Endothelial Cell Polarization and Chemotaxis in a Microfluidic Device

Department of Mechanical Engineering, Stanford University, Stanford, CA, USA.
Lab on a Chip (Impact Factor: 6.12). 08/2008; 8(8):1292-9. DOI: 10.1039/b719788h
Source: PubMed


The directed migration of endothelial cells is an early and critical step in angiogenesis, or new blood vessel formation. In this study, the polarization and chemotaxis of human umbilical vein endothelial cells (HUVEC) in response to quantified gradients of vascular endothelial growth factor (VEGF) were examined. To accomplish this, a microfluidic device was designed and fabricated to generate stable concentration gradients of biomolecules in a cell culture chamber while minimizing the fluid shear stress experienced by the cells. Finite element simulation of the device geometry produced excellent agreement with the observed VEGF concentration distribution, which was found to be stable across multiple hours. This device is expected to have wide applicability in the study of shear-sensitive cells such as HUVEC and non-adherent cell types as well as in the study of migration through three-dimensional matrices. HUVEC were observed to chemotax towards higher VEGF concentrations across the entire range of concentrations studied (18-32 ng mL(-1)) when the concentration gradient was 14 ng mL(-1) mm(-1). In contrast, shallow gradients (2 ng mL(-1) mm(-1)) across the same concentration range were unable to induce HUVEC chemotaxis. Furthermore, while all HUVEC exposed to elevated VEGF levels (both in steep and shallow gradients) displayed an increased number of filopodia, only chemotaxing HUVEC displayed an asymmetric distribution of filopodia, with enhanced numbers of protrusions present along the leading edge. These results suggest a two-part requirement to induce VEGF chemotaxis: the VEGF absolute concentration enhances the total number of filopodia extended while the VEGF gradient steepness induces filopodia localization, cell polarization, and subsequent directed migration.

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    • "Best characterized is the chemotactic response of microvessel endothelial cells to acidic fibroblast growth factor (aFGF), where the chemotactic index was estimated as 1.5, thus chemotactic directed motion dominated over random motility (Stokes et al., 1991). More recent studies using microfluidic chambers indicated that a VEGF gradient of approximately 20 mg/ml is needed to elucidate directional migration of cells (Chen et al., 2007; Shamloo et al., 2008). To put this value in perspective, 40–100 mg/ml is the estimated bulk concentration of VEGF needed to saturate cell surface VEGF receptors in vitro (Chen et al., 2007). "
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    ABSTRACT: Vasculogenesis, the assembly of the first vascular network, is an intriguing developmental process that yields the first functional organ system of the embryo. In addition to being a fundamental part of embryonic development, vasculogenic processes also have medical importance. To explain the organizational principles behind vascular patterning, we must understand how morphogenesis of tissue level structures can be controlled through cell behavior patterns that, in turn, are determined by biochemical signal transduction processes. Mathematical analyses and computer simulations can help conceptualize how to bridge organizational levels and thus help in evaluating hypotheses regarding the formation of vascular networks. Here, we discuss the ideas that have been proposed to explain the formation of the first vascular pattern: cell motility guided by extracellular matrix alignment (contact guidance), chemotaxis guided by paracrine and autocrine morphogens, and sprouting guided by cell-cell contacts.
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    • "For example, with the Dunn chamber, the linear chemotactic gradient was originally created in a glass bridge between two concentric wells, and the microfluidic version included a source/sink construct to generate the chemotactic gradient in a microfabricated device (Shamloo et al. 2008; Cheng et al. 2007; Abhyankar et al. 2006; Diao et al. 2006). To slow down the decay of the gradient, microcapillaries (Shamloo et al. 2008), hydrogels (Cheng et al. 2007), and membranes (Abhyankar et al. 2006; Diao et al. 2006) were used to serve as high fluidic resistances to minimize convective transport and maintain a diffusion-dominating environment. "
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    ABSTRACT: Creating and maintaining a precise molecular gradient which is stable in space and time are essential to studies of chemotaxis. This paper describes a simple, compact, and user-friendly microfluidic device using a passive pumping method to drive the liquid flow to generate a stable concentration gradient. A fluidic circuit is designed to offset the effects of the pressure imbalance between the two inlets. After loading approximately the same amount of culture media containing different concentrations of a certain chemotactic agent into the two inlet reservoirs, a linear concentration gradient will be automatically and quickly established at the downstream. Our device takes advantage of passive pumping and is compact enough to fit into a Petri dish, which is an attractive feature to biologists. Furthermore, this microfluidic gradient generator offers a platform for a facile way of long-term imaging and analysis using high resolution microscopy.
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    • ") (Shamloo et al., 2008) which allowed us to directly image the migration of cells exposed to a defined gradient of chemoattractant for >12 hr. The establishment and maintenance of a linear soluble gradient in this device was confirmed using fluorescent dextran (Figure 3B) Based on the fluorescence intensity profile, this equates to a slope of 43.6%/mm across the cell culture chamber. "
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    ABSTRACT: Lamellipodia are sheet-like, leading edge protrusions in firmly adherent cells that contain Arp2/3-generated dendritic actin networks. Although lamellipodia are widely believed to be critical for directional cell motility, this notion has not been rigorously tested. Using fibroblasts derived from Ink4a/Arf-deficient mice, we generated a stable line depleted of Arp2/3 complex that lacks lamellipodia. This line shows defective random cell motility and relies on a filopodia-based protrusion system. Utilizing a microfluidic gradient generation system, we tested the role of Arp2/3 complex and lamellipodia in directional cell migration. Surprisingly, Arp2/3-depleted cells respond normally to shallow gradients of PDGF, indicating that lamellipodia are not required for fibroblast chemotaxis. Conversely, these cells cannot respond to a surface-bound gradient of extracellular matrix (haptotaxis). Consistent with this finding, cells depleted of Arp2/3 fail to globally align focal adhesions, suggesting that one principle function of lamellipodia is to organize cell-matrix adhesions in a spatially coherent manner.
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