(776d) Spatially Separated Chemokine Gradients and Their Effects on Single Cell Migration

A variety of physiological and pathophysiological processes including immune responses, metastasis, and stem cell differentiation are governed by an intricate network of endogenously produced cytokines. In vivo, these signals are often present as gradients permeating through the three-dimensional (3D) tissue and their concentrations as well as spatial diffusion are critical in providing adequate guidance for directional cell migration. Several in vitro approaches such as Boyden chambers or microfluidic platforms are well established for quantitative as well as qualitative analysis of cell motility; however, require a predetermined optimum chemokine concentration for each cell type. Additionally, these devices are prone to temporally diminishing gradient or provide chemotactic observations in response to a fixed slope of the gradient limiting the ability to recreate in vivo like conditions. To date, there is no efficient method to determine the effects of gradient concentrations (e.g. chemotaxis vs fugetaxis) on three dimensional cellular migration. Here, we demonstrate a novel microfluidic strategy that allows creation of gradients with varying slopes in the y-direction as a result of orthogonal gradient in the x-direction. This approach allows real-time observations of transendothelial migration (TEM) and chemotaxis under several different levels of chemotactic stimulation. The multifold advantages of the proposed strategy include the ability to: 1) observe cytokine activated stimulation of endothelial cells in response to different chemokine concentrations and the subsequent effects on homing of immune/cancer cells, 2) identify the optimum chemoattractant concentration governing chemotaxts and TEM, 3) correlate effects of the slope of gradient on cell motility, and 4) observe real-time migration behaviors through a 3D ECM environment at the single cell level. More interestingly, the orthogonal gradient technique allows the possibility of investigation chemotaxis and chemorepulsion (fugetaxis) in a single platform. Using this device, we exposed metastatic breast cancer cells to a known chemoattractant, stromal derived factor 1α (SDF-1α). We show that the tumor cells only extravasate and migrate towards gradients that are generated with a maximum concentration of 350-300 ng/mL, which closely resembles previously reported concentrations. This device is versatile and may be implemented in a variety of studies, including leukocyte recruitment, stem cell differentiation and migration, and pharmaceutical screening.