Isolation and Culture of Human Endometrial Organoids

Endometrial/decidual/carcinoma tissues were chopped using scalpels into approximately 0.5 mm³ cubes and enzymatically digested in 20-30 mL 1.25U/mL Dispase II (Sigma, D4693)/ 0.4mg/mL collagenase V (Sigma, C-9263) solution in RPMI 1640 medium (ThermoFisher Scientific, 21875-034)/10% FCS (Biosera, FB-1001) with gentle shaking at 37°C for 30-60 min. The supernatant was passed through one or more 100 μm cell sieves (Corning, 431752) and the sieve washed several times with medium. The flow-through was collected for stromal cell culture in Advanced DMEM/F12 (ThermoFisher Scientific, 12634010) +10%FBS+pen/strep (Sigma, P0781) +L-glutamine (Sigma, 25030-024) for several days and subsequent analysis. The sieves were inverted over a petri dish and retained glandular elements were backwashed from the sieve membranes, pelleted by centrifugation and resuspended in ice cold Matrigel (Corning, 536231) at a ratio of 1:20 (vol:vol). 20 μL drops of Matrigel-cell suspension were plated into 48-well plate (Costar, 3548), allowed to set at 37°C and overlaid with 250 μL organoid Expansion Medium (ExM). See Supplementary Table 3 for ExM composition. The medium was changed every 2-3 d. Cultures were passaged by manual pipetting every 7-10 d. For freezing organoids, Matrigel was removed using Cell Recovery Solution (Corning, 354253) and organoids were resuspended in Recovery cell culture freezing medium (ThermoFisher Scientific, 12648-010). A step-by-step protocol of the derivation and maintenance of human endometrial organoid cultures can be found at Nature Protocol Exchange59 (link).