A microphysiological model of the human placental barrier

Abstract

During human pregnancy, the fetal circulation is separated from maternal blood in the placenta by two cell layers - the fetal capillary endothelium and placental trophoblast. This placental barrier plays an essential role in fetal development and health by tightly regulating the exchange of endogenous and exogenous materials between the mother and the fetus. Here we present a microengineered device that provides a novel platform to mimic the structural and functional complexity of this specialized tissue in vitro. Our model is created in a multilayered microfluidic system that enables co-culture of human trophoblast cells and human fetal endothelial cells in a physiologically relevant spatial arrangement to replicate the characteristic architecture of the human placental barrier. We have engineered this co-culture model to induce progressive fusion of trophoblast cells and to form a syncytialized epithelium that resembles the syncytiotrophoblast in vivo. Our system also allows the cultured trophoblasts to form dense microvilli under dynamic flow conditions and to reconstitute expression and physiological localization of membrane transport proteins, such as glucose transporters (GLUTs), critical to the barrier function of the placenta. To provide a proof-of-principle for using this microdevice to recapitulate native function of the placental barrier, we demonstrated physiological transport of glucose across the microengineered maternal-fetal interface. Importantly, the rate of maternal-to-fetal glucose transfer in this system closely approximated that measured in ex vivo perfused human placentas. Our "placenta-on-a-chip" platform represents an important advance in the development of new technologies to model and study the physiological complexity of the human placenta for a wide variety of applications.

Figure 1

A. Schematic of a human fetus and placenta within the uterine cavity. The placenta is anchored to the uterine wall and connected to the developing fetus via the umbilical cord. B. Cross-sectional view of the placenta illustrates the placental cotyledons. Each cotyledon consists of a stem chorionic villus and its branches. These villi are in direct contact with maternal blood in the intervillous space. C. The maternal intervillous space is separated from the lumen of the fetal capillary by the maternal-fetal interface composed of the syncytiotrophoblast, basal lamina, and villous endothelial cells. D. The three-dimensional microarchitecture of the placental barrier is reconstituted within our microengineered system. The placenta-on-a-chip consists of upper and lower microchannels separated by a thin, semipermeable membrane. Trophoblast cells are cultured in the upper microchannel on the apical side of the membrane, and villous endothelial cells are grown in the lower microchannel on the basal side of the membrane.

Figure 2

A. Three-dimensional rendering of the microengineered placental barrier. The trophoblast and endothelial cell populations are stained for E-cadherin (red) and VE-cadherin (green), respectively. Nuclei are shown in blue. Scale bar: 30 μm. B. Cross-sectional view of the same microengineered barrier. Scale bar: 30 μm. C. The trophoblast cells form a continuous network of epithelial adherens junctions (E-cadherin, red). Nuclei are stained with DAPI (blue). Scale bar: 20 μm. D. The placental villous endothelium also displays intact cell-cell junctions (VE-cadherin, red). Green and blue show actin and nuclear staining, respectively. Scale bar: 20 μm. E. The trophoblast cells produce laminin (green) during culture in our microdevice. The image shows cells on Day 6. Scale bar: 30 μm. F. Cross-sectional view of laminin deposition. Laminin is stained green, and trophoblast nuclei are labeled in blue. The location of the cell culture membrane is indicated by the dotted line. Scale bar: 10 μm.

Figure 3

A. Trophoblast cells cultured under dynamic flow conditions in the placenta-on-a-chip show widespread microvilli formation on the apical cell surface. Scale bar: 15 μm. B. Three-dimensional rendering of microvilli on the surface of BeWo cells in the placenta-on-a-chip. Scale bar: 20 μm. C. Microvilli in BeWo cells cultured in Transwell appear finer and significantly less abundant. Scale bar: 15 μm. D. Quantification of relative fluorescence of microvilli F-actin (green) in static and dynamic culture conditions. Cell nuclei were counterstained with DAPI (blue). The intensity of fluorescence generated by microvilli was increased substantially when the trophoblast cells were grown under flow conditions in the placenta-on-a-chip, as compared to static Transwell culture (****, p<0.0001).

Figure 4

Trophoblast syncytialization. A. In the human placenta, cytotrophoblast cells in early gestation go through a process of cell fusion to form a multinucleated syncytiotrophoblast. The resulting syncytium makes up the outer lining of the chorionic villi that comes into contact with maternal blood in the intervillous space. B. The BeWo cells cultured in our model exhibit high levels of E-cadherin (red) expression prior to forskolin treatment (left). Continuous epithelial exposure to forskolin over 72 hours induces trophoblasts to undergo cell-cell fusion, leading to nuclear aggregation and deceased expression of epithelial junctions. C. During forskolin-induced syncytialization, the permeability of the microengineered barrier to dextran decreases over time. * represents a statistically significant difference between 24 h and 72h (p<0.05; a.u. represents arbitrary unit), as well as between 48h and 72h (**, p<0.01). D. The BeWo cells produce increasing levels of β-human chorionic gonadotropin (β-HCG) over 120 hours of forskolin treatment (red bars). Hormone secretion by untreated cells is negligible.

Figure 5

A. The syncytial epithelium in our model expresses high levels of GLUT1 transporters (red). Blue shows DAPI staining. Scale bar: 10 μm. B. This cross-sectional view of the trophoblasts shows the asymmetric localization of GLUT1 transporters to the apical membrane. Scale bar: 5 μm. C. Immunofluorescence staining of GLUT1 on the apical side is substantially stronger than that on the basal side (****, p<0.0001). a.u. represents arbitrary unit. D. A concentration gradient of glucose (green dots) is generated across the microengineered placental barrier to drive glucose transport from the maternal to fetal compartments. E. Percent increase in fetal glucose concentration for the bare membrane, trophoblast monoculture, and co-culture conditions (*, p<0.05). F. The percent rate of glucose transfer in the placenta-on-a-chip device is similar to that measured in the perfused ex vivo placenta. The in vitro placental barrier formed in Transwell fails to reconstitute the physiological glucose transport rates.