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. 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 17β-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.