Design of Side-View Particle Image Velocimetry System for Cellular Adhesion Analysis
Design of Side-View Particle Image Velocimetry System for Cellular Adhesion Analysis
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Professor Cheng DongDr. Cheng Dong is the principle investigator of the Cellular Biomechanics Laboratory of the Department of Bioengineering at Pennsylvania State University. His career work focuses on studying the biomechanical, biophysical and biochemical aspects of cellular function in the human circulatory system by biological experimentation and mathematical modeling. Dr. Dong collaborates with investigators at the National Institutes of Health as well as the Colleges of Science and Medicine at Penn State. Most of Dr. Dong’s current research involves the process of cell adhesion, intercellular and intracellular signaling, and cell motility involved in the human inflammatory response and cancer metastasis. His research has been sponsored by the Whitaker Foundation, the American Cancer Society, the National Science Foundation, and the National Institutes of Health. ExtravasationOne of crucial steps in the inflammatory response and cancer metastasis is called extravasation, during which individual leukocytes or tumor cells adhere to the walls of microvascular capillaries and migrate through the endothelium to reach the underlying tissue. The process of cellular adhesion revolves around a complex balance of forces arising from the hemodynamic shearing effects of the surrounding flow and the strength of adhesive bonds between cells and their endothelial substrate. This balance depends strongly on cell deformability and the expression of cell adhesion molecules (CAMs). Cell adhesion to the microvascular endothelium is one of the important initial steps in the extravasation of leukocytes in the inflammatory response and tumor cells in metastasis processes. The process of adhesion involves a complex balance of forces resulting from hydrodynamic shear forces and the adhesive bonds between the adhering cells and the endothelium. Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate the ability of tumor cells and leukocytes to arrest on the endothelium and sustain adhesion [1]. However, the mechanisms involved, especially in tumor cell adhesion, are poorly understood and investigation has been limited by the lack of experimental models that allow quantitative analysis of flow profiles over adherent cells. In vivo studies [2-5] have made clear that leukocytes and metastatic tumor cells employ vastly different mechanisms to adhere to a vessel wall prior to extravasation. In the inflammatory response, leukocytes adhere to the vascular endothelium [6-8] and significant progress has been made toward understanding how human neutrophils (PMNs), which comprise 50-70% of circulation leukocytes, role along the endothelium before forming a firm bond resistant to flow shear stresses. Inflammatory cytokines (e.g. including interleukin-1 (IL-1)) are known to induce expression of cell adhesion molecules on endothelial cells. Studies have shown that the selectins are necessary for the initial rolling of leukocytes on activated endothelium and that L-selectins on leukocytes and E- and P-selectins on endothelial cells participate in these interactions [9, 10]. Firm adhesion, or prolonged shear-resisted attachment requires the role of integrins which are expressed on the leukocyte (Mac-1 or LFA-1) and on the endothelial cells (ICAM-1) [11]. Studies [12, 13] have suggested that LFA-1 to ICAM-1 adhesion is more important in the initial endothelial capture of PMNs, while the interaction between Mac-1 and ICAM-1 functions to form the shear-resisted bonds that stabilize PMN-endothelium adhesion. Less is known about how tumor cells bind to the endothelium. It has been noted that the initial arrest of metastasizing tumor cells on the endothelium does not exhibit “leukocyte-like rolling” [14]. In vivo work [4] has suggested that melanoma cells can become arrested by a chemoattraction mechanism. Despite the fact that leukocytes seem to utilize different mechanisms of adhesion, many of the same endothelial surface molecules that mediate leukocyte adhesion also mediate tumor cell adhesion to the endothelium [5] (e.g.ICAM-1, VCAM-1 (vascular adhesion molecule), E-selectin, and P-selectin). In addition, ligands for inducible endothelial adhesion molecules have been identified on various types of tumor cells [15]. PMN-Tumor Cell InteractionsAlthough the involvement of PMNs in tumor metastasis is still uncertain, there have been reports of tumor cells exploiting leukocytes to enhance binding and metastasis [16, 17]. Investigations have been made into the abilities of lymphocytes [18], natural killer cells [19], and monocyte/macrophages [20-22] to mediate tumor cell adhesion. PMNs have been shown to promote tumor adhesion and transendothelial migration under certain conditions[23]. In vivo studies with rat hepatocarcinoma cells [24] and andadenocarcinoma cells [16] together suggested that PMNs enhance the ability of tumor cells to bind to endothelium and enhance the overall metastatic invasiveness of tumors. A recent in vitro study [25] suggests that PMNs influence tumor cell adhesion and migration under flow conditions, but not under static conditions when tumor cells are allowed to adhere in the absence of shear forces. Thus, PMNs have been proposed to play a role in the initial arrest of tumor cells, compensating for the lack of tumor cells to undergo “leukocyte-like rolling.” Although the precise mechanism that PMNs play in mediating tumor cell extravasation is unclear, PMNs have been found to activate specific transduction pathways and alter gene expression in tumor cells, yet their roles are not clearly understood. Role of Fluid Stresses and Cell Deformation in AdhesionThe roles of fluid shear stresses and cell deformation in cell adhesion and cell rolling have been studied extensively [26-31]. In vivo studies [27, 