Identification of optimal conditions for human placental explant culture and extracellular vesicle release.

Extracellular vesicles (EVs) can mediate intercellular communication, including signaling between the placenta and maternal tissues. Human placental explant culture is a versatile model system to investigate placental function. We performed systematic studies in different tissue culture media types and oxygen tensions to identify a defined serum-free culture condition that supports high trophoblast viability and metabolism, as well as the release of similar populations of EVs, compared to traditional undefined conditions that contain media additives potentially contaminated with exogenous EVs. We also determined the time frame in which trophoblast viability and functionality remain optimal. Multiplex vesicle flow cytometry with classical EV and placenta-specific markers revealed three separate populations of explant-derived EVs: small CD63 EVs; large PLAP+ EVs; and CD63-/PLAP- EVs. These culture and analytical approaches will enable modeling of short-term effects of environmental perturbations associated with pregnancy complications on placental function and EV release.