**7. Outlook—Future Directions**

In recent years a number of excellent studies have yielded in an increasing body of valuable knowledge, substantiating an important influence of maternal blood flow on the (patho-)physiology of different trophoblast subtypes. The application of different flow culture systems, including customized micro-scale 3D flow chambers, has revealed substantial differences of trophoblasts cultured under flow, when compared to cells under static conditions (Figure 2).

(2) Pump

(3) Chamber

(4) Tubings

(5) Culture medium

(6) Microscope







on endothelial cells, this aspect has not yet been considered for trophoblast culture so far.

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


Besides fluidic flow, a low oxygen environment is another crucial key regulator of trophoblastand 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



**Figure 2.** Influence of fluidic flow on different trophoblast subtypes. Fluidic flow regulates differentiation and physiology of both, the syncytiotrophoblast (STB) and extravillous trophoblasts (EVTs). During syncytialization, cell-cell contact proteins, such as desmoplakin and E-cadherin are downregulated, and the structural protein ezrin relocalizes from the basal to the apical side. Formation and appearance of microvilli on the apical surface as well as accumulation of lipid droplets in the STB are influenced by fluidic flow. Moreover, fluidic flow affects expression and localization of GLUT1, secretion of hCG as well as conversion of cortisol to cortisone. In the EVT subpopulation, formation of filopodia, and hence migratory behavior is regulated by fluidic flow as well.

Some important key parameters should be considered when setting up a trophoblast flow system \*:

	- a single reservoir intercalated in a circulating flow system
	- a single reservoir with fresh medium and a tank for consumed medium
	- additional reservoirs with buffer between pump and chamber to damp flow
	- Peristaltic pump for circulating flow loop systems
	- Syringe pump or electropneumatic pump for one-time inlet-to-outlet flow systems
	- Rotating wall vessel bioreactor (rotation is responsible for distribution of medium)
	- Commercial flow chamber (laminar or turbulent)
	- Customized Chamber (laminar or turbulent)
	- Chambers created with 3D printers (suitable for sterilization)
	- Size is related to flow rate
