An advanced human in vitro co-culture model for translocation studies across the placental barrier

Abstract

Although various drugs, environmental pollutants and nanoparticles (NP) can cross the human placental barrier and may harm the developing fetus, knowledge on predictive placental transfer rates and the underlying transport pathways is mostly lacking. Current available in vitro placental transfer models are often inappropriate for translocation studies of macromolecules or NPs and do not consider barrier function of placental endothelial cells (EC). Therefore, we developed a human placental in vitro co-culture transfer model with tight layers of trophoblasts (BeWo b30) and placental microvascular ECs (HPEC-A2) on a low-absorbing, 3 µm porous membrane. Translocation studies with four model substances and two polystyrene (PS) NPs across the individual and co-culture layers revealed that for most of these compounds, the trophoblast and the EC layer both demonstrate similar, but not additive, retention capacity. Only the paracellular marker Na-F was substantially more retained by the BeWo layer. Furthermore, simple shaking, which is often applied to mimic placental perfusion, did not alter translocation kinetics compared to static exposure. In conclusion, we developed a novel placental co-culture model, which provides predictive values for translocation of a broad variety of molecules and NPs and enables valuable mechanistic investigations on cell type-specific placental barrier function.

Figure 1

Scheme of the human placental barrier at term and the co-culture translocation model. (a) At the end of pregnancy, molecules or NPs present in the maternal blood stream would have to at least cross the ST layer, a basal lamina and the fetal ECs to reach the fetal circulation. (b) For mechanistic studies on molecule/NP translocation, a co-culture model was established consisting of a confluent layer of trophoblasts (BeWo cells) on the apical side and a monolayer of ECs (HPEC; human placental venous endothelial cells) on the basolateral side of a microporous membrane.

Figure 2

Establishment of the co-culture. (a) Confocal micrographs of BeWo/HPEC co-cultures after 3 d of cultivation on microporous inserts stained for γ-catenin (green, adherence junctions), tubulin (red, microtubule) and Dapi (blue, nuclei). (b,c) Images of BeWo cells (B) and HPECs (C) grown on porous membranes for 3 d and stained for ZO-1 (red, tight junctions) and Dapi (blue, nuclei). (d,e) Barrier formation was determined from day 1 to 4 after cell seeding by TEER measurements (d) and Na-F exclusion assays (e). Data represent the median ± error range (upper and lower limit) of 4 biologically independent experiments with 1 technical replicate each.

Figure 3

Morphological investigation of the co-culture after 3 d of cultivation. (a) Microscopic image of a semithin section of the co-culture (Toluidine Blue O staining). (b–d) TEM micrographs showing cellular protrusions from BeWo cells through the insert pores (b), microvilli on the apical side of the BeWo layer (c) and the formation of close cell-cell contacts by both cell types (c,d). Abbreviations: ECS, extra cellular space; D, desmosome; TJ, tight junction; MV, microvilli.

Figure 4

Translocation of 5 µM Na-F, 5 µM FITC-dextran (40 kDa), 100 µM antipyrine, 100 µM indomethacin, 0.5 mg/ml 49 nm PS NPs and 50 µg/ml 70 nm PS NPs across each monolayer (BeWo, HPEC) and the co-cultivated membrane under static conditions. Cells were cultivated for 3 d on collagen-coated inserts before translocation studies were performed for 24 h with Na-F (a), FITC-dextran (b), 49 nm PS NP (e) and 70 nm PS NP (f) or for 6 h with antipyrine (c) and indomethacin (d). Data represent the median ± error range (upper and lower limit) of 3–4 biologically independent experiments with 1 technical replicate each.

Figure 5

Seeding of the cells on the basolateral side of the insert. (a) Small rubber spacers are placed in each corner of a 6-well plate to avoid direct contact between membrane and lid. (b) 1 ml PBS is added to one well to humidify the surrounding. The reversed insert is placed into this well and 1 × 10⁵ HPECs in 200 µl are added to the membrane. (c) The lid is closed immediately. Adhesion of the drop prevents cell aggregation in the center and medium loss. (d) The insert is placed back into a 12-well plate after 2 h incubation at 37 °C/5% CO₂.