Effect of fluid flow on vascular network
organization in a multi-structural in vitro model
Fabian Stein, Chiara Trenti, Nasim Salehi-Nik and Jeroen Rouwkema
The Vascularization Lab, Department of Biomechanical Engineering, MIRA Institute for Biomedical Technology and Technical
Medicine, University of Twente, 7522NB Enschede, The Netherlands
Presenting Author’s Email: f.stein@utwente.nl
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 . Additionally both hydrogel channels possess together four different diameters to analyze the effect of hy-drogel 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 angiogen-ic factors.The fluid-flow channel and VEGF channel possess a pressure difference , whangiogen-ich supports the diffusion of VEGF through the hydrogel towords the endothelialized channel. Different fluid-flow profiles are applied to the cell seeded channels. The newly formed capillary network are analysed by ImageJ.
ACKNOWLEDGEMENTS
This work is supported by an ERC Consolidator Grant under grant agreement no 724469.
OUTLOOK
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 differ-ent sprouting behaviour or if the already described endothelial plasticity leads to similar results.
RESULTS
The Geltrex® (soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm tumors) based hydro-gel 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 (rat tal) instead of Geltrex®, which could reduce the shrinking phenomenon during polymeriza-tion. The Collagen I coated fluid-flow channels are succesfully endothelialized with HUVECs, who formed a dense monolay-er in the whole innmonolay-er surface area of the channels, which also keep stable after appling of a fluid flow.
Blood Flow
Shear Stress
INTRODUCTION
The process of converting mechanical signals to biochemical and physical change is known as mechanotransduction. Several different key com-penents are included e.g. the cytoskeleton, nuclear lamina, cadherins, integrins, strech activated ion channels and focal adhesions. Due to this signal-ling, maturation, organisation and cell survival is regulated.
The integration of engineered tissues after implan-tation is limited due to the lack of a vascular net-work, vascular networks that are not organized, or networks that lose their initial organization fast. In order to have a better understanding about the vascular organization and maturation of prevascu-larized tissue, different fluid flow profiles are ap-plied to a Endothelial cell containing microfluidic system.
Glycocalyx
Erythrocyte
Endothelial cells Basal membrane Smooth Muscle cells Tunica Adventitia
(incl. Myofibroblast, Fibroblasts)
Perycyte
Endothelial Tip cell
Fluid-Flow channel Fluid-Flow channel Hydrogel-Channel VEGF-Channel+ HUVECs (Collagen I)
Endothelial Stalk cell DII4 DII4 Notch Notch Target Genes VEGF VEGFR-2 Cytoskeleton Adherence Junctions Tight Junctions Focal Adhesions Surface Proteins NO Angiopoietin II PDGF Angiopoietin I PDMS Pillar Hydrogel-Channel VEGF channel
