scRNA-seq of organoids derived from fetal uterine epithelial primary samples
scRNA-seq of organoids derived from fetal uterine epithelial primary samples
Fetal uterine organoids were derived from 12 PCW (Hrv_276_ORG) and 17 PCW (Hrv_277_ORG) fetal reproductive tract samples, (developing uterus, cervix, and vagina) following tissue dissociation. The single-cell suspensions were washed in Advanced DMEM/F12 (Gibco, 12634-010), centrifuged and the cell pellet resuspended in Matrigel (Corning, 356231), at around a 1:3 ratio (pellet volume : Matrigel volume).  The organoids were cultured within 25 µL Matrigel domes in 48-well tissue treated plates covered by 250 µL of basal endometrial organoid media (Advanced DMEM/F12 (Gibco, 12634-010), 1% GLUTAMAX  (Gibco, 35050061), 1% Insulin-Transferrin-Selenium (ITS -G)(Gibco, 41400045), 100 µg/mL Primocin (Invivogen, ant-pm-1), 1X B27-Vitamin A (Life Technologies, 12587010), 1x N2 (Life Technologies, 17502048), 1.25 mM N-Acetyl-L-cysteine (Sigma-Aldrich, A9165-5G), 2 mM Nicotinamide (Sigma, N0636-100G), 2 ng/mL Recombinant Human FGF-basic (154 a.a.) (Peprotech, 100-18B), 500 ng/mL recombinant human R-spondin-1 (R&D Systems, 4645-RS-01M/CF), 10 µM SB202190 (p38i) (StemCell Technologies, 72632), 500nM A83-01 (Tocris, 2939), 50 ng/mL recombinant human EGF (Peprotech, AF-100-15), 10 ng/mL recombinant human FGF-10 (Peprotech, 100-26) and 100 ng/mL recombinant human Noggin (Peprotech, 120-10C))140. The media was supplemented with 10 µM of ROCK inhibitor Y-27632 (Millipore, 688000) for the first 2 days of organoid line establishment with full media changes every 2-3 days.  All organoid lines were split and passaged approximately every 5-7 days, depending on their size and density. TrypLE™ Express Enzyme  (Gibco, 12604013) was added to each well, and domes were detached with either a 1000ul tip or cell scraper before being transferred to a 15ml Falcon tube. The organoids were dissociated into cell clumps by forcefully pipetting the solution 15-30 times using a 1000 µL tip, followed by incubation at 37 °C for 6-8 mins. Advanced DMEM/F12 was added at at 1:1 ratio to quench TrypLE™ Express Enzyme volume and  pipetted up/down 10 more times with the 1000 µL tip. Cell suspensions were centrifuged at 800g for 2 min at 4°C. The supernatant was removed as close to the pellet as possible. 30 µL of cold Matrigel per desired dome were added and the pellet was slowly resuspended to evenly distribute the cells. 30ul domes were seeded into 6, 12 or 24 well tissue culture treated plates depending on whether the organoids were being expanded or set-up for drug treatment. The domes were placed in the incubator for 10 min at 37 °C, followed by the addition of basal endometrial organoid media supplemented with 10 µM of ROCK inhibitor Y-27632.   For all drug treatment experiments, organoids were passaged 48 hours before drug addition. Organoids were plated in 30µl domes as described above in two technical replicates, each containing 2 organoid domes. For testing the effect of endocrine disruptors on fetal reproductive organoids, Hrv_276_ORG and Hrv_277_ORG lines were treated with either 10μM of Bisphenol A (BPA) (Sigma, 239658) or 100μM of Benzyl butyl phthalate (BBP) (Sigma, 308501), with controls receiving the  volume of DMSO as BPA/BBP administered. For the endocrine disrupter experiments BPA, BBP or DMSO were added to basal endometrial organoid media (as above), but with a phenol red free DMEM/F12 as the base media (Merck, D6434). Organoids were treated with BPA or BBP for a total of 96 or 144 hours with a full media change every 48 hours. After 96 or 144 hours of drug treatment, organoids were dissociated into a single-cell suspension. Briefly, organoids were collected and washed in ice-cold phenol red free DMEM/F12 (BPA, BBP, DMSO control) and centrifuged at 600g for 6 mins at 4 °C. The supernatant was removed and replaced with TrypLE™ Express Enzyme at a ratio of 500ul per 30ul dome, and pipette mixed with a p1000 tip for 30 times. Organoid suspensions were incubated at 37 °C for 15-25 mins, with manual shaking every 2 mins. Cells were checked at 15 mins then every 5 mins until an adequate single cell suspension was achieved ~70% single cells. Once the cells were sufficiently digested, TrypLE™ was quenched with phenol red free DMEM/F12. Cells were re-centrifuged and media was aspirated leaving ~ 50ul of media. The cell suspensions were then vigorously pipette mixed with a p20 30-60 times. To this suspension 200μl of 1% PBS/BSA was added, thoroughly mixed and passed through a 70μm filter. Live cells were counted using Trypan blue. For the scRNA-seq experiments, cells were loaded according to the manufacturer's protocol for the Chromium Next GEM Single Cell 5′ v2 (DUAL) Kit  (10X Genomics) to attain between 2,000 and 10,000 cells per reaction. Library preparation was carried out according to the manufacturer's protocol.