Matrix directs trophoblast differentiation in a bioprinted organoid model of early placental development

Trophoblast organoids can provide crucial insights into mechanisms of placentation, however their potential is limited by highly variable extracellular matrices unable to reflect in vivo tissues. Here, we present a bioprinted placental organoid model, generated using the first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol (PEG) matrix. Bioprinted or Matrigel-embedded organoids differentiate spontaneously from cytotrophoblasts into two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs). Bioprinted organoids are driven towards EVT differentiation and show close similarity with early human placenta or primary trophoblast organoids. Inflammation inhibits proliferation and STBs within bioprinted organoids, which aspirin or metformin (0.5 mM) cannot rescue. We reverse the inside-out architecture of ACH-3P organoids by suspension culture with STBs forming on the outer layer of organoids, reflecting placental tissue. Our bioprinted methodology is applicable to trophoblast stem cells. We present a high-throughput, automated, and tuneable trophoblast organoid model that reproducibly mimics the placental microenvironment in health and disease. Overall design: ACH-3P first trimester trophoblast cell line was cultured as organoids in a tranditional Matrigel and was in bioprinted as organoids using RASTRUM bioprinter (Inventia Life Science). Bioprinted and matrigel-embedded organoids were dissociated to single cells by accutase. Single-cell RNA sequencing was performed on the single cells extracted from approximately 700 organoids in total.