(346g) A Rapid Method for Label-Free Enrichment of Rare Trophoblast Cells from Cervical Samples

Authors: 
Bailey-Hytholt, C. M., Brown University
Sayeed, S., Brown University
Joseph, R., PerkinElmer
Kraus, M., PerkinElmer
Shukla, A., Brown University
Tripathi, A., Brown University
Extravillous trophoblasts (EVTs), cells that originate from the placenta and invade the endometrium, have the potential to provide the entire fetal genome for prenatal testing. Current prenatal diagnostic techniques, such as the amniocentesis and chorionic villous sampling, are invasive, carrying risk for miscarriage and infection. During trophoblast invasion, EVTs enter the endocervical canal, which can be sampled by a cervical swab. Previous studies have determined the presence of trophoblast cells within these collected cervical samples. However, for downstream prenatal diagnostics, the trophoblast cell needs to be free of any maternal cells or DNA. With the abundance of cervical and mucus content compared to few trophoblast cells in these samples, there is a need for a trophoblast isolation technique. We report a rapid and inexpensive method that would enrich trophoblast cells within clinical cervical samples.

In this study we provided a facile workflow that eliminates at least 90% of cervical cells while capturing at least 70% of fetal trophoblast cells. Our workflow allowed JEG-3 cells (a common trophoblast cell line) and clinical cervical samples taken from 5 to 20 weeks of gestation to settle in a polystyrene well for a variable amount of time. After the incubation time, we removed the supernatant, which contained a large majority of cervical cells. Remaining in the capture well was the enriched population of trophoblast cells. We determined that 4 min of settling before removing the supernatant provided an optimal capture and a 700% purity increase of the sample. After 4 min, the enrichment decreased likely due to cervical mucus settling at longer time points. We further investigated the role of mucus by investigating cervical samples that were washed in either 1x PBS or PreservCyt (the fixative solution cervical samples are collected in). After washing the samples, we observed a decrease in overall enrichment, suggesting the presence of mucus is important for the differential settling and enrichment process. We determined that the initial number of JEG-3 cells incubated with the cervical sample does not affect our enrichment, which is important when the quantity of trophoblasts within cervical samples can vary from patient to patient. Finally, we performed a proof-of-concept test, integrating our enriched trophoblast cell population with an automated cell picker. We successfully isolated single trophoblast cells, both from our JEG-3 incubated sample, and clinical fetal trophoblasts that expressed human leukocyte antigen G (HLA-G). Whole genome amplification and gender polymerase chain reaction (PCR) showed strong Y chromosome signal for the isolated JEG-3 cell, indicating that maternal mucus or cells did not overwhelm the JEG-3 DNA. Ultimately, our workflow can make new isolation techniques, such as cell picking, more efficient and effective towards non-invasive prenatal testing.