a, Growth factors (HGF, PGE2, Y-27632 and nicotinamide) were added as supplements to basal TOM that contains EGF, CHIR99021, R-spondin 1, A83-01 and FGF2 (Supplementary Table 1a, b). Bright-field images of placental digests at passage 1, day 7. The cystic structures that appear with the addition of nicotinamide (red asterisks) are contaminating maternal glandular organoids. Representative images from n = 2. Conditions containing factors that did not show growth are not included. Scale bars, 500 μm. b, Trophoblast organoid cultures at passages 2 and 10 with continuous culture. Representative images from n = 3. Scale bars, 500 μm. c, Analysis of genetic stability of cultures (n = 2) with comparative genomics hybridization (CGH) array. Shown is a representative whole-genome array CGH plot generated with Agilent Cytogenomics software. Genomic DNA from late passage (passage 8) trophoblast organoids is compared to genomic DNA from an early passage (passage 2). Each spot is a single probe. Plotted are the log ratios of the average signal intensity of each probe on the y axis along its position on the chromosomes (1–22, X and Y) on the x axis. A log signal ratio of 0 represents equivalent copy number in the samples. No significant DNA copy number abnormalities were identified. d, Live imaging of trophoblast organoid cultures (n = 2) passaged for more than 6 months and then frozen, thawed and exposed to Mitotracker Red. Functional mitochondria are visible showing that the cells are healthy (white arrowheads). Scale bars, 50 μm (whole organoid) and 10 μm (individual cells). e, Organoids derived from the same placental cell isolate using either trophoblast or decidual organoid medium (TOM or ExM, respectively) demonstrate that matched placental (fetal) and decidual (maternal) organoids can be derived from one sample. Representative bright-field images from n = 3. Scale bars, 500 μm.