Generating controlled molecular gradients in 3D gels

Abstract

A new method for producing molecular gradients of arbitrary shape in thin three dimensional gels is described. Patterns are produced on the surface of the gel by printing with a micropump that dispenses small droplets of solution at controlled rates. The molecules in the solution rapidly diffuse into the gel and create a smooth concentration profile that is independent of depth. The pattern is relatively stable for long times, and its evolution can be accurately described by finite element modeling of the diffusion equation. As a demonstration of the method, direct measurements of protein gradients are performed by quantitative fluorescence microscopy. A complementary technique for measuring diffusion coefficients is also presented. This rapid, flexible, contactless approach to gradient generation is ideally suited for cell culture experiments to investigate the role of gradients of diffusible substances in processes such as chemotaxis, morphogenesis, and pattern formation, as well as for high-throughput screening of system responses to a wide range of chemical concentrations.

Matrices and scaffolds for DNA delivery in tissue engineering

“…Microfabrication techniques, such as three-dimensional printing, laser ablation, and similar procedures are being developed to create scaffolds with controllable feature size, patterned topography, and the ability to reproduce the complexity of several tissues [157][158][159][160]. Inkjet printing, for example, has been applied to pattern and control cell attachment [161].…”

The Response of Dorsal Root Ganglion Axons to Nerve Growth Factor Gradients Depends on Spinal Level

“…Using the guidance assay of Rosoff and associates (2004;2005), as updated by Mortimer and colleagues (2009), DRGs isolated from spinal levels S3-C8 were exposed to identical NGF gradients. The gradient parameters were a steepness of 0.3% per 10 microns, and an NGF concentration of 0.3 nM at the explant.…”

“…Limitations of available guidance assays , such as the difficulty of precisely controlling gradient steepness and concentration at the explant, have precluded systematic assessment to date. We recently introduced a novel guidance assay allowing generation of precisely controlled gradient conditions Rosoff et al, 2005Rosoff et al, , 2004. Using this assay we have shown that, averaging over dorsal root ganglia (DRG) from several spinal levels, the effect of exquisitely small differences in gradient parameters can be seen in differential guidance responses of DRGs to NGF gradients .…”

Analyzing neurite outgrowth from explants by fitting ellipses

“…The gradients were generated using the technology of Rosoff et al (2005). Fractional gradient steepnesses of 0.1%, 0.2%, 0.3% and 0.4% per 10 m were used, at absolute concentrations varying from 0.001 to 100 nM.…”