Derivation and Maintenance of Human Trophoblast Organoids

To obtain trophoblast-enriched cell suspensions, villi from first trimester placental tissue were sequentially digested with 0.2% trypsin-250 (Pan Biotech P10-025100P)/0.02% EDTA (Sigma #E9884)/PBS then collagenase V at 1.0mg/ml (Sigma #C9263) in Hams F12/10% FBS. Both digests were pooled, washed in Advanced DMEM/F12 medium (Gibco #12634-010) and re-suspended in ~10x volume growth-factor-reduced Matrigel (Corning #356231) on ice. 25µL drops were plated per well into a 48-well culture plate (Costar #3548), set at 37°C for 15min and overlaid with 250µL Trophoblast Organoid Medium (TOM, Supplementary Table 1b). Cultures were maintained in 5% CO₂ in a humidified incubator at 37°C. Medium was replaced every 2-3d. Small organoid clusters became visible by ~7d and were passaged when at least 50% had reached a diameter of 200-300µm (usually between 7-10d). Mechanical disruption was achieved with Eppendorf Explorer Plus automatic pipettes on a mix cycle of 99 rounds (x4-5), maximum speed. Organoids from the same sample were initiated and maintained in the absence of each individual component to test its importance. Frozen stocks of organoids were made in 70% TOM, 20% FBS and 10% DMSO freeze medium and stored in liquid nitrogen. A step-by-step protocol of the derivation and maintenance of human trophoblast organoid cultures can be found at Nature Protocol Exchange24 .