Hormone responsiveness and spatial heterogeneityof apical-out endometrial organoids

We have developed apical-out (AO)- endometrial organoids (EMO) that emulate the in vivo archtecture of endometrial epithelium. The AO-EMO exposes the apical surface of the epithelium. To explore the hormone responsiveness and spatial heterogeneity of AO-EMO, we conducted transcriptomic analysis. Overall design: We cultured human endometrial organoids in collagen-based gels (70% type-I collagen and 30% matrigel) for 7-10 days with ECSY medium. The composition of the ECSY medium was as follows: DMEM/F-12 supplemented with 1% ITS-X supplement, 0.15% BSA, 1% Knockout Serum Replacement, 200 mM L-ascorbic acid, 50 ng/ml EGF, 2 µM CHIR99021, 2 µM SB202190, and 10 µM Y27632. Following the ECSY culture, we sequentially introduced 17ß-estradiol(E2), medroxyprogesterone acetate (MPA), 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP), and prolactin. The treatment durations were as follows: 2 days for E2 and 4 days for the combined treatment of E2, MPA, 8-Br-cAMP, and prolactin (EPCP). As a control, we prepared AO-EMO without any hormone treatment as well as AO-EMO treated exclusively with E2. To isolate the outer and inner cells of AO-EMO, the culture medium was aspirated, and PBS was added to wash. Subsequently, TrypLE Express was added to the culture dish, and plates were incubated at 37? for 30 min. Following the incubation, TrypLE was removed and AO-EMO were washed with PBS. The outer cells were detached by pipetting PBS onto the surface of the AO-EMO several times using a manual pipette. We used 4 independent organoids from 4 donors.