Biochemical gradients are ubiquitous in biology. At the tissue level, they dictate differentiation patterning or cell migration. Recapitulating in vitro the complexity of such concentration profiles with great spatial and dynamic control is crucial in order to understand the underlying mechanisms of biological phenomena. Here we describe a microfluidic design capable of generating diffusion-driven, simultaneous or sequential, orthogonal linear concentration gradients in a three-dimensional cell-embedded scaffold. Formation and stability of the orthogonal gradients are demonstrated by computational and fluorescent dextran-based characterizations. We then explore system utility in two biological systems. First, we subject stem cells to orthogonal gradients of morphogens in order to mimic the localized differentiation of motor neurons in the neural tube. Similarly to in vivo, motor neurons preferentially differentiated in regions of high concentration of retinoic acid and smoothened agonist (acting as sonic hedgehog), in a concentration-dependent fashion. We then apply a rotating gradient to HT1080 cancer cells and investigate the change in migration direction as the cells adapt to a new chemical environment. We report that the response time is ~4h. These two examples demonstrate the versatility of this new design that could also prove useful in many applications including tissue engineering and drug screening.
The advent of microfluidic technology allows control and interrogation of cell behavior by defining the local microenvironment with an assortment of biochemical and biophysical stimuli. Many approaches have been developed to create gradients of soluble factors, but the complexity of such systems or their inability to create defined and controllable chemical gradients has limited their widespread implementation. Here we describe a new microfluidic device which employs a parallel arrangement of wells and channels to create stable, linear concentration gradients in a gel region between a source and a sink well. Pressure gradients between the source and sink wells are dissipated through low resistance channels in parallel with the gel channel, thus minimizing the convection of solute in this region. We demonstrate the ability of the new device to quantitate chemotactic responses in a variety of cell types, yielding a complete profile of the migratory response and representing the total number of migrating cells and the distance each cell has migrated. Additionally we show the effect of concentration gradients of the morphogen Sonic hedgehog on the specification of differentiating neural progenitors in a 3-dimensional matrix.
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