32] of leukocyte-endothelial adhesion as well as CFD (computational fluid dynamics) studies [33-35] employing biomechanical models have found that the shearing drag force on leukocytes adhering on the endothelium is affected by the incoming wall shear force as well as the degree of deformation. Cells deform viscoelastically, and are often modeled [36] as standard linear viscoelastic solids, with series elastic and viscous elements in parallel with an additional elastic solid. The endothelial contact area of the leukocytes under adhesion in these studies was found to increase under periods of increasing shear forces, suggesting an increase in viable interactions among adhesion molecules. The computational models have also suggested that the effective drag forces acting on adhering cells is reduced following deformation, thus offering another enhancement of the ability of cells to maintain adhesion under shear flow as a result of deformation. Interestingly, the modeling studies by Liu et. al. [28, 37-39] have noted that deformation may increase as a result of changes in the Reynolds number with maximum deformation occurring at high Reynolds numbers. Another study [26] found that circulating erythrocytes managed to increase cell deformation. Although the roles of shear stresses in the flow have been analyzed extensively, little attention has been made to the role of shear rates and differing fluid viscosities in deformation. Increases in the Reynolds number within identical microvascular geometry reflects changes in the fluid velocity and shear rate as well as the viscosity [40]. Thus, it is possible to alter the Reynolds number, while maintaining constant incoming shear forces. Clearly, there must be more investigation into the issue of how fluid stresses affect cell adhesion before a more complete understanding can be realized. pathways and alter gene expression in tumor cells, yet their roles are not clearly understood. Use of Side-View Flow Chamber in Cell Adhesion Studies
A side-view flow chamber was developed by Dong and colleagues [41] to visualize cell surface adhesion and deformation in a microcapillary (700x550 µm2). It consisted of two chromium plated 45° prisms that redirected incident light from a condenser in a phase-contrast Nikon microscope. This setup removes the limitation of top-down illumination, allowing cells to be visualized and studied in a fashion that is not possible with conventional parallel plate chambers. Among other applications, the side-view flow chamber has been used to measure the effects of flow on cell-surface adhesion strength and the cell-substrate contact interface [31, 35].
Role of PIV in Quantitative Fluid Mechanics
Particle Image Velocimetry (PIV) is a quantitative field measurement technique that involves using laser illuminated tracer particles to visualize and quantify flow structure. Generally, for two dimensional flow data, sets of two closely spaced frames are compared using a cross correlation algorithm that grids the image field into interrogation windows within which correlation functions are calculated:
where X is the spatial coordinate in the image plane and s is the spatial coordinate in the correlation plane. I1(X) and I2(X) represent the intensities of the image field at each of the two frames. Usually, this equation is altered to account for the discrete pixel elements within the interrogation windows. For a window of M x N pixels, the correlation function is:
where x and y form the special coordinates in the correlation plane and i and j are the pixel indices within the image windows. The maximum of the correlation function, R(s)max within a given interrogation window theoretically corresponds to the given displacement within the associated spatial region of tracer particles during the time delay between frame acquisitions. It is normally advantageous to have a high density of tracer particles so that accurate measurements within each interrogation window can be achieved. In addition, higher resolution profiles can be achieved by minimizing the size, and thus maximizing the number of interrogation windows. In recent years, PIV techniques have been extended to the microscale, proving highly useful for acquiring microscale flow data [42-45] in microfluidic studies. However, the limitations of microscale optics have limited the acquisition of microscale flow data to 2 dimensional planar representations. In addition, the volume illumination element of microscale PIV limits the signal quality that can be achieved, often necessitating data processing techniques prior to analysis. Detailed Problem DescriptionCell adhesion to the microvascular endothelium is one of the important initial steps in the extravasation of leukocytes in the inflammatory response and tumor cells in metastasis processes. It involves a complex balance of forces resulting from hydrodynamic shear forces and the adhesive bonds between the adhering cells and the endothelium. Experimental models that mimic the flow conditions in microcapillaries have suggested that the local shear stresses and shear rates can mediate the ability of tumor cells and leukocytes to arrest on the endothelium and sustain adhesion [1]. However, the mechanisms involved, especially in tumor cell adhesion, are poorly understood and investigation has been limited by the lack of experimental models that allow quantitative analysis of flow profiles over adherent cells. Future investigation will be aided with the development of a system capable of acquiring quantitative flow profiles over adherent cells. This document reports the construction of an in vitro model capable of acquiring such data by combining the techniques of side-view imaging and particle image velocimetry (PIV), thus providing a means for future study of the hydrodynamic forces underlying cellular adhesion. Currently, cancer metastasis is the leading cause of death among cancer patients. Over one million people are diagnosed with cancer each year |
