The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.

• Keywords
• LAT1, P‐gp, co‐culture, endothelial cell, placental barrier, polarized monolayer, primary trophoblast, transport,
• MeSH
• Models, Biological MeSH
• Biological Transport MeSH
• Human Umbilical Vein Endothelial Cells * metabolism   MeSH
• Inulin metabolism   MeSH
• Isoquinolines MeSH
• Coculture Techniques MeSH
• Leucine metabolism   MeSH
• Humans MeSH
• Maternal-Fetal Exchange * MeSH
• ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism   MeSH
• Placenta * metabolism   MeSH
• Rhodamine 123 metabolism   MeSH
• Pregnancy MeSH
• Trophoblasts * metabolism   MeSH
• Check Tag
• Humans MeSH
• Pregnancy MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Inulin MeSH
• Isoquinolines MeSH
• Leucine MeSH
• lucifer yellow MeSH Browser
• ATP Binding Cassette Transporter, Subfamily B, Member 1 MeSH
• Rhodamine 123 MeSH

Lamivudine is one of the antiretroviral drugs of choice for the prevention of mother-to-child transmission (MTCT) in HIV-positive women. In this study, we investigated the relevance of drug efflux transporters P-glycoprotein (P-gp) (MDR1 [ABCB1]), BCRP (ABCG2), MRP2 (ABCC2), and MATE1 (SLC47A1) for the transmembrane transport and transplacental transfer of lamivudine. We employed in vitro accumulation and transport experiments on MDCK cells overexpressing drug efflux transporters, in situ-perfused rat term placenta, and vesicular uptake in microvillous plasma membrane (MVM) vesicles isolated from human term placenta. MATE1 significantly accelerated lamivudine transport in MATE1-expressing MDCK cells, whereas no transporter-driven efflux of lamivudine was observed in MDCK-MDR1, MDCK-MRP2, and MDCK-BCRP monolayers. MATE1-mediated efflux of lamivudine appeared to be a low-affinity process (apparent Km of 4.21 mM and Vmax of 5.18 nmol/mg protein/min in MDCK-MATE1 cells). Consistent with in vitro transport studies, the transplacental clearance of lamivudine was not affected by P-gp, BCRP, or MRP2. However, lamivudine transfer across dually perfused rat placenta and the uptake of lamivudine into human placental MVM vesicles revealed pH dependency, indicating possible involvement of MATE1 in the fetal-to-maternal efflux of the drug. To conclude, placental transport of lamivudine does not seem to be affected by P-gp, MRP2, or BCRP, but a pH-dependent mechanism mediates transport of lamivudine in the fetal-to-maternal direction. We suggest that MATE1 might be, at least partly, responsible for this transport.

• MeSH
• ATP-Binding Cassette Transporters metabolism   MeSH
• Biological Transport physiology   MeSH
• Cell Line MeSH
• Madin Darby Canine Kidney Cells MeSH
• Rats MeSH
• Lamivudine metabolism   MeSH
• Humans MeSH
• ATP Binding Cassette Transporter, Subfamily B metabolism   MeSH
• Placenta metabolism   MeSH
• Rats, Wistar MeSH
• Multidrug Resistance-Associated Protein 2 MeSH
• Organic Cation Transport Proteins metabolism   MeSH
• Multidrug Resistance-Associated Proteins metabolism   MeSH
• Dogs MeSH
• Pregnancy MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Dogs MeSH
• Pregnancy MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• ATP-Binding Cassette Transporters MeSH
• ABCC2 protein, human MeSH Browser
• Lamivudine MeSH
• ATP Binding Cassette Transporter, Subfamily B MeSH
• Multidrug Resistance-Associated Protein 2 MeSH
• Organic Cation Transport Proteins MeSH
• Multidrug Resistance-Associated Proteins MeSH