(F-201) Cell derived extracellular matrix modulates response to fluid shear stress

Introduction:: Endothelial cell (EC) morphology is impacted by local mechanical cues including wall shear stress and the mechanical properties and architecture of the substrate¹. Compliant substrates have been shown to impact endothelial cell morphology and promote Notch1 signaling¹, while fiber orientation induces cell elongation and directed migration^2,3. Cell derived matrix (CDM) has been shown to retain the mechanical properties and fibrous architecture of native extracellular matrix (ECM)⁴ and to present native adhesion ligands⁵. Furthermore, hemodynamic shear stress induces EC elongation and alignment^2,6. To recapitulate hemodynamic stresses ex vivo, cone and plate rheometers have been developed to impart consistent wall shear stress across a large surface area⁶, to allow biochemical assays that require significant input mass, though most rheometers include a glass or tissue culture plastic substrate. Here, we present a custom, low-overhead cone and plate rheometer that allows the application of hemodynamic shear stress to cells cultured on compliant substrates, including CDM. We implement the platform in a study that seeks to decouple the morphological impacts of flow and substrate fiber alignment by comparing polarity markers in endothelial cells on isotropic substrates, CDM, and aligned CDM with and without exposure to flow.

Materials and Methods:: The components for the novel cone and plate rheometer were modeled in Fusion 360 and printed on an Ultimaker Cura S5 with Tough PLA. To allow the cone to spin smoothly on the dish, a 50 μm thick Teflon disk was glued to the center of each dish with cyanoacrylate superglue. Two different substrates were used in this study: a 5μg/mL fibronectin coating and a CDM substrate grown from wild type human dermal fibroblasts (HDF-WT) that were allowed to grow CDM for 1 week before decellularization. After preparing the coated plate, human aortic endothelial cells (HAECs) were plated between passages 7 and 9 at 25%-50% confluence and allowed to grow to confluence in the prepared 10 cm dishes. Once cells reach confluence, the dish is inserted into the rheometer to apply flow. A short ramp up period of 30 minutes total of 2.5 dynes/cm² and 5 dynes/cm² shear were applied initially before increasing to the total shear of 10 dynes/cm² for 24 hours. Cells were imaged with phase contrast before and after flow. The flow profile was visualized by using 50 μm diameter fluorescent microspheres and running the rheometer on a widefield microscope.

Results, Conclusions, and Discussions:: Results and Discussion: The flow system consists of a 3D-printed housing, cone, and control box (Fig. 1A) and is driven by an Arduino controlled motor. The cone design included a PDMS outer sleeve and an aluminum ball bearing to limit the compression of the PDMS (Fig. 1B). The applied flow profile was validated using fluorescent microbeads resulting in an observed flow that was largely concentric with few deviations for the 3D printed cone (Fig 1C). Shear stress applied to cells cultured on tissue culture plastic coated with fibronectin resulted in some cell detachment near the center of the cone, and cells aligned in a consistent direction after 24hrs of flow (Fig. 1D, 1E). When plated on CDM, HAECs aligned with the local structure of the CDM (Fig. 1F), and after shear was applied, some regions of cells appeared to align with flow, while others remained aligned with the CDM (Fig. 1G).

Conclusion: The results from this study demonstrate that the 3D printed cone rheometer is effective at generating physiologically relevant wall shear stress to EC and that the modular design allows for shear stress to be applied to substrates with varying architectures and material properties. At 24 hours, EC alignment appears to be fiber orientation dominated, with the orientation of the HAECs after flow more closely resembling pre flow alignment on CDM rather than the direction of flow. Future work will involve staining both conditions for golgi polarization and compare with CDM grown from flow conditioned HDF-WT.


Acknowledgements (Optional): : This work has been supported by NIH R35GM142944 (WJP), T32 GM133393 (SEK), NIH T32HL69768 (ELD, CW), and NIH F31HL162462 (ELD)

References (Optional): : ¹ Kretschmer, M., et al. (2023). "Matrix stiffness regulates Notch signaling activity in endothelial cells." Journal of Cell Science 136(2).

² Yi, B., et al. (2020). "Stiffness of the aligned fibers affects structural and functional integrity of the oriented endothelial cells." Acta Biomaterialia 108: 237-249

³ McCoy, M. G., et al. (2018). "Collagen Fiber Orientation Regulates 3D Vascular Network Formation and Alignment." ACS Biomaterials Science & Engineering 4(8): 2967-2976.

⁴ Girardeau-Hubert, S., et al. (2022). "Impact of microstructure on cell behavior and tissue mechanics in collagen and dermal decellularized extra-cellular matrices." Acta Biomaterialia 143: 100-114.

⁵ Eyckmans, J., et al. (2011). "A Hitchhiker's Guide to Mechanobiology." Developmental Cell 21(1): 35-47.

⁶Dewey, C. F., et al. (1981). "The Dynamic Response of Vascular Endothelial Cells to Fluid Shear Stress." Journal of Biomechanical Engineering 103(3): 177-185.