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

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.

Development and validation of real-time recombinase polymerase amplification-based assays for detecting HPV16 and HPV18 DNA

“…Patients with a positive or suspicious result underwent cytological screening. Exfoliated cells were rinsed with 20 mL of ThinPrep PreserveCyt (Hologic, Marlborough, MA, USA) fixing solution immediately after removal of the brush from the cervix and the samples were stored at 4°C ( 51 ). These patients underwent colposcopy examination and two biopsy specimens were collected from adjacent sites in each patient.…”

Advances in Biosensors and Diagnostic Technologies Using Nanostructures and Nanomaterials

“…[457] Similar principles have also been applied to the sorting and analysis of rare bacterial cells within blood and urine, as well as fetal cells in prenatal diagnostics. [387,388,414,458,459] Rare cell biosensors illustrate some of the more creative nanotechnology approaches that can overcome limitations of many traditional instruments and diagnostic techniques.…”

Preparation of Tissues and Heterogeneous Cellular Samples for Single-Cell Analysis

“…Different cell types settle and adhere differently in in vitro environments, due to a combination of material interaction and mechanical properties. Bailey-Hytholt et al recently demonstrated effective rare cell selection via different settling behavior of cells in polystyrene wells [63]. This approach was used in a prenatal testing workflow for the purpose of separating rare extravillous trophoblasts from infants in utero from maternal cervical cells in cervical swabs.…”