A Microfluidic Platform to Quantify Spatio-Temporal Diffusion of Chemo-Gradients Within 3D Scaffolds: Applications in Axonal Biology

Axonal outgrowth and guidance play an important role in wiring the developing and regenerating nervous system. The critical role of biomolecular gradients in facilitating this axonal sensitivity and directionality along specific trajectories needs to be elucidated for designing effective therapeutic treatments under injury or disease conditions. However, previous in vitro approaches based on micropipette assay or gel-turning assay proved to be unsuitable or inefficient for precise generation and quantification of diffusive gradients. In this study, we utilized a microfluidic device to generate and quantify physiologically-relevant biomolecular gradients in a simple and reliable manner. Using a combination of computational and experimental techniques, we designed and developed a microfluidic platform to study the synergistic effects of 3D scaffold concentration (0 - 3 mg/mL), molecular weight of the diffusing molecule (1-1000 kDa) as well as its dosage (0.1-10 æM), on gradient generation and steady-state spatio-temporal evolution. The device was fabricated using standard soft-lithography techniques, and has three separate chambers, flanked by two media channels on the sides. The scaffold (gel) of interest was filled in the left and right chambers (L = 3.6 mm, thickness = 150 æm), and the biomolecule of interest was loaded in the middle chamber to facilitate diffusion through the gel on both sides. The channels on both sides act as sink for the diffusing biomolecule, creating a gradient across the 3D gel. Two different types of scaffolding materials were used in these studies -- collagen-1 or matrigel®. The viscosities of these gels at various concentrations were obtained from commercial vendors, and diffusion coefficients of biomolecules within these gels calculated using the Stokes-Einstein equation. Computational simulations were performed using the finite element methods (COMSOL® Multiphysics), to obtain a gradient profile across the chamber in all three dimensions. The numerical gri