title: Establishment of a confluent monolayer model with human primary trophoblast cells: novel insights into placental glucose transport.
abstract: Using optimized conditions, primary trophoblast cells isolated from human term placenta can develop a confluent monolayer in vitro, which morphologically and functionally resembles the microvilli structure found in vivo.
  
  
  
  We report the successful establishment of a confluent human primary trophoblast monolayer using pre-coated polycarbonate inserts, where the integrity and functionality was validated by cell morphology, biophysical features, cellular marker expression and secretion, and asymmetric glucose transport.
  
  
  
  Human trophoblast cells form the initial barrier between maternal and fetal blood to regulate materno-fetal exchange processes. Although the method for isolating pure human cytotrophoblast cells was developed almost 30 years ago, a functional in vitro model with primary trophoblasts forming a confluent monolayer is still lacking.
  
  
  
  Human term cytotrophoblasts were isolated by enzymatic digestion and density gradient separation. The purity of the primary cells was evaluated by flow cytometry using the trophoblast-specific marker cytokeratin 7, and vimentin as an indicator for potentially contaminating cells. We screened different coating matrices for high cell viability to optimize the growth conditions for primary trophoblasts on polycarbonate inserts. During culture, cell confluency and polarity were monitored daily by determining transepithelial electrical resistance (TEER) and permeability properties of florescent dyes. The time course of syncytia-related gene expression and hCG secretion during syncytialization were assessed by quantitative RT-PCR and enzyme-linked immunosorbent assay, respectively. The morphology of cultured trophoblasts after 5 days was determined by light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Membrane makers were visualized using confocal microscopy. Additionally, glucose transport studies were performed on the polarized trophoblasts in the same system.
  
  
  
  During 5-day culture, the highly pure trophoblasts were cultured on inserts coated with reconstituted basement membrane matrix . They exhibited a confluent polarized monolayer, with a modest TEER and a size-dependent apparent permeability coefficient (Papp) to fluorescently labeled compounds (MW ∼400-70 000 Da). The syncytialization progress was characterized by gradually increasing mRNA levels of fusogen genes and elevating hCG secretion. SEM analyses confirmed a confluent trophoblast layer with numerous microvilli, and TEM revealed a monolayer with tight junctions. Immunocytochemistry on the confluent trophoblasts showed positivity for the cell-cell adhesion molecule E-cadherin, the tight junction protein 1 (ZO-1) and the membrane proteins ATP-binding cassette transporter A1 (ABCA1) and glucose transporter 1 (GLUT1). Applying this model to study the bidirectional transport of a non-metabolizable glucose derivative indicated a carrier-mediated placental glucose transport mechanism with asymmetric kinetics.
  
  
  
  The current study is only focused on primary trophoblast cells isolated from healthy placentas delivered at term. It remains to be evaluated whether this system can be extended to pathological trophoblasts isolated from diverse gestational diseases.
  
  
  
  These findings confirmed the physiological properties of the newly developed human trophoblast barrier, which can be applied to study the exchange of endobiotics and xenobiotics between the maternal and fetal compartment, as well as intracellular metabolism, paracellular contributions and regulatory mechanisms influencing the vectorial transport of molecules.
  
  
  
  Not applicable.
  
  
  
  This study was supported by the Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Switzerland, and the Swiss National Science Foundation (grant no. 310030_149958, C.A.). All authors declare that their participation in the study did not involve factual or potential conflicts of interests.
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Xiao
authors.initials: X
authors.lastname: Huang
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Lüthi
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Edgar C
authors.initials: EC
authors.lastname: Ontsouka
authors.affiliation: Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland.
authors.forename: Sampada
authors.initials: S
authors.lastname: Kallol
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Department of Obstetrics and Gynecology, University Hospital, University of Bern, Bern, Switzerland.
authors.forename: Marc U
authors.initials: MU
authors.lastname: Baumann
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Department of Obstetrics and Gynecology, University Hospital, University of Bern, Bern, Switzerland.
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Surbek
authors.affiliation: Swiss National Center of Competence in Research, NCCR TransCure, University of Bern, Bern, Switzerland Institute of Biochemistry and Molecular Medicine, Faculty of Medicine, University of Bern, Buehlstrasse 28, CH-3012 Bern, Switzerland christiane.albrecht@ibmm.unibe.ch.
authors.forename: Christiane
authors.initials: C
authors.lastname: Albrecht
mesh_terms: D001692:Biological Transport / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D005260:Female; D005947:Glucose / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008431:Maternal-Fetal Exchange / Q000235:genetics / Q000502:physiology; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D010920:Placenta / Q000378:metabolism* / Q000648:ultrastructure; D011247:Pregnancy; D014327:Trophoblasts / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D005947:Glucose
keywords: confluent monolayer; glucose transport; polarized trophoblasts; polycarbonate inserts; primary trophoblasts; transepithelial electrical resistance