Ultra low cell attachment 6 well plates

NHDFs were purchased from Lonza Bioscience company and cultured in fibroblast basic medium-2 (FBM-2) with a FGM-2 SingleQuot Kit supplement (Lonza Bioscience Company, Singapore). Cell (passage 4-7)-seeded gelatin–PEG and elastin hybrid hydrogels were prepared. The precursor solution was made by dissolving the elastin–PEG powder into gelatin–PEG solution with 0.1 w/v % Irgacure 2959 at 37 °C as the gelatin–PEG precursor was purified and stored in PBS (mentioned in Section 2.1) at –80 °C. Cell-seeded constructs were made from 100 μL aliquots of the cells in a suspension of gelatin–PEG and elastin–PEG to give a final cell density of 2 × 10⁶ cells/mL (Figure 1B). The cell bearing solution was deposited into a flat-bottom 96-well plate as the mold. After UV photopolymerization, the cell-seeded hydrogels were transferred into ultra-low cell attachment 6-well plates (Corning, New York, NY, USA), washed with PBS and immersed in the culture medium. Cell encapsulated elastin–PEG only hydrogel (45 mg/mL) was prepared by the same method as gelatin/elastin hybrid PEG hydrogels.

hEPSCs and hESCs were differentiated into trophoblastic spheroids (BAP‐EB) as previously described^[25] with slight modifications. Briefly, the cells were digested and seeded in AggreWell 400 plates (Stemcell Technologies) at a density of 150 cells/EB for hEPSCs or 200 cells/EB for hESCs. After 24 h, the EBs were transferred to ultra‐low cell attachment 6‐well plates (Corning). The differentiation medium consisted of mouse embryonic fibroblast conditioned medium: MEF‐CM supplemented with 10 ng mL⁻¹ BMP4 (R&D), 1 µm A83‐01 (Stemgent), and 0.1 µm PD173074 (Stemgent). In some experiments, the trophoblast differentiation was conducted in monolayer. The cells were digested and seeded onto Matrigel‐coated plates (Corning) at 0.025 × 10⁶ cells/cm² in MEF‐CM. The differentiation medium was changed from the next day onwards. In some experiments, HES medium (knockout DMEM, 15% KOSR, 1× glutamine, 1× penicillin–streptomycin, 1× NEAA, and 0.1 µm 2‐mercaptoethanol) was used as the differentiation medium with supplementation of different doses of BAP: high (BMP4: 10 ng mL⁻¹, A83‐01: 1 µm, PD173074: 0.1 µm), mid (BMP4: 0.5 ng mL⁻¹, A83‐01: 0.5 µm, PD173074: 0.1 µm), and low (BMP4: 0.5 ng mL⁻¹, A83‐01: 0.1 µm, PD173074: 0.1 µm).