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.

Department of Pharmacology and Toxicology Faculty of Pharmacy in Hradec Kralove Charles University Hradec Kralove Czech Republic

Institute of Biochemistry and Molecular Medicine Faculty of Medicine University of Bern Bern Switzerland

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Burton GJ, Fowden AL. The placenta: a multifaceted, transient organ. Philos Trans R Soc Lond Ser B Biol Sci. 2015;370(1663):20140066. doi:10.1098/rstb.2014.0066 PubMed DOI PMC

Burton GJ, Fowden AL, Thornburg KL. Placental origins of chronic disease. Physiol Rev. 2016;96(4):1509‐1565. doi:10.1152/physrev.00029.2015 PubMed DOI PMC

Benirschke K, Driscoll SG. The pathology of the human placenta. In: Strauss F, Benirschke K, Driscoll SG, eds. Placenta. Springer; 1967:97‐571. doi:10.1007/978-3-662-38455-8_2 DOI

Midgley AR, Pierce GB, Deneau GA, Gosling JRG. Morphogenesis of Syncytiotrophoblast in vivo: an autoradiographic demonstration. Science. 1963;141(3578):349‐350. doi:10.1126/science.141.3578.349 PubMed DOI

Myllynen P, Pasanen M, Vähäkangas K. The fate and effects of xenobiotics in human placenta. Expert Opin Drug Metab Toxicol. 2007;3(3):331‐346. doi:10.1517/17425255.3.3.331 PubMed DOI

Koren G, Ornoy A. The role of the placenta in drug transport and fetal drug exposure. Expert Rev Clin Pharmacol. 2018;11(4):373‐385. doi:10.1080/17512433.2018.1425615 PubMed DOI

Ewing G, Tatarchuk Y, Appleby D, Schwartz N, Kim D. Placental transfer of antidepressant medications: implications for postnatal adaptation syndrome. Clin Pharmacokinet. 2015;54(4):359‐370. doi:10.1007/s40262-014-0233-3 PubMed DOI PMC

Vahakangas K, Myllynen P. Experimental methods to study human transplacental exposure to genotoxic agents. Mutat Res. 2006;608(2):129‐135. doi:10.1016/j.mrgentox.2006.02.014 PubMed DOI

Vargesson N. Thalidomide‐induced teratogenesis: history and mechanisms. Birth Defects Res Part C Embryo Today Rev. 2015;105(2):140‐156. doi:10.1002/bdrc.21096 PubMed DOI PMC

Rubinchik‐Stern M, Eyal S. Drug interactions at the human placenta: what is the evidence? Front Pharmacol. 2012;3:126. doi:10.3389/fphar.2012.00126 PubMed DOI PMC

Mitchell AA, Gilboa SM, Werler MM, Kelley KE, Louik C, Hernández‐Díaz S. Medication use during pregnancy, with particular focus on prescription drugs: 1976‐2008. Am J Obstet Gynecol. 2011;205(1):51.e1‐51.e8. doi:10.1016/j.ajog.2011.02.029 PubMed DOI PMC

Schmidt A, Morales‐Prieto DM, Pastuschek J, Fröhlich K, Markert UR. Only humans have human placentas: molecular differences between mice and humans. J Reprod Immunol. 2015;108:65‐71. doi:10.1016/j.jri.2015.03.001 PubMed DOI

Schmidt A, Schmidt A, Markert UR. The road (not) taken – placental transfer and interspecies differences. Placenta. 2021;115:70‐77. doi:10.1016/j.placenta.2021.09.011 PubMed DOI

Wadman M. FDA no longer has to require animal testing for new drugs. Science. 2023;379(6628):127‐128. doi:10.1126/science.adg6276 PubMed DOI

Aengenheister L, Keevend K, Muoth C, et al. An advanced human in vitro co‐culture model for translocation studies across the placental barrier. Sci Rep. 2018;8(1):5388. doi:10.1038/s41598-018-23410-6 PubMed DOI PMC

Göhner C, Svensson‐Arvelund J, Pfarrer C, et al. The placenta in toxicology. Part IV Toxicol Pathol. 2014;42(2):345‐351. doi:10.1177/0192623313482206 PubMed DOI PMC

Elzinga FA, Khalili B, Touw DJ, et al. Placenta‐on‐a‐Chip as an in vitro approach to evaluate the physiological and structural characteristics of the human placental barrier upon drug exposure: a systematic review. J Clin Med. 2023;12(13):4315. doi:10.3390/jcm12134315 PubMed DOI PMC

Morales‐Prieto DM, Chaiwangyen W, Ospina‐Prieto S, et al. MicroRNA expression profiles of trophoblastic cells. Placenta. 2012;33(9):725‐734. doi:10.1016/j.placenta.2012.05.009 PubMed DOI

