The multinucleated syncytiotrophoblast covers the human placenta and releases large quantities of extracellular vesicles (EVs) into the maternal blood. These EVs are lipid enclosed packages of cellular contents that are implicated in the fetal regulation of maternal adaptation or mal-adaptation to pregnancy. We pioneered the use of placental explants to generate EVs but are not amenable to long-term manipulations. Here we investigate an alternative model capable of stable generation of placental EVs.
Trophoblast cells were obtained from term placentae following sequential Dispase digestion. Concentrations and sizes of EVs were determined by Nanoparticle tracking analysis (NTA). Purity and viability were determined by FACs analysis (w6/32 and propidium iodide negative, respectively). Results were expressed as mean ± SEM.
There were approximately 2.51e+06 ± 0.23 cells isolated/gram of placenta (n = 4). Of these, 91.3 % ± 2.23 were viable but 3.7 % ± 1.45 (n = 3) were not cytotrophoblasts (w6/32+). By day three of culture, syncytialisation of the trophoblasts was obvious with loss of adjoining plasma membranes visualized by PKH67 staining.
NTA analysis of media, sampled every second day, indicated 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. 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.
It has been long known that mononuclear trophoblasts spontaneously form syncytial structures during in vitro culture. Here we show preliminary evidence that the EVs produced by such cultures are similar in quantity to those produced by placental explants. Unlike explants, we have maintained some of these cultures with minimal contamination for at least 35 days and therefore they may be a useful tool for the study of placental EVs.