DMEM/F-12

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge	Refresh medium every 2 days Passage organoids every 14 days

Upstream tips
Defrost the growth factor reduced Matrigel in advance, on ice or in fridge
Protocol tips
Refresh medium every 2 days Passage organoids every 14 days

Publication protocol

Organoid culture from human endometrium: Endometrial biopsies were obtained from patients undergoing laparoscopy for benign gynecological conditions. The normal phenotype of the endometrial tissue specimens was confirmed by expert histopathologists. The study was approved by the UZ Leuven Ethical Committee (S59006) and informed written consent was obtained from each patient. The women were of reproductive age (30-42 years old) and were not taking any hormones (and determined to be in the follicular/proliferative phase by histological dating). The tissue samples were minced and incubated for 1.5-2.5 hrs at 37°C with collagenase IV (1 mg ml-1; Life Technologies). In addition, mechanical dissociation was applied by vigorous pipetting using a fire- polished Pasteur pipet every 30 min. Subsequently, the suspension was centrifuged and the pellet resuspended and incubated in TrypLE with Y27632 (15 min at 37°C). After centrifugation, the cell pellet was resuspended in 70% Matrigel in DMEM/F12, and plated as described above for mouse. Human organoid culture medium contained N-acetyl-L-cysteine (1.25 mM; Sigma-Aldrich), the p38 inhibitor SB202190 (10 μM; Sigma-Aldrich) and 17β-estradiol (E2, 2 nM; Sigma-Aldrich), supplementary added to the mouse EMO medium. Medium was refreshed every 2 days and organoids were enzymatically dissociated and passaged every 14 days at an average ratio of 1:5, according to the same procedure as described above for mouse EMOs. Unless otherwise stated, organoids of low passage number (P3-P5) were used for all experiments described.

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Manufacturer protocol

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