Placental Drug Transport-on-a-Chip: A Microengineered In Vitro Model of Transporter-Mediated Drug Efflux in the Human Placental Barrier

Abstract

The current lack of knowledge about the effect of maternally administered drugs on the developing fetus is a major public health concern worldwide. The first critical step toward predicting the safety of medications in pregnancy is to screen drug compounds for their ability to cross the placenta. However, this type of preclinical study has been hampered by the limited capacity of existing in vitro and ex vivo models to mimic physiological drug transport across the maternal-fetal interface in the human placenta. Here the proof-of-principle for utilizing a microengineered model of the human placental barrier to simulate and investigate drug transfer from the maternal to the fetal circulation is demonstrated. Using the gestational diabetes drug glyburide as a model compound, it is shown that the microphysiological system is capable of reconstituting efflux transporter-mediated active transport function of the human placental barrier to limit fetal exposure to maternally administered drugs. The data provide evidence that the placenta-on-a-chip may serve as a new screening platform to enable more accurate prediction of drug transport in the human placenta.

Keywords: drug transport; glyburide; placenta-on-a-chip.

Figure 1. The human placenta and placenta-on-a-chip

A. Three-dimensional cross section of the human placenta illustrates cotyledons, which contain chorionic villi bathed in the maternal blood in the intervillous space. B. A zoomed-in view highlights the fetal capillaries that are located within the chorionic villi. C. The placental barrier that separates the maternal intervillous space from the fetal capillary lumen consists of two cell types: the syncytiotrophoblast and fetal endothelial cells separated by a thin interstitial tissue layer. D–E. The placenta-on-a-chip is a microengineered model designed to recapitulate the multi-layered three-dimensional architecture of the placental barrier. Within the device, trophoblast and endothelial cells are co-cultured on the opposite sides of a thin porous polymeric membrane. During culture, the viability of the cells is maintained by continuous low of culture medium on both sides of the membrane.

Figure 2. Microengineered in vitro placental barrier

A. BeWo cells cultured on the upper side of the porous membrane form a continuous epithelial barrier. Red shows E-cadherin staining. Scale bar: 55 µm. B. Human placental villous endothelial cells (HPVECs) are grown to confluence on the lower side of the membrane. Widespread expression of VE-cadherin (green) illustrates the structural integrity of the endothelial tissue. Scale bar: 36 µm. C. The microengineered placental barrier effectively prevents the transfer of FITC-inulin from the maternal microchannel to the fetal compartment, demonstrating appropriate barrier function. D. Dense microvilli cover the surface of the BeWo cell population, as illustrated by F-actin staining (green). Scale bar: 20 µm. E. A cross-sectional view of actin-stained BeWo cells reveals the apical microvilli projections protruding from the cell body. Scale bar: 6 µm. F. Extended forskolin treatment induces a significant loss of intercellular junctions (red) and syncytialization-like cell fusion in BeWo cells over the course of 72 hours. Scale bars: 55 µm. Blue shows nuclear staining. G. During image analysis of E-cadherin staining, individual cells are pseudo-colored to delineate cell-cell junctions and to quantify cell area during forskolin treatment. The average area of BeWo cells increases over 72 hours, illustrating progressive cell fusion in the trophoblast population. * and ** indicate p < 0.05 and p < 0.01, respectively. H. Another method for image analysis of cell fusion is to segment DAPI-stained nuclei of BeWo cells and then measure the area of the segmented nuclei. The average size of cell nuclei increases during forskolin treatment. *** and **** indicate p < 0.001 and p < 0.0001, respectively.

Figure 3. Drug transport in the placenta-on-a-chip

A. The kinetics of drug transport across the microengineered placental barrier is examined by perfusing the maternal microchannel with fluorescently labeled substrate and measuring fluorescence in the perfusates. B. Transfer of fluorescein-heparin from the maternal compartment to the fetal chamber is evaluated by measuring mean fluorescence intensity (MFI) of perfusate collected from the fetal microchannel. Acellular devices allow a significant fraction of maternally administered heparin to move across the membrane into the fetal compartment, whereas the barrier covered with trophoblast cells and endothelial cells (co-culture) effectively blocks heparin transport. ** shows p < 0.01. C. BeWo cells exhibit widespread expression of BCRP (red). Blue shows cell nuclei. Scale bar: 36 µm. D. BCRP is localized to the maternal-facing microvillous membrane of BeWo cells. **** shows p < 0.0001. Scale bar: 36 µm. F. In the co-culture model, a continuous increase in the fluorescence of maternal perfusate indicates transporter-mediated efflux of glyburide back into the maternal circulation. The fluorescence of perfusate from the fetal compartment remains low and constant, showing limited transfer. G. Under endothelial cell monoculture conditions, maternal glyburide concentration is decreased. **** indicates p < 0.0001. H. Microscopic analysis illustrates accumulation of glyburide in the BeWo cell monolayer, but no visible fluorescence in the HPVEC population. **** indicates p < 0.0001.

Figure 4. Inhibition of BCRP

A. Addition of Ko143, a BCRP inhibitor, results in decreased maternal glyburide levels as compared to the control perfusion. This is attributable to inhibition of efflux function of BCRP. B. The levels of fetal glyburide in the inhibitor condition are not significantly different from those in the control group without the inhibitor. Passage of glyburide to the fetal compartment remains very low over the course of drug perfusion in both groups. C. Increased drug accumulation in BeWo cells due to reduced activity of BCRP is evident from fluorescence imaging of the cells. D. In the presence of the inhibitor, the cells show significantly higher levels of fluorescence, suggesting increased intracellular drug retention. **** indicates p < 0.0001.