(229bn) Leukocyte Purification and 3D Migration from a Drop of Whole Blood

The directional migration of leukocytes in response to soluble chemokine gradients is the basis of multiple physiological and pathophysiological processes including wound repair, immune response, and several other homeostatic phenomena. Current in vitro approaches developed to characterize the behavior of primary leukocyte cells rely on fractionation procedures to obtain white blood cells from whole blood (e.g. centrifugation, chemical lysis, or fluorescence assisted cell sorting). While these techniques efficiently deplete unwanted cell populations, they are prone to activating leukocytes and altering their native phenotypes. Furthermore, due to the low abundance of leukocytes in whole blood, these procedures can be time consuming, laborious, and may not be suitable for small sample volumes. Recently, microfluidic approaches have also been utilized to achieve highly precise fractionation of whole blood. Using these devices, researchers have been able to analyze the migratory and chemotactic behavior of leukocytes along two dimensional surfaces. To adequately mimic the three-dimensional in vivo environment, we have developed an integrated platform that combines whole blood sorting and hemodynamic focusing to enhance leukocyte-endothelial cell interactions for subsequent chemokine stimulated adhesion and transendothelial migration (TEM). Once through the endothelium, the leukocytes can migrate towards a chemokine gradient that is generated through a biomimetic three-dimensional extracellular matrix. The unique design of this device preferentially limits migration to single cells allowing high resolution and real-time observations of native cell behaviors. We have characterized the separation technique using finite element simulations, and verified the efficiency using fluorescent microparticles with radii similar to red blood cells and leukocytes. Furthermore, we have recently demonstrated the efficiency of our single cell migration device (S.A. Roberts, Anal. Chem. 2016). While we utilize this platform primarily to study leukocyte TEM behavior, the integration of cell isolation with 3D migration makes the design highly versatile making it suitable for many applications, including liquid biopsies and pharmaceutical screenings.