Preparation of extravillous trophoblast from the basal plate of term human placenta

Abstract

Introduction: Placenta accreta-related pathologies are characterized by excessive trophoblast invasion of the uterus. Evidence suggests that the trophoblast-decidual interaction is abnormal in these pathologies. We decided to isolate extravillous trophoblast (EVT) from term placenta as a precursor to obtaining and investigating these cells in accreta-related pathologies. Methods: Basal plate tissue (2-3 mm) from normal term placenta was digested using trypsin, collagenase or dispase, individually or in combination. After digestion cells were spun on a 30%:60% discontinuous Percoll gradient and the interface layer was collected. Cells were incubated with a mAb to HLA-G (clone MEM/G9) coupled to phycoerythrin (PE; Abcam) for 30min, washed, then incubated for a further 15minwith anti-PEmagnetic microbeads. Positive selection of HLA-G-labeled cells was performed on an AutoMACS magnetic cell separator (Miltenyi). Cell fractions were analyzed by flow cytometry using PE and the dead cell stain, SYTOX Green, as markers. Results: Use of collagenase or dispase in DMEM/F12 for digestion produced a significant degree of cell death whereas there was minimal cell death with these agents when digested in HBSS. A combination of 0.25% trypsin, 0.1% dispase, 0.1% collagenase IV in HBSS (3 x 20min) gave the best digestion as assessed by number of cells released and percentage of dead cells (1.6 0.4%; mean SEM, n1⁄43). Optimal anti-HLA-G dilutionwas 1:500. HLA-G-labeled cells comprised 0.9 0.3% of total after digestion, 23.7 5.7% after gradient separation and 94.3 2.5% following magnetic bead separation. Of the non-PE (HLA-G) labeled cells remaining in final fraction, 5.9 1.0% were SYTOX Green-stained suggesting that the primary contaminating component is a dead cell population. Conclusions: The method outlined here selects for a fraction highly enriched in the EVT-specific marker, HLA-G. This technique appears to isolate EVT cells from third trimester placenta and will be of value in the investigation of accreta-related pathologies.