In vitro modelling of the human embryo implantation niche
In vitro modelling of the human embryo implantation niche
At the end of the culture, each implantation site was dissected using precision forceps and scissors in warm DMEM. The implantation site was washed with PBS devoid of Mg2+ and Ca2+. Enzymatic digestion was achieved using 50 µl of AccuMAX (Merck, SCR006) for 7 minutes at 37°C. AccuMAX contains collagenases which degrades the hydrogel releasing stromal and embryo cells. Single cell dissociation was achieved by manual pipetting using a micropipetter (Origio, MXL3-STR-CGR) and micropipetter flexible tips with decreasing apertures from 250 µm diameter (Origio, MXL3-275) to 50 µm diameter (MXL3-50, Origio). The reaction was stopped by transferring cells to ice cold PBS devoid of Mg2+ and Ca2+ and supplemented with 10% FBS.  Embryos were cultured from 5 d.p.f. as above until 14 d.p.f. At the end of the culture, each implantation site was dissected using precision forceps and scissors in warm DMEM. The implantation site was washed with PBS devoid of Mg2+ and Ca2+. Enzymatic digestion was achieved using 50 µl of AccuMAX (Merck, SCR006) for 7 minutes at 37°C. AccuMAX contains collagenases which degrades the hydrogel releasing stromal and embryo cells. Single cell dissociation was achieved by manual pipetting using a micropipetter (Origio, MXL3-STR-CGR) and micropipetter flexible tips with decreasing apertures from 250 µm diameter (Origio, MXL3-275) to 50 µm diameter (MXL3-50, Origio). The reaction was stopped by transferring cells to ice cold PBS devoid of Mg2+ and Ca2+ and supplemented with 10% FBS. Following this, cells from each implantation site cells were labelled using the 10X Genomics Chromium 3' CellPlex Kit Set A (10x Genomics Chromium PN-1000261). Pooled single-cell suspension samples were loaded onto a Chromium Next GEM Chip G (10X Genomics® Chromium Next GEM Single Cell 3' Reagents Kits v3.1). Reverse transcription and library construction were carried out according to the manufacturer's instructions. Libraries were sequenced on Illumina NovaSeq 6000 S2 lane at the Cancer Research Cambridge Institute Genomics Facility. Samples were sequenced using a paired-end sequencing run with: read 1: 28 cycles; i7 index: 10 cycles, i5 index: 10 cycles; read 2: 90 cycles The scaffolds were stimulated hormonally in two steps to mimic the proliferative phase and mid-secretory phases of the human endometrial cycle. In the first step, the scaffolds were stimulated with 10 nM β-oestradiol (E2) for 4 days as follows: 250 μL of organoid medium (+ 2% FBS, + 10 nM E2) in the upper compartment; 1 mL of stromal medium (2% FBS, + 10 nM E2) in the lower compartment. Media were replaced after 48 hours. In the second step, scaffolds were stimulated with full hormonal stimulation for 8 days with 10 nM β-oestradiol (E2), 1 µM progesterone (P4) and 1 µM 8-bromoadenosine 3′, 5′-cyclic monophosphate (cAMP) as follows: 250 μL of organoid medium (+ 2% FBS, + 10 nM E2, + 1 µM P4 and 1 µM cAMP) in the upper compartment, 1 mL of stromal medium (2% FBS, + 10 nM E2, + 1 µM P4 and 1 µM cAMP) in the lower compartment. Media were replaced every 48 hours. After incubation, only the recovered blastocysts that fully expanded (79%) were taken forwards and transferred onto endometrial scaffolds. For some embryos, the zona pellucida was removed by briefly treating the embryos with acidic Tyrode's solution (Origio REF10605000A). Human embryo medium (detailed below) to be added onto the upper compartment and medium to be added to lower compartment of the transwell (stromal + 2% charcoal stripped FBS supplemented with 10 nM β-oestradiol (E2) and 1 µM progesterone (P4)) were pre-equilibrated in an incubator for 3 hours before transfer of the embryo. We devised the composition of the upper medium and named as Human Embryo Medium (HEM). HEM-1 was used for the culture of embryos from 5 d.p.f. to 9 d.p.f.; HEM-2 was used for the culture of embryos from 9 d.p.f. to 12 d.p.f.; HEM-3 was used for the culture of embryos from 12 d.p.f. to 14 d.p.f. HEM-1 (300 µL per scaffold) contains the following: Advanced DMEM/F12 (Gibco, 12634-010) supplemented with 1 x B27 (Gibco, 12587-010), 1 x N2 supplement (Gibco, 17502-048), 2 mM L-glutamine, 0.2 % Penicillin-Streptomycin (10000 U/mL, Gibco, 15140122), 1.25 mM N-Acetyl-L-cysteine (NAC, Sigma, A9165), 10 mM Nicotinamide (Sigma, N0636), 2% charcoal stripped FBS (Sigma Aldrich, F6765), 5% Human Serum Albumin (Vitrolife 10064), 0.5 mg/mL hyaluronan (Abcam, Ab143634), 10 nM β-oestradiol (E2), 1 µM progesterone (P4), 20 ng/mL Prolactin (Peprotech 100-07), 1 µg/mL human Chorionic Gonadotropin (hCG, RP-1484, Alpha Diagnostic Intl) and 20 ng/mL human Placental Lactogen (hPL, R&D 5757-PL/CF), 50 ng/mL 1 IGF-1 (Cell Guidance Systems GFH34AF) and 50 ng/mL epidermal growth factor (EGF, Peprotech, AF-100-15). As nutrient requirements of embryos increase, HEM-2 (350 µL) is introduced at 9 d.p.f. and contains all compounds present in HEM-1 with the following modifications or addition: charcoal stripped FBS concentration increased from 2% to 5%; 7.5 mM glucose (Sigma, G8644); 1X Insulin-Transferrin-Selenium-Ethanolamine (Gibco, 51500-056), essential (1X) and non-essential amino acids (1X) (Gibco 11130-036 and Gibco 11140-035). Upon embryo attachment, 10 µM of ROCK inhibitor (Y-27632) was added to HEM-2. HEM-3 (400 µL) is introduced at 12 d.p.f. and contains all compounds present in HEM-2 with the following modifications: charcoal stripped FBS concentration increased from 5% to 10%; Human Serum Albumin concentration increased from 5% to 10%; 10 µM of ROCK inhibitor. HEM-2 and HEM-3 require pH adjustments using 1M sodium bicarbonate. Half volume of each HEM medium was replaced daily with fresh medium (medium can be made and stored at +4C for 4 days). Lower compartment of the transwell culture system contains 1 mL of stromal medium (2% charcoal stripped FBS, with 10 nM E2 and 1 µM P4) throughout the entire culture. Culture conditions were 37°C, 20% O2 and 5% CO2. Following this, cells from each implantation site cells were labelled using the 10X Genomics Chromium 3' CellPlex Kit Set A (10x Genomics Chromium PN-1000261).  Pooled single-cell suspension samples were loaded onto a Chromium Next GEM Chip G (10X Genomics® Chromium Next GEM Single Cell 3' Reagents Kits v3.1). Reverse transcription and library construction were carried out according to the manufacturer's instructions.