Corning® 1L DMEM (Dulbecco’s Modified Eagle’s Medium)/F12 50:50 Mix

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the Matrigel in advance on ice or in fridge	Change media every 3 days Harvest organoids after 21 days of culture

Upstream tips
Defrost the Matrigel in advance on ice or in fridge
Protocol tips
Change media every 3 days Harvest organoids after 21 days of culture

Publication protocol

Oviductal organoids: Oviducts from adult female mice were collected for epithelial isolation. Briefly, both oviducts were placed on a clean Petri dish containing Dulbecco phosphate buffer saline (DPBS) to remove any excess vascular and connective tissues. The oviducts were then uncoiled under stereo microscope and microdissecetd into 1-2 mm small pieces and digested in enzyme solution (10 mg/ml Pronase from Streptomyces griseus (Sigma-Aldrich) and 0.5 mg/ml DNase I (Sigma-Aldrich) at 4°C for 13-14 hours. Enzymatic digestion was stopped by adding 10% v/v fetal bovine serum (FBS) (Bovogen) in the enzyme solution and the epithelium was then squeezed out from the oviductal pieces by using fine needles under stereo microscope. Isolated epithelial tubes were collected in the collection tube and a single cell suspension was prepared by repeated pipetting. These epithelial cells were then washed twice with DMEM-F12 media containing 5% FBS, 1% L-glutamine (HyClone) and 1% penicillin-streptomycin (Thermo Fisher Scientific). After washing, differential attachment was performed by incubating cells in a 10 cm cell culture plate containing the above mentioned DMEM-F12 media in an incubator at 37°C with 5% CO2. After 2 hours of incubation, media was collected in a 15 mL tube and centrifuged at 1500 rpm for 5 minutes to pellet the epithelial cells. The epithelial cell pellet was resuspended in 30% media and 70% Matrigel and was placed as 50 μl droplets in each well of a 24-well cell culture plate. The Matrigel with cells was allowed to solidify by at 37°C for 20 minutes. Each well of the culture plate was overlaid with 75% oviductal epithelial (ode) culture media (Dulbecco’s modification of Eagle’s medium/Ham’s F-12 50/50 mix media (DMEM-Ham’s F12) without L-glutamine (Mediatech, cat. no. 15-090), supplemented with 2% Ultroser G serum substitute (USG; Pall Corporation), 1% penicillin-streptomycin (Thermo Fisher Scientific) and 25% WNT3A-RSPO3-NOGGIN conditioned media (WRN-CM). WRN-CM was prepared from L-WRN cells (ATCC® CRL-3276) by following previously described protocol (Miyoshi and Stappenbeck, 2013). This media was further supplemented with several signaling modulators and growth factors, such as mouse epidermal growth factor (EGF; 100 ng/ml; Sigma-Aldrich), fibroblast growth factor 10 (FGF 10, 100ng/ml; Peprotech), nicotinamide (1mM; Sigma) and TGFBR1 kinase inhibitor IV (SB-431542; 0.5 μM; Selleckchem). ROCK inhibitor (Y-27632; 10 μM; TOCRIS) was added to the media only during the first 3 days of culture and passaging. Media was changed every 3 days. After 21 days in culture, organoids were harvested for further processing. During organoid development the images were captured by JuLi™ Stage Real-Time Cell History Recorder (NanoEnTek).

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