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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session A

Poster I4 - Creating an In Vitro Model of a Cerebral Blood Vessel to Study Endothelial Function

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cerebral cavernous malformation (CCM) is a vascular disease that affects the central nervous system (CNS), often compromising the control of vascular integrity through lesion formation. CCM affects approximately 1 in every 100-200 individuals and approximately 75% of patients with CCM experience symptoms (Cavernous Malformations, 2019). The cerebral microvasculature is a major component of the blood brain barrier (BBB) and has a crucial role in the regulation of cerebral perfusion (Brain Microcirculation, n.d.). The BBB is characterized by extremely small spaces between endothelial cells known as tight junctions. These junctions allow for regulation of molecule transport between the vasculature and the brain, protecting the cerebral tissue from toxins and allowing for control of homeostasis of the CNS (Daneman & Prat, 2015). The endothelial cells composing the BBB are responsive to shear stress. Exposure to fluid shear stress can impact endothelial cell morphology and function by increasing cell permeability and destabilizing cell-to-cell junctions, possibly contributing to vessel remodeling (Walsh et al., 2011). This project aims to study endothelial cell response to shear stress exposure and its role in CCM. A loss-of-function mutation in the kriv intertrapped 1 (KRIT-1), Malcaverin, and PDCD10 genes is linked to the formation of CCM (Riolo et al., 2021). We hypothesize that KRIT-1 mutated endothelial cell exposure to shear stress initiates CCM formation in cerebral blood vessels. In this project, we demonstrate the fabrication of a cerebral blood vessel mimic in a hydrogel material and endothelial cell responsiveness to flow, two key steps towards testing this hypothesis.

Materials and

Methods:  Hyaluronic acid crosslinked with added norbornene groups (NorHA) was the polymer chosen for hydrogel synthesis. The NorHA was synthesized using an aqueous esterification route via a reaction with carbic anhydride (Galarraga et al., 2023). A 1.5-weight % NorHA solution in DI water was formulated with LAP, DTT, and RGD (Figure 1). A 36 x 44 x 3.4 mm resin mold for the hydrogel was 3D printed using a stereolithography (STL) printer. Two 22 gauge needles were inserted into the mold before it was filled with 330 mg of polydimethylsiloxane (PDMS) solution and allowed to cure overnight. The device was punctured with an 8 mm biopsy punch and plasma bonded to a glass slide (Figure 2). After hydrogel synthesis, it was pipetted into the mold’s center and UV crosslinked at 10 mW/cm2 for 60 s (Figure 3).
Wild-type bovine aortic endothelial cells (BAECS) were cultured in 2D on culture-treated plastic in a T-75 flask until they reached confluence (105 cells/cm2). The BAECS were passaged, resuspended, and counted using a hemocytometer. The BAECS were then plated on a 75 x 38 mm, 1.01 +/- 0.05 mm thick glass slide and again given time to reach confluence. A parallel plate flow chamber was assembled to pump fluid over the BAECS plated on the glass slide to mimic the shear stress of blood flow, 10 dyn/cm2 (Figure 4). Flow was applied to the system for 6 hours, and the change in the morphology of the cells was studied.

Results, Conclusions, and Discussions: A 36% NorHA modification percentage was confirmed using NMR spectroscopy, 4% shy of the attempted modification (Figure 5). Successful hydrogel crosslinking on the PDMS device was completed using this NorHA supply and the materials listed prior.
Ongoing work is being performed to show wild-type BAECs aligning and elongating in the direction of the flow. We expect that after the flow is applied, the distribution of angle alignment of the cells will indicate that the cells have become more aligned.
The successful crosslinking of a hydrogel within the PDMS device and parallel plate flow chamber experiment will make it possible to line the hydrogel with wild-type BAECS and apply flow to the model. This will allow for comparison of the morphology of wild-type BAECS with and without exposure to shear stress.
By transfecting endothelial cells with silencing RNA (siRNA) to modify the expression of KRIT-1, then injecting these cells in a 3D, perfusable, hydrogel model of a capillary to expose them to fluid flow, we can study these effects in vitro. Proper alignment of endothelial cells to the hydrogel model with a microfluidics setup to allow it to be perfusable is also a future work in the project. Additionally, a fluorescein isothiocyanate (FITC)-conjugated dextran permeability assay will compare the permeability of each experimental group (Pauty et al., 2017). If future data from this project supports the hypothesis that endothelial cell exposure to shear stress increases cell permeability after a KRIT-1 mutation, then the claim that shear stress heightens the progression of CCM can be supported. A long-term goal would be to target shear stress-induced endothelial cell signaling pathways with therapeutic approaches such as focused ultrasound or the development of pharmacological agents (DiStefano & Glading, 2020).

Acknowledgements (Optional):  We would like to thank the University of Virginia's Biomedical Engineering Department and Dr. Brian Helmke, Dr. Chris Highley, Natasha Claxton, Tanya Cruz, Anna Dugan, Emily Ferrarese, Remington Martinez, Joy Bethea, Grace Ford, and Amalie Harrison for their support.