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Cardiovascular Engineering

NovelMicrofluidics System to Investigate Blood Rheology and Endothelial Cell Behavior

(G-256) Novel Microfluidics System to Investigate Blood Rheology and Endothelial Cell Behavior

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row G - Poster # 256

Presenting Author(s)

Michael A. Daanen (he/him/his)

Undergraduate Researcher
Bucknell University
Sherrills Ford, North Carolina, United States

Co-Author(s)

NB

Naafay Bajwa

Student Researcher
Bucknell University, United States

Primary Investigator(s)

OB

Olivia Boerman

Assistant Professor
Bucknell University
Lewisburg, Pennsylvania, United States

Introduction:: Blood pressure, flow, and arterial shear stress all impact endothelial cell activity, arterial remodeling, and angiogenesis [1,2]. One of the most fundamental examples of this phenomenon is the arterial exposure of in-vivo shear stresses from normal pulsatile blood flow leading to vascular endothelial cell alignment in the direction of flow. Furthermore, abnormal flow, such as the flow due from implanted artificial circulation devices, can expose endothelial cells to supraphysiological arterial shear stresses and cause microcirculatory dysfunction, leading to a number of pathophysiologies including organ dysfunction and mortality [3]. While much research has been done to characterize endothelial cell behavior and endothelial cell dysfunction, most of this research has been done under static conditions, failing to account for the effects of flow on endothelial cell behavior. Furthermore, the biophysiological thresholds at which shear levels begin to give rise to endothelial cell dysfunction are largely unknown. The purpose of this research is to validate a microfluidic technology capable of emulating arterial environments, allowing for a tightly controlled mechanistic investigation of the interactions between blood flow, arterial shear stress, and endothelial cell activity.

Materials and Methods::

A microfluidic device was designed, capable of generating biologically relevant shear stresses. The microfluidic device was developed using the Computer Aided Design (CAD) Software OnShape. Specifications of the device have been deliberately omitted to protect intellectual property, as it is currently undergoing the patent process. Generated shear stress, shear homogeneity, and laminar flow within the flow chamber were verified with COMSOL Multiphysics. When performing live cell tests, coverslips were coated with a 10 microgram/mL concentration of human recombinant fibronectin, expressed in HEK 293 cells, to allow for cellular adherence. The fibronectin was set for 45 minutes before being seeded with 10x10^6 human umbilical vein endothelial cells (HUVECs) until 100% confluency was reached. The confluent coverslips were then placed into the microfluidic device within an incubator at 37° C and 5% CO2. Experiments were conducted recirculating complete vascular media at 4 and 8 Pa of shear stress, representing average and maximum shear under vascular conditions. Cells were imaged with Fullbright microscopy for 24 hours, and directionality of endothelial cells was analyzed using the Directionality tool in Fiji, a package of ImageJ. Directionality of cells was compared statistically before and after flow using a t-test. A p-value of less than 0.05 was considered to be significant.

Results, Conclusions, and Discussions::

In both the 4 Pa and 8 Pa studies, endothelial cellular alignment was observed with the direction of flow, with alignment occurring within 18 hours at 4 Pa of shear stress and 8 hours at 8 Pa of shear stress (Figures 1B &1D). For the 4 Pa study, average directionality was found to be 49.22° with a standard deviation of 23.86° before flow, and average directionality was 71.39° with a standard deviation of 7.88° after flow. For the 8 Pa study, average directionality was 43.06° with a standard deviation of 24.36° before flow, and 60.40° with a standard deviation of 19.83° after flow. Directionality was statistically compared before and after flow for 4 Pa and 8 Pa, both yield p-values < 0.001, indicating a statistically significant difference in directionality and alignment with flow. Alignment of the endothelial cells with the directionality of flow within the microfluidic device was determined to be indicative of a successful replication of arterial environments, as cultured endothelial cell behavior was observed to mimic the in-vivo alignment response to pulsatile blood flow. Alignment occurred roughly twice as fast when shear stress doubled, indicating a direct relationship between shear stress magnitude and time of alignment. Endothelial cells align in the presence of shear stresses in an effort to mitigate intercellular forces. As such, alignment likely occurs at a greater speed under higher-shear situations to preserve cellular integrity and disperse the larger intercellular forces experienced in the presence of larger shear stresses.

The results of this research indicate the successful development of a technology to mimic arterial conditions. Such a technology can be used for a widespan of endothelial cell behavior and rheological studies, including the elucidation of biophysiological thresholds for abnormal endothelial cell behavior, simulation of supraphysiological environments experienced due to implanted artificial circulation devices, and measuring the rate of endothelial cell alignment under varying magnitudes of arterial shear stress.

Acknowledgements (Optional): :

References (Optional): :

1) Haga, J.H.; Li, Y.-S.J.; Chien, S. Molecular Basis of the Effects of Mechanical Stretch on Vascular Smooth Muscle Cells. Journal of Biomechanics 2007, 40, 947– 960. 2) Li, Y.-S.J.; Haga, J.H.; Chien, S. Molecular Basis of the Effects of Shear Stress on Vascular Endothelial Cells. Journal of Biomechanics 2005, 38, 1949–1971, doi:10.1016/j.jbiomech.2004.09.030. 3) De Backer, D., Orbegozo Cortes, D., Donadello, K., & Vincent, J. L. (2014). Pathophysiology of microcirculatory dysfunction and the pathogenesis of septic shock. Virulence, 5(1), 73–79. 4) Nigro, P., Abe, J., & Berk, B. C. (2011). Flow shear stress and atherosclerosis: a matter of site specificity. Antioxidants & redox signaling, 15(5), 1405–1414.