Investigating Mechanical Stimulation on Angiogenesis for Vascularized Brain Models

<b><u>Introduction</u></b>: Neurodegenerative disorders are caused by the progressive loss of structural brain tissue and the death of neurons, which lead to devastating conditions and diseases such as Alzheimer's disease (AD). Although the mechanisms underlying AD have been extensively studied, there are currently no treatments proven to stop or reduce neurodegeneration. One of the main contributing reasons is that the current models poorly represent the physiology of the human brain. While brain organoids hold great potential for understanding the development of neurodegeneration, current organoid models still have important limitations, the absence of functional vascularization. This limitation not only restricts the supply of nutrient transport, delivery of oxygen, and waste removal to/from organoids but also hinders the recapitulation of the blood-brain barrier (BBB), an essential component in AD progression. Despite a wide range of biochemical and biological approaches to promote vascularization, there remains a need for fully perfusable systems developed in a controlled manner. While mechanical cues are as important as biochemical factors in developing vascularized organoids, they are much less studied due to the complexity of platforms. There is thus a critical need for a bioengineering approach to control mechanical cues and study their impacts on the vascularization of brain organoids, with the ultimate goal of developing fully vascularized AD models.<br/><br/><b><u>Methods</u></b>: Our lab has previously demonstrated that a magnetically actuated matrix (MagMA) embedded in an extracellular matrix can be controlled by a permanent magnet, enabling non-invasive mechanical stimulation. We integrated MagMA into a 3D tubular perfusable vasculature on a microfluidics platform to investigate the impacts of mechanical stimulations on angiogenesis and vascularization of brain organoids. The embedded MagMA was fabricated by mixing iron microparticles with poly(dimethyl siloxane) (PDMS). This microfluidics platform is composed of two hollow cylindrical vessels (600 µm in diameter) lined with human umbilical vein endothelial cells (HUVECs), positioned on either side of the brain organoid culture site. Programmable mechanical stimulation on this platform was achieved by embedding MagMA into the collagen-based hydrogel bed. This straightforward 3D platform allows for studying the effects of dynamic mechanical stimulation on angiogenesis and vessel alignment.<br/><br/><b><u>Results</u></b><b>: </b>We demonstrated that our microfluidics platform is capable of forming perfusable feed vasculature in a 3D setting after 5 days of cell culture. Additionally, we showed that this platform can effectively investigate the effects of controlled mechanical stimulation on angiogenic sprouting from existing endothelial channels. As a proof of concept, we studied the impacts of different regimes of mechanical stimulation using embedded MagMA on angiogenic sprouting. We examined the effects of duration and frequency of mechanical deformation on the formation and alignment of sprouts from endothelial channels using deep learning-based image processing algorithms (Angio-Net). Furthermore, we characterized the effects of mechanical stimulation on molecular permeability in the vessels, a key application for BBB models. Two molecular weights (10 and 40 kDa) of FITC-Dextran were used to model vessel permeability for both small and large molecules. Molecular permeability was investigated via confocal imaging of FITC-Dextran molecules.<br/><br/><b><u>Conclusion</u></b><b>: </b>We have developed a novel platform for investigating the impact of controlled mechanical stimulation on the formation of new vessels in a 3D setting. Our microfluidic platform has the potential to generate new fundamental knowledge on the sole effects of mechanical factors in the formation and alignment of angiogenesis, with the ultimate goal of developing fully vascularized brain organoids.