Abstract
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.
Highlights
During trophoblast invasion Extravillous trophoblasts (EVTs) enter the endocervical canal[5], which can be sampled by a cervical swab
In this study we provide an optimized workflow for enriching trophoblast cells from a heterogeneous cervical cell population
Using JEG-3 cells, we conclude that enrichment is possible with removing at least 90% of squamous cervical cells while capturing at least 70% of JEG-3 cells at an optimal 4 min settling time
Summary
During trophoblast invasion EVTs enter the endocervical canal[5], which can be sampled by a cervical swab. With the frequency of one EVT per 2,000 cervical cells[5], novel isolation methods are needed for downstream testing to provide a quality sample that is not overwhelmed by maternal cells. Common cell separation techniques are based on cell density, size, shape, piezoelectric effects, electric capacitance, magnetic susceptibility, hydrodynamic properties, and affinity to antibodies[11,12]. Many of these techniques are not suitable for capturing EVTs with minimal cell loss or equipped to handle the cervical matrix[13]. Our strategy allows enrichment to a degree that improves the ability to pick and isolate a single trophoblast cell while effectively removing maternal contamination. We used cervical cells from clinical Papanicolaou (Pap) tests stored in
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