Background: Human placental syncytiotrophoblast releases large amounts of extracellular vesicles (EVs) into maternal blood throughout gestation. These EVs are hypothesised to contribute to the significant physiological adaptations of the maternal body to pregnancy. As the syncytiotrophoblast is a giant non-replicating cell, placental explants are often used as rich a source of EVs but explants are short-lived and not amenable to long-term studies such as those involving genetic manipulation. Here we investigate the ability of an alternative model, syncytialisation of primary mononuclear trophoblasts and investigate the production of EVs.
Methods: Trophoblast cells were isolated from term placentae and their purity and viability determined by FACs analysis. Concentrations and sizes of EVs were determined by Nanoparticle tracking analysis (NTA). DNA in the EVs was analysed by Qubit, TapeStation and long range PCR. Results were expressed as mean ± SEM.
Results: Approximately 90% of the isolated trophoblasts were viable and they were 97.3 % ± 1.45 (n = 3) pure. There was no significant change in the rate of micro-EVs (p = 0.45, n = 4) or nano-EVs (p = 0.15, n = 4) produced from days 3-12 of culture. Cultured trophoblasts produced equivalent quantities of nano-EVs but significantly less micro-EVs (p = <0.0001, n = 4) per cm2 of syncytiotrophoblast than placental explants. Qubit assays indicated that there was less DNA in micro-EVs than nano-EVs at day 3 (p = 0.0488, n = 3) but not at days 5 (p = 0.8810, n = 3) or 7 (p = 0. 0.8106, n = 3). TapeStation analysis indicated DNA as large as 15,000 bp was present in Nano-EV while long range PCR shows Micro-EVs contained intact CSH1 genes (~7,000 bp).
Conclusions: Here we show preliminary evidence that the EVs produced by primary cultures of trophoblasts are similar in quantity and quality to those produced by placental explants. Unlike explants, which routinely survive for only 3-4 days, we have maintained our cultures for up to 35 days. These results suggest that primary cultures of trophoblasts may be a viable model to study production and manipulation of placental EVs long-term.