Wang JD, Douville NJ, Takayama S, ElSayed M. Quantitative analysis of molecular absorption into PDMS microfluidic channels. Ann Biomed Eng. 2012;40(9):1862‐1873. doi:10.1007/s10439-012-0562-z PubMed DOI

Melhem H, Kallol S, Huang X, et al. Placental secretion of apolipoprotein A1 and E: the anti‐atherogenic impact of the placenta. Sci Rep. 2019;9(1):1‐12. doi:10.1038/s41598-019-42522-1 PubMed DOI PMC

Huang X, Lüthi M, Ontsouka EC, et al. Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport. Mol Hum Reprod. 2016;22(6):442‐456. doi:10.1093/molehr/gaw018 PubMed DOI

Fuenzalida B, Cantin C, Kallol S, et al. Cholesterol uptake and efflux are impaired in human trophoblast cells from pregnancies with maternal supraphysiological hypercholesterolemia. Sci Rep. 2020;10(1):5264. doi:10.1038/s41598-020-61629-4 PubMed DOI PMC

Fuenzalida B, Kallol S, Zaugg J, et al. Primary human trophoblasts mimic the preeclampsia phenotype after acute hypoxia‐reoxygenation insult. Cells. 2022;11(12):1898. doi:10.3390/cells11121898 PubMed DOI PMC

Baudin B, Bruneel A, Bosselut N, Vaubourdolle M. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc. 2007;2(3):481‐485. doi:10.1038/nprot.2007.54 PubMed DOI

Huang X, Anderle P, Hostettler L, et al. Identification of placental nutrient transporters associated with intrauterine growth restriction and pre‐eclampsia. BMC Genomics. 2018;19(1):173. doi:10.1186/s12864-018-4518-z PubMed DOI PMC

Fuenzalida B, Yañez MJ, Mueller M, Mistry HD, Leiva A, Albrecht C. Evidence for hypoxia‐induced dysregulated cholesterol homeostasis in preeclampsia: insights into the mechanisms from human placental cells and tissues. FASEB J. 2024;38(2):e23431. doi:10.1096/fj.202301708RR PubMed DOI PMC

Poulsen MS, Rytting E, Mose T, Knudsen LE. Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion. Toxicol In Vitro. 2009;23(7):1380‐1386. doi:10.1016/j.tiv.2009.07.028 PubMed DOI

Pemathilaka RL, Caplin JD, Aykar SS, Montazami R, Hashemi NN. Placenta‐on‐a‐chip: in vitro study of caffeine transport across placental barrier using liquid chromatography mass spectrometry. Glob Challenges. 2019;3(3):1800112. doi:10.1002/gch2.201800112 PubMed DOI PMC

Christensen HN. Role of amino acid transport and countertransport in nutrition and metabolism. Physiol Rev. 1990;70(1):43‐77. doi:10.1152/physrev.1990.70.1.43 PubMed DOI

Kim DK, Kanai Y, Choi HW, et al. Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells. Biochim Biophys Acta Biomembr. 2002;1565(1):112‐122. doi:10.1016/S0005-2736(02)00516-3 PubMed DOI

Zaugg J, Albrecht C. Assessment of placental sodium‐independent leucine uptake and transfer in trophoblast cells. Methods Mol Biol. 2024;2728:105‐121. doi:10.1007/978-1-0716-3495-0_9 PubMed DOI

Jansson T, Scholtbach V, Powell TL. Placental transport of leucine and lysine is reduced in intrauterine growth restriction. Pediatr Res. 1998;44(4):532‐537. doi:10.1203/00006450-199810000-00011 PubMed DOI

Fuenzalida B, Kallol S, Lüthi M, Albrecht C, Leiva A. The polarized localization of lipoprotein receptors and cholesterol transporters in the syncytiotrophoblast of the placenta is reproducible in a monolayer of primary human trophoblasts. Placenta. 2020;105:50‐60. doi:10.1016/j.placenta.2021.01.019 PubMed DOI

Benson K, Cramer S, Galla H‐J. Impedance‐based cell monitoring: barrier properties and beyond. Fluids Barriers CNS. 2013;10(1):5. doi:10.1186/2045-8118-10-5 PubMed DOI PMC

Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER Measurement Techniques for In Vitro Barrier Model Systems. SLAS Technol. 2015;20(2):107‐126. doi:10.1177/2211068214561025 PubMed DOI PMC

Bertrand CA, Durand DM, Saidel GM, Laboisse C, Hopfer U. System for dynamic measurements of membrane capacitance in intact epithelial monolayers. Biophys J. 1998;75(6):2743‐2756. doi:10.1016/S0006-3495(98)77718-5 PubMed DOI PMC

