymatic digestion was stopped by adding 10% v/v fetal bovine serum (FBS) (Bovogen), and uterine epithelium was collected after passing through 70 μm cell strainer. Cells were then washed with DMEM-F12 media containing 5% FBS, 1% L-glutamine (Sigma-Aldrich) and 1% penicillin-streptomycin (Thermo Fisher Scientific) by centrifugation at 1500 rpm for 5 minutes. After washing cells were incubated for 2 hours in a cell culture plate with above-mentioned FBS containing DMEM-F12 media for differential attachment.
After differential attachment, uterine epithelial cells were resuspended in 30% media and 70% matrigel and was placed as 50 μl droplets in each well of a 24-well cell culture plate. Matrigel droplets were incubated at 37°C for 20 minutes for solidification and were then overlaid with uterine organoid culture medium as described previously (Syed et al., 2020). The uterine organoid culture medium contained 25% Wnt3A-R-spondin3-noggin conditioned media (WRN-CM) and 75% DMEM-F12, supplemented with 1% Glutamax (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific) and additional growth factors such as 2% B27 (Thermo Fisher Scientific), 1% N2 (Thermo Fisher Scientific), 1% insulin-transferrin-selenium (ITS) (Sigma-Aldrich), 50 ng/ml mouse EGF (Sigma-Aldrich), 50ng/ml human FGF10 (Peprotech), 1mM nicotinamide (Sigma-Aldrich), 0.5 μM A83-01 (TGFβ/Alk inhibitor) (Tocris) and 10 μM Y-27632 dihydrochloride (Rock inhibitor) (Tocris). ROCK inhibitor (Y-27632; 10 μM; TOCRIS) was only added to the media during the first 3 days of culture. Media was changed every 3-4 days and after 12 days organoids were harvested for further processing.


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