A Rapid Method for Label-Free Enrichment of Rare Trophoblast Cells from Cervical Samples

Extravillous trophoblasts (EVTs) have the potential to provide the entire fetal genome for prenatal testing. Previous studies have demonstrated the presence of EVTs in the cervical canal and the ability to retrieve a small quantity of these cells by cervical sampling. However, these small quantities of trophoblasts are far outnumbered by the population of cervical cells in the sample, making isolation of the trophoblasts challenging. We have developed a method to enrich trophoblast cells from a cervical sample using differential settling of the cells in polystyrene wells. We tested the addition of small quantities of JEG-3 trophoblast cell line cells into clinical samples from standard Pap tests taken at 5 to 20 weeks of gestation to determine the optimal work flow. We observed that a 4 min incubation in the capture wells led to a maximum in JEG-3 cell settling on the surface (71 ± 10% of the initial amount added) with the removal of 91 ± 3% of the cervical cell population, leading to a 700% enrichment in JEG-3 cells. We hypothesized that settling of mucus in the cervical sample affects the separation. Finally, we performed a proof-of-concept study using our work flow and CyteFinder cell picking to verify enrichment and pick individual JEG-3 and trophoblast cells free of cervical cells. Ultimately, this work provides a rapid, facile, and cost-effective method for enriching native trophoblasts from cervical samples for use in subsequent non-invasive prenatal testing using methods including single cell picking.