Effect of fluid flow on vascular network organization in a multi-structural in vitro model

Introduction:
Engineered tissues offer a great promise to the field of medicine as an alternative for donor tissues for which the supply is not meeting the demands. However, the integration of engineered tissues after implantation is limited due to the lack of a vascular network. Currently, strategies to include vascular networks rely on the spontaneous organization of vascular cells, or on the patterning of these cells. However, this results in either vascular networks that are not organized, or networks that lose their initial organization fast.1-3 In this project we will use interstitial flow as one of the main cues to control vascular organization and maturation in hydrogel-based tissues.

Aim:
To develop a microfluidic system to evaluate the effect of fluid flow profiles on vascular organization and maturation.

Materials and Methods:
We use a microfluidic 5-channel PDMS system that was developed in our group. The hydrogel channels are flanked by media channels and PDMS pillars to contain the Collagen I (5 mg/mL) (see Scheme 1). Additionally both hydrogel channels possess together four different diameters to analyze the effect of hydrogel thickness on endothelial cell sprouting. The media channels are coated with 0.1% Collagen I to improve the cell attachment and seeded with Human Umbilical Vein Endothelial Cells (HUVECs). One channel is filled with VEGF (50 ng/mL), which is known as one of the main angiogenic factors.4 Different fluid-flow profiles are applied to the cell seeded channels 24 hours later. The newly formed capillary network are analysed by ImageJ.

Results and Conclusions:
The Geltrex® (soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm tumors) based hydrogel channels shrink rapidly during the polymerization process, which further led to the formation of deep pores between the pillars. Due to the presence of the pores, the formation of a smooth HUVEC monolayer is disturbed. Therefore, it is better to use Collagen I hydrogel instead of Geltrex®, which could reduce the shrinking phenomenon during polymerization. Based on various fluid-flow profiles, hydrogel thicknesses and diffusion of VEGF within the hydrogel, different sprouting of HUVECs into the Collage I hydrogel channel is observed.

Future Plans:
Gradients of stiffness of different hydrogels (Collagen I, Geltrex®) will be generated and used in a designed mold with a 3-Channel system. To mimic the physiological state, different Endothelial cell types (e.g. HUVECs, HMECs, HIAEC) will be integrated into the fluid flow channels. This will allow us to see if different endothelial cell origins leads to a different sprouting behaviour or if the already described endothelial plasticity leads to similar results.

References:
1. Levenberg et. al., Engineering vascularized skeletal muscle tissue. Nat Biotechnol, 2005. 23(7): 879-84.
2. Rivron et. al., Tissue deformation spatially modulates VEGF signaling and angiogenesis. Proc Natl Acad Sci U S A, 2012. 109(18): 6886-91.
3. Rivron et. al., Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation. Proc Natl Acad Sci U S A, 2012. 109(12): 4413-8.
4. Shibuya M., Vascular endothelial growth factor and its receptor system: physiological functions in angiogenesis and pathological roles in various diseases. J Biochem, 2013. 153(1):13-9.
Acknowledgements
This work is supported by an ERC Consolidator Grant under grant agreement no