Single cell RNA sequencing of endometrial organoids in the IVMC protocol
Single cell RNA sequencing of endometrial organoids in the IVMC protocol
Endometrial organoids were derived from biopsies of women in the secretory phase of the cycle undergoing IVF at the Bourn Hall Fertility Clinic with approval from East of England Cambridge Central Research Ethics Committee (17/EE/01551 IRAS 225205). The transfer and use of all the endometrial tissue samples together with the established endometrial organoid cultures for this study are authorized under a Material Transfer Agreements (MTA) between the University of Cambridge and the Friedrich Miescher Institute for Biomedical Research (Agreements No. G119629 and G108313). Endometrial organoids from donors B050, B066 and B080 were used for this experiment.  They were subjected to the in vitro menstrual cycle (IVMC) protocol. Organoids were dissociated into single cells and alive cells were FACS sorted at the following timepoints: before breakdown (hormonal withdrawal 48h), 12h after breakdown (post-breakdown 12h), 24h after breakdown (post-breakdown 24h), 48h after breakdown (post-breakdown 48h). Estrogen was re-introduced, and samples were collected at 24h (E2 differentiation 24h) and 48h after estrogen treatment (E2 differentiation 48h).  Endometrial organoids were collected and Matrigel was removed using Cell Recovery Solution (Corning, 354253) for 50 min maximum on ice. They were then washed with cold PBS pH (7.4) (GIBCO, 10010-015) and mechanically broken by automatic (400 times) and manual (100 times) pipetting. Organoids were first incubated with pre-warmed accutase cell detachment solution (Gibco, A11105-01) for 5 minutes at 37oC and then incubated with collagenase V (Sigma-Aldrich, C-9263) diluted in 10% FBS/Advanced DMEM/F12 for 20 minutes at 37oC. The digest was passed through a 40 μm nylon mesh cell strainer to ensure single cell suspension, and the flow through was collected in falcon round bottom tubes (Corning, 352235). Cell pellets were diluted in EOM, and cells were counted using the NucleoCounter® NC-202™ Automated Cell Counter (Chemometec, NC-200). Organoids growing from single cells were supplemented with 10µM Y-27632 until the formation of cystic structures. Endometrial organoids were grown at 37°C in a humidified atmosphere of 5% CO2. They were cultured in Matrigel (Corning, 356231) droplets supplemented with endometrial organoid medium (EOM).  The medium was refreshed every 2-3 days, and the organoids were passaged at an average ratio of 1:3 every 5-7 days. The organoid suspension was centrifuged for 6 minutes at 600×g between passaging steps. The passaged organoid pellet was resuspended in ice cold Matrigel droplets, allowed to solidify at 37°C for 15-30 minutes, and covered with EOM.  Endometrial organoid cultures were first dissociated into single cells which were cultured until organoid formation. To mimic the proliferative phase, organoids were then primed with 500 nM β-estradiol (E2) for 48 h followed by treatment with 80 μM progesterone, 100 μg/ml cAMP and 100 mg/ml prolactin in the presence of 500 nM β-estradiol for four additional days to simulate the secretory phase. Hormonal treatment was then withdrawn for 48h, and organoids were collected for scRNAseq (Hormonal Withdrawal 48h).  Organoids were then subjected to mechanical breakdown with automatic (300 times) and manual (100 times) pipetting. Organoid fragments were then replated and maintained without hormones for 48h to simulate the post-breakdown phase. Organoids were collected at post-breakdown 12h, post-breakdown 24h and post-breakdown 48h. Finally, organoids were re-exposed to E2 for another 48h to simulate the transition back to the proliferative phase and they were collected at E2-differentiation 24h and E2-differentiation 48h for scRNAseq. The nucleic acid extraction was performed following the manufacturer's instructions (Chromium Single Cell 3' v3.1, 10X Genomics) The libraries were constructed following the manufacturer's instructions (Chromium Single Cell 3' v3.1, 10X Genomics)