A microfluidics assay to study invasion of human placental trophoblast cells

Abstract

Pre-eclampsia, fetal growth restriction and stillbirth are major pregnancy disorders throughout the world. The underlying pathogenesis of these diseases is defective placentation characterized by inadequate invasion of extravillous placental trophoblast cells into the uterine arteries. How trophoblast invasion is controlled remains an unanswered question but is influenced by maternal uterine immune cells called decidual natural killer cells. Here, we describe an in vitro microfluidic invasion assay to study the migration of primary human trophoblast cells. Each experiment can be performed with a small number of cells making it possible to conduct research on human samples despite the challenges of isolating primary trophoblast cells. Cells are exposed to a chemical gradient and tracked in a three-dimensional microenvironment using real-time high-resolution imaging, so that dynamic readouts on cell migration such as directionality, motility and velocity are obtained. The microfluidic system was validated using isolated trophoblast and a gradient of granulocyte-macrophage colony-stimulating factor, a cytokine produced by activated decidual natural killer cells. This microfluidic model provides detailed analysis of the dynamics of trophoblast migration compared to previous assays and can be modified in future to study in vitro how human trophoblast behaves during placentation.

Keywords: human; microfluidics; placentation; trophoblast.

Figure 1.

Trophoblast invasion. The placenta implants into the maternal decidua during the first trimester of pregnancy. Fetal extravillous trophoblasts (EVTs) detach from the implanting placenta and invade the maternal decidua to remodel uterine spiral arteries. Maternal leucocytes present at the maternal–fetal interface, including decidual natural killer (dNK) cells, may regulate trophoblast invasion and transformation of the spiral arteries by secreting cytokines such as GM-CSF. (Online version in colour.)

Figure 2.

Microfluidics as a model for trophoblast invasion. EVTs are isolated from first trimester placentas, stained with a cell tracker and embedded in growth factor-reduced Matrigel in the central hydrogel channel. (a) A constant flow of medium is applied in the two side channels, one with (channel A) and without hrGM-CSF (channel B) to create a gradient of the cytokine across the hydrogel channel. Individual cell tracks are generated from time lapse microscopy. (b) To confirm the purity of cells embedded in the microfluidic device, EVT were immune-stained for HLA-G (green) and the nucleus of each cell stained with DAPI dye. (Online version in colour.)

Figure 3.

Image acquisition and analysis of individual cell tracks. (a) Primary EVTs embedded in Matrigel are subjected to a chemical gradient of hrGM-CSF for 12 h. EVTs are tagged with a fluorescent cell tracker and imaged at five positions using an LSM 700 confocal microscope in the x, y and z-direction. Images are processed with TrackMate (FIJI) and converted into cells tracks and then quantified into velocity, directionality and motility. The track colours are automatically generated by TrackMate and have no significance. (b) Directionality is the ratio of the net distance a cell migrates over the total distance. (c) Motility is found by subtracting the number of cells migrating upstream towards channel A (N_A) by the number of cells migrating downstream (N_B) away and dividing this by the total number of cells (N_t). (Online version in colour.)

Figure 4.

Characterization of the chemical gradient across the hydrogel channel during constant fluid flow. (a) Fluorescein dextran of similar molecular weight to human GM-CSF was added to the medium in channel A, constant fluid flow of 50 µl h⁻¹ per channel was applied, and the gradient was investigated for 12 h. The gradient gradually builds up over a time period of 1 h and is stable for at least 12 h. (b) Computational model for gradient generation demonstrates similar gradient at the device mid-plane at steady state to experimentally observed gradient 12 h. (c) Plot of fluorescein intensity across device, with width 1300 µm demonstrates that linear gradient is established after 1 h. (d) Plot of mean intensity and standard deviation (black bars) for four independent devices demonstrates linear gradient in the central hydrogel region, with width 900 µm (each triangular post is 200 µm in length) is similar to the computationally predicted gradient (red line). C/C₀ is concentration of tracer normalized by the concentration in the media channel. (Online version in colour.)

Figure 5.

Recombinant hrGM-CSF stimulates the migration of human extravillous trophoblasts (EVTs). (a) Cell tracks and polar histograms show the random migration of EVTs in the control compared to directed migration when 10 ng ml⁻¹ GM-CSF is added to channel A. A representative experiment is shown out of five performed. Only cells that migrated more than 30 µm were analysed. Cells migrating towards channel A (hrGM-CSF) and towards channel B (no hrGM-CSF) are coloured blue and red, respectively. Polar histograms visualize the number of cells that migrate in different directions (inner radial axis). The outer radial axis represents migration angles. (b) The difference of cell fractions migrating towards and away from hrGM-CSF was quantified. Significantly more cells migrate towards GM-CSF when exposed to 10 ng ml⁻¹ hrGM-CSF (n = 5). (c) Trophoblast cells migrate with increased directionality in the presence of GM-CSF. *p < 0.05 calculated using the unpaired t-test. (Online version in colour.)

Figure 6.

GM-CSF produced by dNK cells after activation of KIR2DS1. To activate KIR2DS1, dNK cells are stimulated with plate-bound (a) mAb, EB6 or (b) negative control IgG. The levels of GM-CSF are measured by ELISA and show a significantly increased production of GM-CSF by dNK cells from donors with the KIR2DS1 gene but not those who lack it. *p < 0.05, ns—not significant, determined using the Wilcoxon rank-sum test. (Online version in colour.)

Figure 7.

GM-CSF produced by dNK cells after activation of KIR2DS1 stimulates trophoblast migration. Trophoblast migration measured with a chemical gradient of supernatants from dNK cells after (a) KIR2DS1+ dNK cells activated by mAb, EB6 (supernatant I), anti-GM-CSF neutralizing antibody (supernatant II) or negative control (supernatant III). (b) Trophoblast cells migrate with increased directionality towards supernatants produced by activated KIR2DS1+ dNK cells. Directed migration is reduced when anti-GM-CSF neutralizing antibody is added. (c) A large fraction of cells migrate towards increasing concentrations of supernatants produced by activated KIR2DS1+ dNK cells. *p < 0.05 calculated using one-way analysis of variance (ANOVA) and Dunn's multiple comparisons test. (Online version in colour.)