Small-molecule axon-polarization studies enabled by a shear-free microfluidic gradient generator

Abstract

A deep understanding of the mechanisms behind neurite polarization and axon path-finding is important for interpreting how the human body guides neurite growth during development and response to injury. Further, it is of great clinical importance to identify diffusible chemical cues that promote neurite regeneration for nervous tissue repair. Despite the fast development of various types of concentration gradient generators, it has been challenging to fabricate neuron friendly (i.e. shear-free and biocompatible for neuron growth and maturation) devices to create stable gradients, particularly for fast diffusing small molecules, which typically require high flow and shear rates. Here we present a finite element analysis for a polydimethylsiloxane/polyethylene glycol diacrylate (PDMS/PEG-DA) based gradient generator, describe the microfabrication process, and validate its use for neuronal axon polarization studies. This device provides a totally shear-free, biocompatible microenvironment with a linear and stable concentration gradient of small molecules such as forskolin. The gradient profile in this device can be customized by changing the composition or width of the PEG-DA barriers during direct UV photo-patterning within a permanently bonded PDMS device. Primary rat cortical neurons (embryonic E18) exposed to soluble forskolin gradients for 72 hr exhibited statistically significant polarization and guidance of their axons. This device provides a useful platform for both chemotaxis and directional guidance studies, particularly for shear sensitive and non-adhesive cell cultures, while allowing fast new device design prototyping at a low cost.

Cells and Organs on Chip—A Revolutionary Platform for Biomedicine

“…Excellent optical transparency is prime advantage of PDMS that enabled real-time monitoring of nitric oxide production and variation in pulmonary vascular resistance in a microfluidic model and cell morphology, tissue repair and reorganization. [79][80][81] Moreover, control of cellular parameters is another important phenomenon in designing OOC devices and recent advances in microfabrication techniques have significant contribution toward efficient monitoring and control of cellular responses and study of broad array of physiological factors that wasn't possible with 3D static cultures. Electrical, chemical, mechanical and optical probes for direct visualization and quantitative analysis of cellular biochemistry, gene expression, structure and mechanical responses also can be integrated into virtually any microfabricated cell culture devices and more relevant data can be obtained with these advanced OOC devices.…”

A thin permeable-membrane device for single-molecule manipulation

“…Because of the advantages accrued by avoiding direct flow in the measurement region, permeable-membrane devices have been developed for a variety of soft matter, chemical, or biological studies. [4][5][6][7][8][9][10] However, they have not previously been developed for single-molecule manipulation instruments.…”

“…4,6 Permeable membranes in this geometry have been fabricated by cross-linking poly(ethylene glycol) diacrylate (PEG-DA) with patterned UV light. [7][8][9][10] This horizontal geometry does not hinder highresolution optical methods because the membranes do not obstruct transmitted light. Also, with this design, one can easily array several separate lanes; particularly, one can surround a central analysis channel with two flow channels.…”

“…4,6,8 Additionally, PEG's biocompatibility allows the study of biomolecules or living cells. [7][8][9][10][11][12][13] However, prior implementations of this device design resulted in flow cells whose vertical thicknesses were too large to permit close approach of external apparatus from both sides. This has precluded their use in instruments that require close access to a sample from both the top and bottom.…”

A hybrid microfluidic device for on-demand orientation and multidirectional imaging of C. elegans organs and neurons

“…Recent development of non-flowing gradient generators include static gradients generated by diffusion of molecules through porous nitrocellulose 7 or polyester 38 membranes, agarose hydrogel, 15,39,40 collagen, 10,41 polyethylene glycol (PEG) hydrogels, 42,43 through micro-jet array perfusion channels with minimal flow, 44,45 or through in situ biofabricated biopolymer membranes. 46,47 By restricting convective flow with membranes or hydrogels while allowing diffusion of small molecules to generate chemical gradients, flow-free and diffusion-based static gradient generators are able to decouple cell motion of nonadherent cells from flow.…”