Materno-fetal nutrient transfer across primary human trophoblast layer.

Huang, Xiao; Lüthi, Michael; Ontsouka, Edgar; Baumann, Marc Ulrich; Surbek, Daniel; Albrecht, Christiane (September 2014). Materno-fetal nutrient transfer across primary human trophoblast layer. Placenta, 35, A97-A98. Elsevier

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MATERNO-FETAL NUTRIENT TRANSFER ACROSS PRIMARY HUMAN
TROPHOBLAST MONOLAYER
Objectives: Polarized trophoblasts represent the transport and metabolic
barrier between the maternal and fetal circulation. Currently human
placental nutrient transfer in vitro is mainly investigated unidirectionallyon cultured primary trophoblasts, or bidirectionally on the Transwell®
system using BeWo cells treated with forskolin. As forskolin can induce
various gene alterations (e.g. cAMP response element genes), we aimed to
establish a physiological primary trophoblast model for materno-fetal
nutrient exchange studies without forskolin application.
Methods: Human term cytotrophoblasts were isolated by enzymatic
digestion and Percoll® gradient separation. The purity of the primary cells
was assessed by flow cytometry using the trophoblast-specific marker
cytokeratin-7. After screening different coating matrices, we optimized the
growth conditions for the primary cytotrophoblasts on Transwell/ inserts.
The morphology of 5 days cultured trophoblasts was determined by
scanning electron microscopy (SEM) and transmission electron microscopy
(TEM). Membrane makers were visualized using confocal microscopy.
Additionally transport studies were performed on the polarized
trophoblasts in the Transwell® system.
Results: During 5 days culture, the trophoblasts (>90% purity) developed
a modest trans-epithelial electrical resistance (TEER) and a sizedependent
apparent permeability coefficient (Papp) to fluorescently
labeled compounds (MW ~400-70’000D). SEM analyses confirmed a
confluent trophoblast layer with numerous microvilli at day six, 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 ZO-1, and the
membrane proteins ABCA1 and Na+/K+-ATPase. Vectorial glucose and
cholesterol transport studies confirmed functionality of the cultured
trophoblast barrier.
Conclusion: Evidence from cell morphology, biophysical parameters and
cell marker expressions indicate the successful and reproducible establishment
of a primary trophoblast monolayer model suitable for transport
studies. Application of this model to pathological trophoblasts will help to
better understand the mechanism underlying gestational diseases, and to
define the consequences of placental pathology on materno-fetal nutrient
transport.

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