Exploring Endothelial States: How Chemical Fixation Does Not Adversely Affect Aspects of Vascular Function in Complex in Vitro Models with Sickle Cell Disease
Authors: Mejia-Jaramillo Daniel , Fibben Kirby S , Sencer Jacob , Lam Wilbur 
Published: December 2024

In hematological diseases such as Sickle Cell disease, genetic abnormality causes biophysical changes among red blood cells that lead to vaso-occlusive crises via blockages in the blood vessels. In human patients with Sickle Cell Disease, building a physiological in vitro model has proven challenging. This undergrad research project focuses on using an ‘endothelialized’ microfluidic microvasculature model partnered with whole blood samples from patients with Sickle Cell Disease to further investigate these unique interactions. The aim of this project is to research these specific interactions applied to both a healthy endothelium and a fixed one when using blood samples from patients with Sickle Cell disease. This microfluidic has the capability of recapitulating and integrating the pathophysiological process developed within the body, in particular the microvascular. Once the cells become confluent and healthy, patients blood samples are obtained in sodium citrate and can be recalcified and prefused in order to restart the clotting cascade within the created endothelium. This process allows CD41 and CD45 to be tagged, imaged and analyzed using iCLOTS to quantify area and occlusion. This model has been well characterized for a healthy endothelium and has been shown to work for patient specific drug diagnostics. Here, this abstract highlights how this model could be translated for a fixed endothelium and furthermore how it allows for translating the microvascular-on-a-chip to the clinical lab. To produce a chemically preserved (fixed) endothelium, 4% Paraformaldehyde (PFA) is perfused within the device. A fixed endothelium will retain its ability to support platelet adhesion when human whole blood is flowed through the channel (Figure 1A). These devices lined with a fixed endothelium support the adhesion of platelets to the P-selectin membrane proteins (Figure 1B). The potential clinical value of a fixed device can be demonstrated through a non-significant difference in occlusion of platelets and white blood cells within minutes of using µL of the same patient sample whole blood when compared to a non-fixed device (Figure 1C). A 2x2 matrix is utilized to analyze the attenuation of occlusion across a fixed and healthy endothelium and compared with control blood and patient blood. Based on the results, the amount of tagged CD41 in both fixed and healthy endothelium devices were shown to be the same across the Sickle population and a non-Sickle healthy control population (Figure 1D). The microfluidic devices demonstrated consistent levels of platelet adhesion with minimal variability across experiments indicating a consistent level of interaction between the platelets and endothelium regardless of whether control blood or blood from individuals with Sickle Cell disease was used. The undergraduate research is ongoing, focusing on quantifying occlusion through a microvascular-on-a-chip model. By using a fixed endothelium, it can enable the development of more reliable and robust assays for analyzing occlusion with conditions that mimic the vasculature, which could benefit clinical laboratories and point-of-care settings in the future.