Isolating and Culturing Trophoblasts

Primary cells of the second trimester were purchased (Cat number 7120, ScienCell, Carlsbad, CA). As demonstrated in our previous study, EVs exert different effects on early stage trophoblast vs. term trophoblast cell cultures. Specifically, in HP women, treatment with EVs decreased apoptosis and induced higher migration in early-stage trophoblasts compared with untreated. Conversely, exposure to EVs obtained from women with GVCs increased term trophoblast apoptosis and inhibited early-stage trophoblast migration when compared to cells exposed to HP EVs (Shomer et al., 2013 (link)). Because GVCs begin earliest at 20 weeks of gestation (Peracoli et al., 2019 (link)), we preferred to study the effects of EVs on early stage trophoblasts and therefore did not culture HVT from term placentas. HVT were grown in a recommended trophoblast medium (50%, catalog number 7121, Sciencell) supplemented with Dulbecco’s Modified Eagle Medium–high glucose (22%, Biological Industries, Israel), F-12 (HAM) nutrient mixture (22%, Biological Industries, Israel), fetal calf serum (FCS) (10%, Biological Industries, Israel), and penicillin G sodium salt (10,000 units/mL) - streptomycin sulfate (10 mg/ml)–nystatin (1,250 units/mL) solution (1%, Biological Industries, Israel). The cells were grown at 37°C, 5% CO2. Passages 4-8 were used for experiments. HVT cell cultures were labeled with anti-hPL to ensure quality control of their contents. As in our previous studies, greater than 90% labeling with anti-hPL was considered a pure trophoblast culture (Shomer et al., 2013 (link)).