Moore JW. Membranes, Ions and Impulses. A Chapter of Classical Biophysics. Kenneth S. Cole. University of California, Berkeley, 1968. x + 572 pp., illus. $15. Biophysics Series, Vol. 1. Science (80‐). 1969;163(3864):268‐268. doi:10.1126/science.163.3864.268 DOI

Alper SL, Sharma AK. The SLC26 gene family of anion transporters and channels. Mol Asp Med. 2013;34(2–3):494‐515. doi:10.1016/j.mam.2012.07.009 PubMed DOI PMC

Jauniaux E, Gulbis B. In vivo investigation of placental transfer early in human pregnancy. Eur J Obstet Gynecol Reprod Biol. 2000;92(1):45‐49. doi:10.1016/S0301-2115(00)00424-3 PubMed DOI

Miller R, Mace K, Polliotti B, DeRita R, Hall W, Treacy G. Marginal transfer of ReoPro™ (abciximab) compared with immunoglobulin G (F105), inulin and water in the perfused human placenta in vitro. Placenta. 2003;24(7):727‐738. doi:10.1016/S0143-4004(03)00101-2 PubMed DOI

McConkey CA, Delorme‐Axford E, Nickerson CA, et al. A three‐dimensional culture system recapitulates placental syncytiotrophoblast development and microbial resistance. Sci Adv. 2016;2(3):e1501462. doi:10.1126/sciadv.1501462 PubMed DOI PMC

Vandré DD, Ackerman WE, Kniss DA, et al. Dysferlin is expressed in human placenta but does not associate with Caveolin1. Biol Reprod. 2007;77(3):533‐542. doi:10.1095/biolreprod.107.062190 PubMed DOI

Lennon NJ, Kho A, Bacskai BJ, Perlmutter SL, Hyman BT, Brown RH. Dysferlin interacts with annexins A1 and A2 and mediates Sarcolemmal wound‐healing. J Biol Chem. 2003;278(50):50466‐50473. doi:10.1074/jbc.M307247200 PubMed DOI

Bansal D, Miyake K, Vogel SS, et al. Defective membrane repair in dysferlin‐deficient muscular dystrophy. Nature. 2003;423(6936):168‐172. doi:10.1038/nature01573 PubMed DOI

Drwal E, Rak A, Tworzydło W, Gregoraszczuk EŁ. “Real life” polycyclic aromatic hydrocarbon (PAH) mixtures modulate hCG, hPL and hPLGF levels and disrupt the physiological ratio of MMP‐2 to MMP‐9 and VEGF expression in human placenta cell lines. Reprod Toxicol. 2020;95:1‐10. doi:10.1016/j.reprotox.2020.02.006 PubMed DOI

Huppertz B, Bartz C, Kokozidou M. Trophoblast fusion: Fusogenic proteins, syncytins and ADAMs, and other prerequisites for syncytial fusion. Micron. 2006;37(6):509‐517. doi:10.1016/j.micron.2005.12.011 PubMed DOI

Schneider H, Panigel M, Dancis J. Transfer across the perfused human placenta of antipyrine, sodium, and leucine. Am J Obstet Gynecol. 1972;114(6):822‐828. doi:10.1016/0002-9378(72)90909-X PubMed DOI

Conings S, Amant F, Annaert P, Van Calsteren K. Integration and validation of the ex vivo human placenta perfusion model. J Pharmacol Toxicol Methods. 2017;88:25‐31. doi:10.1016/j.vascn.2017.05.002 PubMed DOI

Rozance PJ. Maternal amino acid supplementation for intrauterine growth restriction. Front Biosci. 2011;S3(2):162. doi:10.2741/s162 PubMed DOI PMC

Pedroso J, Zampieri T, Donato J. Reviewing the effects of l‐leucine supplementation in the regulation of food intake, energy balance, and glucose homeostasis. Nutrients. 2015;7(5):3914‐3937. doi:10.3390/nu7053914 PubMed DOI PMC

Kalhan SC. Protein metabolism in pregnancy. Am J Clin Nutr. 2000;71(5):1249S‐1255S. doi:10.1093/ajcn/71.5.1249s PubMed DOI

Grasso S, Palumbo G, Rugolo S, Cianci A, Tumino G, Reitano G. Human fetal insulin secretion in response to maternal glucose and leucine administration. Pediatr Res. 1980;14(5):782‐783. doi:10.1203/00006450-198005000-00016 PubMed DOI

Hayashi K, Jutabha P, Kamai T, Endou H, Anzai N. LAT1 is a central transporter of essential amino acids in human umbilical vein endothelial cells. J Pharmacol Sci. 2014;124(4):511‐513. doi:10.1254/jphs.13255SC PubMed DOI

Gaccioli F, Aye ILMH, Roos S, et al. Expression and functional characterisation of system L amino acid transporters in the human term placenta. Reprod Biol Endocrinol. 2015;13(1):57. doi:10.1186/s12958-015-0054-8 PubMed DOI PMC

