Neurite growth in 3D collagen gels with gradients of mechanical properties

Abstract

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.

Acute and Chronic Neural Stimulation via Mechano-Sensitive Ion Channels

“…2A). 33 The substrate can also be patterned with proteins to create protein gradients 34,35 with different stiffness 36 and topographies such as grooves to influence neuronal growth. 37,13,38 This method offers much higher throughput compared to the aforementioned techniques which assess mechanically evoked responses one cell at a time.…”

Whole-organ engineering with natural extracellular materials

“…Cell migration occurring along a gradient, such as that which occurs during chemotaxis (chemical gradient), haptotaxis (cellular adhesion site gradients) and durotaxis (rigidity gradients), can therefore be manipulated based upon the design of the ECM [39,72]. Further studies in 3D have been able to demonstrate more physiologically relevant applications by creating matrices with natural materials that take advantage of durotaxis [73,74].…”

“…Natural crosslinkers, such as genipin, have already shown improved biocompatibility over glutaraldehyde in tissue fixation [135][136][137]. Other groups have demonstrated incorporation of genipin into natural ECM hydrogels [73,138] and natural electrospun nanofibers [53]. With improved nanofiber production methods, more complex 3D electrospinning techniques can be used to find better ways to integrate a variety of fabricated scaffold layers on the mesoscopic scale.…”

Hydrogel discs for digital microfluidics

“…25,28,31 For example, microchannel systems are well suited for very low ($nl) sample volumes. This property makes microchannel enzyme microreactors useful for analytical applications, but such systems are often not easily adapted for use for preparative applications.…”

“…[22][23][24] There has been great enthusiasm for combining hydrogels with microchannel-based labon-a-chip systems. [25][26][27][28][29][30][31][32][33][34] For example, microfluidic systems are useful for reagent delivery and collection of products from hydrogel-based enzyme microreactors. 25 Likewise, microfluidic systems for culturing cells encapsulated in hydrogel structures are useful because fluidic ports are a) Lindsey K. Fiddes and Vivienne N. Luk contributed equally to this work.…”