Hormone responsiveness and spatial heterogeneityof apical-out endometrial organoids. 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 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.

本研究构建了模拟子宫内膜上皮体内组织结构的顶向外（apical-out, AO）子宫内膜类器官（endometrial organoids, EMO）。该AO-EMO可暴露上皮细胞的顶侧表面。为探究AO-EMO的激素应答特性与空间异质性，本研究开展了转录组学分析。
实验总体设计：本研究使用ECSY培养基，在由70%Ⅰ型胶原与30%基质胶（matrigel）组成的胶原基凝胶中培养人子宫内膜类器官，培养时长为7至10天。ECSY培养基的组分如下：DMEM/F-12培养基中添加1% ITS-X添加剂、0.15%牛血清白蛋白（bovine serum albumin, BSA）、1% Knockout血清替代物（Knockout Serum Replacement）、200 mM L-抗坏血酸、50 ng/ml表皮生长因子（epidermal growth factor, EGF）、2 μM CHIR99021、2 μM SB202190及10 μM Y27632。完成ECSY培养基培养后，我们依次向培养体系中加入17β-雌二醇（17β-estradiol, E2）、醋酸甲羟孕酮（medroxyprogesterone acetate, MPA）、8-溴腺苷3',5'-环一磷酸（8-bromoadenosine 3′,5′-cyclic monophosphate, 8-Br-cAMP）及催乳素。处理时长设置如下：仅用E2处理时长为2天；E2、MPA、8-Br-cAMP与催乳素联合处理（简称EPCP）的时长为4天。作为对照，本研究设置了未进行任何激素处理的AO-EMO对照组，以及仅用E2处理的AO-EMO对照组。为分离AO-EMO的外层与内层细胞，我们先吸弃培养基，加入磷酸盐缓冲液（phosphate buffered saline, PBS）进行洗涤。随后向培养皿中加入TrypLE Express解离液，将培养板置于37℃孵育30分钟。孵育结束后移除TrypLE解离液，用PBS洗涤AO-EMO。通过使用手动移液器向AO-EMO表面反复吹打PBS，即可分离得到外层细胞。本研究使用来自4名供体的4个独立类器官样本。