Poster BB20 - Development of Development of a Sexually Dimorphic in vitro Placenta Barrier Model

Introduction: Previous in vitro placental barrier (PB) models have been established; however, no current models consider the impact of fetal sex.1 This is partly due to a heavy reliance on immortalized cell lines, which typically are only male cells. The placenta originates from the embryo and, therefore, has sexually dimorphic properties. Thus, there is a need for a physiologically relevant in vitro PB model that utilizes human placental cells to study sexual dimorphic placental function. Male and female trophoblasts have different chromosomes and gene expressions, including genes related to glucocorticoid receptors, cytokine receptors, and efflux transporters.2,3 Previous in vitro PB models have used primary and immortalized cells. However, no current models account for the role of fetal sex.4,5 To elucidate the sexually dimorphic functions of the PB, male and female human trophoblast stem cells (hTSCs) and human umbilical vein endothelial cells (HUVECs) will be incorporated in an in vitro PB model. To first establish and validate this model, BeWos, an immortalized male trophoblast cell line, were used. This study shows proper barrier function and hormone secretion with BeWo cells, laying the groundwork for incorporating sex-specific hTSCs and translating this model into a sexually dimorphic PB model.

Materials and

Methods: The model consists of three layers: the syncytiotrophoblast (SCT), cytotrophoblast (CT), and endothelial cell layers (Figure 1). On Day 0, male HUVECs were seeded at a density of 1x106 cells/mL on the basolateral side of a 12-well transwell insert with 0.4 µm pores. On day 4, the middle layer was manufactured by casting male BeWo cells encapsulated in 5% (w/v) GelMa at a cell density of 1x105 cells/mL. All samples were UV crosslinked for 2 minutes after casting. BeWos were then seeded on top of the hydrogel layer at a cell density of 1x106 cells/mL to form the SCT layer. Immunostaining of ZO-1 and DAPI was performed to determine the monolayer formation of BeWos and HUVECs on day 10. The samples were then imaged using laser scanning confocal microscopy. The model's hormone function was determined by measuring hCG secretions via ELISA. Cells were seeded at 1x106 cells/mL on a 12-well transwell and cultured in their respective media. Cell media was collected from the apical compartment and replaced on days 1, 3, and 5.

Results, Conclusions, and Discussions: Results and

Discussion: The integrity of the cell monolayer was confirmed through fluorescent imaging, which revealed that the BeWo and HUVEC cells formed a continuous and intact layer within the in vitro placenta barrier model. This successful formation of the cell monolayer demonstrates the model's efficacy in replicating the physiological structure of the placental barrier. BeWo cells exhibited higher hCG secretion compared to HUVECs and the BeWo-HUVEC co-culture. The measurement of hCG secretion from the cells revealed that BeWo cells, when cultured alone, demonstrated a more robust production of hCG. hCG is secreted by trophoblast cells and promotes angiogenesis in HUVECs. These results align with previous studies, suggesting that the model functions in a biomimetic fashion.5 Future experiments will be conducted using undifferentiated hTSCs to construct the CT layer and differentiated hTSCs to construct the ST layer. The incorporation of hTSCs will allow to evaluate the role of fetal sex in placental function. Barrier integrity is not expected to be effected by fetal sex. It is hypothesized that the female PB model will exhibit increased hCG secretions compared to the male PB model.

Conclusion: These results demonstrate a biomimetic, functional placental barrier model manufactured using male BeWos. Future work studies will incorporate male and female hTSCs and HUVECs to translate this model into a sexually dimorphic in vitro PB model, further elucidating the PB's sexually dimorphic characteristics.

Acknowledgements (Optional):

References: 1. Arumugasaamy, N. et al. Adv. Drug Deliv. Rev. (2020) 2. Braun, A. E. et al. Biol. Sex Differ. (2022).
3. Powell, T. L. et al. BBA-Mol. Cell Biol. Lipids (2021). 4. Walker, N. et al. Biol. Reprod. (2017).
5. Nishiguchi, A. et al. Biomaterials (2019). 6. Arumugasaamy, N. et al. Ann. Biomed. Eng. (2018).