Okamoto Y, Sakata M, Ogura K, et al. Expression and regulation of 4F2hc and hLAT1 in human trophoblasts. Am J Physiol Physiol. 2002;282(1):C196‐C204. doi:10.1152/ajpcell.2002.282.1.C196 PubMed DOI

Zaugg J, Huang X, Ziegler F, et al. Small molecule inhibitors provide insights into the relevance of LAT1 and LAT2 in materno‐foetal amino acid transport. J Cell Mol Med. 2020;24(21):12681‐12693. doi:10.1111/jcmm.15840 PubMed DOI PMC

Ritchie JWA, Taylor PM. Role of the system L permease LAT1 in amino acid and iodothyronine transport in placenta. Biochem J. 2001;356(3):719‐725. doi:10.1042/0264-6021:3560719 PubMed DOI PMC

Ohgaki R, Ohmori T, Hara S, et al. Essential roles of L‐type amino acid transporter 1 in Syncytiotrophoblast development by presenting Fusogenic 4F2hc. Mol Cell Biol. 2017;37(11):e00427‐16. doi:10.1128/MCB.00427-16 PubMed DOI PMC

Aye ILMH, Waddell BJ, Mark PJ, Keelan JA. Placental ABCA1 and ABCG1 transporters efflux cholesterol and protect trophoblasts from oxysterol induced toxicity. Biochim Biophys Acta Mol Cell Biol Lipids. 2010;1801(9):1013‐1024. doi:10.1016/j.bbalip.2010.05.015 PubMed DOI

Maldonado‐Estrada J, Menu E, Roques P, Barré‐Sinoussi F, Chaouat G. Evaluation of cytokeratin 7 as an accurate intracellular marker with which to assess the purity of human placental villous trophoblast cells by flow cytometry. J Immunol Methods. 2004;286(1–2):21‐34. doi:10.1016/j.jim.2003.03.001 PubMed DOI

Quan L, Ohgaki R, Hara S, et al. Amino acid transporter LAT1 in tumor‐associated vascular endothelium promotes angiogenesis by regulating cell proliferation and VEGF‐A‐dependent mTORC1 activation. J Exp Clin Cancer Res. 2020;39(1):266. doi:10.1186/s13046-020-01762-0 PubMed DOI PMC

Ceckova‐Novotna M, Pavek P, Staud F. P‐glycoprotein in the placenta: expression, localization, regulation and function. Reprod Toxicol. 2006;22(3):400‐410. doi:10.1016/j.reprotox.2006.01.007 PubMed DOI

Ho H, Zhang E. P‐glycoprotein efflux transporter: a key to pharmacokinetic modeling for methadone clearance in fetuses. Front Pharmacol. 2023;14:1182571. doi:10.3389/fphar.2023.1182571 PubMed DOI PMC

Han LW, Gao C, Mao Q. An update on expression and function of P‐gp/ABCB1 and BCRP/ABCG2 in the placenta and fetus. Expert Opin Drug Metab Toxicol. 2018;14(8):817‐829. doi:10.1080/17425255.2018.1499726 PubMed DOI PMC

Mark PJ, Waddell BJ. P‐glycoprotein restricts access of cortisol and dexamethasone to the glucocorticoid receptor in placental BeWo cells. Endocrinology. 2006;147(11):5147‐5152. doi:10.1210/en.2006-0633 PubMed DOI

Iwahana M, Utoguchi N, Mayumi T, Goryo M, Okada K. Drug resistance and P‐glycoprotein expression in endothelial cells of newly formed capillaries induced by tumors. Anticancer Res. 1998;18(4C):2977‐2980. PubMed

Cordon‐Cardo C, O'Brien JP, Casals D, et al. Multidrug‐resistance gene (P‐glycoprotein) is expressed by endothelial cells at blood‐brain barrier sites. Proc Natl Acad Sci. 1989;86(2):695‐698. doi:10.1073/pnas.86.2.695 PubMed DOI PMC

Nasim F, Schmid D, Szakács G, et al. Active transport of rhodamine 123 by the human multidrug transporter P‐glycoprotein involves two independent outer gates. Pharmacol Res Perspect. 2020;8(2):e00572. doi:10.1002/prp2.572 PubMed DOI PMC

Fontaine M, Elmquist WF, Miller DW. Use of rhodamine 123 to examine the functional activity of P‐glycoprotein in primary cultured brain microvessel endothelial cell monolayers. Life Sci. 1996;59(18):1521‐1531. doi:10.1016/0024-3205(96)00483-3 PubMed DOI

Unadkat J, Dahlin A, Vijay S. Placental drug transporters. Curr Drug Metab. 2004;5(1):125‐131. doi:10.2174/1389200043489171 PubMed DOI