(6) Microscope


\* after setup, flow systems should be calibrated prior to each experiment

Besides fluidic flow, a low oxygen environment is another crucial key regulator of trophoblast- and placenta development. In the first trimester of human gestation, prior to fully established uteroplacental blood flow, the mean pO2 in the developing placenta is suggested to be less than 20mmHg. At the beginning of the second trimester, when extravillous trophoblast plugs almost completely disintegrate, pO2 rises to values above 50 mmHg and stays at about this level until delivery [40,41]. Thus, future trends in trophoblast flow culture should incorporate appropriate oxygen conditions in the experimental setup. Moreover, a physiological pulsatile blood flow, leading to temporal and spatial variations of the wall shear stress can be considered to result in different phenotypes and functions of cells. While studies in the field of vascular tissue engineering increasingly acknowledge the importance of temporal and spatial variations of the wall shear stress on endothelial cells, this aspect has not yet been considered for trophoblast culture so far.

Since composition of the used culture medium and serum supplementation will greatly influence the trophoblastic phenotype, care should be taken in medium selection to meet specific requirements of different trophoblast subtypes. Recent studies suggest that extravillous trophoblasts penetrate and invade uterine glands (endoglandular trophoblasts), which are thereby opened towards the intervillous space from the very beginning of pregnancy onwards [2]. Hence, studying the consequences of uterine secretion products on trophoblasts cultured under flow could be another exciting field of research. Implementation of such specific culture media should be thoroughly characterized by continuous monitoring of component consumption by the cells.

Investigation of the "trophoblast mechanosome" will be another intriguing future direction, which should include gestational age-dependent variations, fetal sex, and maternal confounders such as smoking, diabetes, and/or obesity. Characterization of the trophoblastic mechanosome and pathologic aberrations thereof could be subject to therapeutic interventions. However, development of such drugs should not only include the therapeutic potential, but also the consequences on human pregnancy. Transfer of mechanosome modulators through the placental barrier may have adverse effects on the fetoplacental circulation, leading for example to disruption of the balanced pressure gradient between the maternal and fetal circulations [7].

**Author Contributions:** writing—original draft preparation, B.A.B., J.G., and M.G.; writing—review and editing, B.A.B., J.G., and M.G.; visualization, B.A.B., J.G.; supervision, M.G.; funding acquisition, M.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** M. Gauster was supported by the Austrian Science Fund (FWF): P 29639, P 33554, I 3304, and through the PhD program Inflammatory Disorders in Pregnancy (DP-iDP) by the Austrian Science Fund (FWF): Doc 31-B26. Moreover, M. Gauster was supported by funds of the Oesterreichische Nationalbank (Austrian Central Bank, Anniversary Fund, project number: 18175).

**Acknowledgments:** Open Access Funding by the Austrian Science Fund (FWF).

**Conflicts of Interest:** The authors declare no conflict of interest.
