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

Trophoblast organoids offer a unique opportunity to investigate mechanisms orchestrating placental growth and development during pregnancy. However, many organoid cultures rely on extracellular matrix reagents that are highly variable and unable to be tuned to reflect different tissues. Here we describe a new bioprinted placental organoid model using immortalised first trimester trophoblast cell line, ACH-3P, and a synthetic polyethylene glycol matrix. Bioprinted organoids were comprehensively compared to classical Matrigel-embedding using functional assays, immunofluorescence microscopy, transcriptomic and proteomic analysis. Organoids spontaneously differentiated from cytotrophoblasts into the two major subtypes: extravillous trophoblasts (EVTs) and syncytiotrophoblasts (STBs), with bioprinted organoids driven towards EVT differentiation. Bioprinted organoids were exposed to inflammation and treated with aspirin or metformin to assess their effects on trophoblast organoid formation and viability. Further, we reversed the inside-out architecture of ACH-3P organoids by transferring immature organoids to suspension culture. Organoid suspension caused STB to form a syndecan-1+ outer layer on the periphery of organoids, reflecting placental tissue. We present an alternative trophoblast organoid model with potential for further tuning to accurately reflect the placental microenvironment and provide more reliable insights into early placental development. 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.