Establishment of an in vitro placental barrier model cultured under physiologically relevant oxygen levels

Abstract

The human placental barrier facilitates many key functions during pregnancy, most notably the exchange of all substances between the mother and fetus. However, preclinical models of the placental barrier often lacked the multiple cell layers, syncytialization of the trophoblast cells and the low oxygen levels that are present within the body. Therefore, we aimed to design and develop an in vitro model of the placental barrier that would reinstate these factors and enable improved investigations of barrier function. BeWo placental trophoblastic cells and human umbilical vein endothelial cells were co-cultured on contralateral sides of an extracellular matrix-coated transwell insert to establish a multilayered barrier. Epidermal growth factor and forskolin led to significantly increased multi-nucleation of the BeWo cell layer and increased biochemical markers of syncytial fusion, for example syncytin-1 and hCGβ. Our in vitro placental barrier possessed size-specific permeability, with 4000-Da molecules experiencing greater transport and a lower apparent permeability coefficient than 70 000-Da molecules. We further demonstrated that the BeWo layer had greater resistance to smaller molecules compared to the endothelial layer. Chronic, physiologically low oxygen exposure (3–8%) increased the expression of hypoxia-inducible factor 1α and syncytin-1, further increased multi-nucleation of the BeWo cell layer and decreased barrier permeability only against smaller molecules (457 Da/4000 Da). In conclusion, we built a novel in vitro co-culture model of the placental barrier that possessed size-specific permeability and could function under physiologically low oxygen levels. Importantly, this will enable future researchers to better study the maternal–fetal transport of nutrients and drugs during pregnancy.

Modeling the Progression of Placental Transport from Early‐ to Late‐Stage Pregnancy by Tuning Trophoblast Differentiation and Vascularization

“…BeWo b30 cells were differentiated by first culturing them in expansion medium (DMEM supplemented with FBS (10%) and penicillin–streptomycin (1%)) and then supplementing the media with forskolin (50 × 10 −6 m ) and EGF (50 ng mL −1 ) for 2 days, where media was changed daily. [ 22 ]…”

“…Both cell types were differentiated according to previously established protocols. [15,22] Briefly, PSCs were immediately seeded in differentiation medium and cultured for 8 days before analysis. BeWo b30 cells were first expanded for 2 days and then treated with 50 × 10 −6 m forskolin and 50 ng mL −1 of epidermal growth factor (EGF) for 2 days of differentiation [22] (Figure 2).…”

Modeling the human placental barrier to understand Toxoplasma gondii´s vertical transmission

“…Please note that T. gondii’s infection has been assayed for an array of intermediate host-derived trophoblasts. These include, but are not limited to, mice ( Wang et al, 2018 ) and sheep ( Fernández-Escobar et al, 2021 ). Varying results regarding infectivity have been obtained, likely reflecting host-specie and parasite-strain specific dynamics.…”

“…HUVECs cells have also been used to demonstrate that T. gondii induces the remodeling of the endothelial cytoskeleton and alteration of the cell barrier function ( Franklin-Murray et al., 2020 ).In addition, infection of bovine derived vein endothelial cells (BUVECs) displays altered progression through the cell cycle ( Velásquez et al., 2019 ), with increased host cell proliferation and an enhanced number of multinucleated cells. HUVEC are also frequently applied in the development of more complex placental models, resembling the fetal compartment, from 2D co cultures ( Wong et al., 2020 ), to organ-on-a-chip systems ( Lee et al., 2016 ).…”

Guidance document on Good Cell and Tissue Culture Practice 2.0 (GCCP 2.0)

“…In addition, the height/level of medium above cells determines the accessibility to oxygen and should be recognized as an important factor for cell culture (Al-Ani et al, 2018). The use of closed cell handling chambers to provide constant physiologically relevant oxygen can improve cell function (Mount et al, 2019;Wong et al, 2020).…”