# Pneumatically Actuated Microfluidic Platform for Reconstituting 3D Vascular Tissue Compression
## 2 Results and Discussion  
### Microfluidic Chip Design and Experimental Design  
Previous studies generated perfusable microvessel networks in a microfluidic device through the co-culture of human umbilical vein endothelial cells (HUVECs) and stromal cells (lung fibroblasts (LFs)) [2]. Endothelial cell sprouts induced by stromal cells resulted in end-to-end perfusible lumina, allowing for uninterrupted fluid flow through the vessel networks. While the single-layer design can reconstitute three-dimensional vessel networks and induce interstitial flow, it cannot induce biomechanical stress in the generated vascular network. The proposed platform included an additional layer to incorporate a pneumatic actuation chamber, allowing for the exertion of compressive forces on the vascular channel (Figure 1A,B).  
The microfluidic chip components were fabricated through soft-lithographed PDMS. The microfluidic chip was composed of an air channel and cell culture compartments. In the cell culture layer, the chip consisted of five parallel channels; a central channel, two stromal cell culture channels, and two media channels. All channels were 800 \(\upmu\)m wide and 70 \(\upmu\)m in height (Figure 1C). One hundred micrometer micropost gaps facilitated the compartmentalization of stromal and endothelial culture channels during initial fluid and cell patterning, allowing for vasculogenic endothelial organization. The PDMS membrane between the air channel and cell culture layer was 70 \(\upmu\)m high (Figure 1D-F) and was actuated using a CellASIC pump (Merck KGaA, Darmstadt, Germany) (Figure 1A). Induced air pressures were held for one minute for each experimental test condition (Figure 1G,H). Prior to testing on live microvascular tissues, the compression mechanism was tested on a channel filled with a 0.5 mg/mL FITC-dextran suspension in Phosphate-Buffered Saline (PBS). Increases in pressure resulted in a decrease of fluorescence along the sagittal axis of the channel, corresponding to the displacement of beads with the PDMS membrane deflection (Figure 2A,B). In the absence of applied pressure, the fluorescence intensity was uniform throughout the channel. The decreased fluorescence profile indicates the gap between the deflected PDMS membrane and glass substrate, and the decrease in fluorescence represents the deflection of the PDMS membrane in accordance with an increasing applied pressure.  
Figure 1: Multilayer microfluidic device for mimicking solid-stress-induced blood vessel compression and collapse in vitro. (**A**) Schematic diagram of the experimental setup. (**B**) Schematic illustration of the pneumatically actuated microfluidic platform for studying blood vessel compression. (**C**) Magnified rendered image in the cell culture layer and 3D vascularized microvessels characterized by immunofluorescence and vasculogenesis on day 5. Scale bar: 100 \(\upmu\)m. (**D**) Configuration of the microfluidic device with the air channel in the “OFF” state, representing that there was no applied pressure. (**E**) Configuration of the microfluidic device with the air channel in the “ON” state, where air pressure was applied to the channel. Polydimethylsiloxane (PDMS) membrane deflection could induce in vitro blood vessel compression in a controlled manner. (**F**) Cross-sectional SEM image of the device. (**G**,**H**) Bright-field microscopy images of in vitro blood vessels in the device at 0 psi and 2.5 psi, respectively.