A Macroscopic Diffusion-Based Gradient Generator to Establish Concentration Gradients of Soluble Molecules Within Hydrogel Scaffolds for Cell Culture.

Anusha Dravid,Zaid Aqrawe,Darren Svirskis,Brad Raos,Simon J O'Carroll,Sam Parittotokkaporn

doi:10.3389/fchem.2019.00638

Abstract

Concentration gradients of soluble molecules are ubiquitous within the living body and known to govern a number of key biological processes. This has motivated the development of numerous in vitro gradient-generators allowing researchers to study cellular response in a precise, controlled environment. Despite this, there remains a current paucity of simplistic, convenient devices capable of generating biologically relevant concentration gradients for cell culture assays. Here, we present the design and fabrication of a compartmentalized polydimethylsiloxane diffusion-based gradient generator capable of sustaining concentration gradients of soluble molecules within thick (5 mm) and thin (2 mm) agarose and agarose-collagen co-gel matrices. The presence of collagen within the agarose-collagen co-gel increased the mechanical properties of the gel. Our model molecules sodium fluorescein (376 Da) and FITC-Dextran (10 kDa) quickly established a concentration gradient that was maintained out to 96 h, with 24 hourly replenishment of the source and sink reservoirs. FITC-Dextran (40 kDa) took longer to establish in all hydrogel setups. The steepness of gradients generated are within appropriate range to elicit response in certain cell types. The compatibility of our platform with cell culture was demonstrated using a LIVE/DEAD® assay on terminally differentiated SH-SY5Y neurons. We believe this device presents as a convenient and useful tool that can be easily adopted for study of cellular response in gradient-based assays.

Highlights

The in vivo microenvironment is modulated by the precise orchestration between a multitude of chemical, physical, and biological stimuli influencing cellular behavior
We have described a modified PDMS diffusion chamber that can establish concentration gradients of soluble molecules within AG and AG-Col matrices
We have reported the fabrication and characterization of a PDMS diffusion-based gradient generator, allowing maintenance of concentration gradients within AG and AG-Col scaffolds for a period of at least 96 h

Summary

Introduction

The in vivo microenvironment is modulated by the precise orchestration between a multitude of chemical, physical, and biological stimuli influencing cellular behavior. Diffusion Gradients in AG and AG-Col Scaffolds gradients are known to direct migration of human neutrophils (Vasaturo et al, 2012), whilst concentration gradients of neurotrophic factors and sonic hedgehog (Shh) have a proven role in axonal guidance (Xu et al, 2018). Due to the complexity of manipulating the in vivo environment and associated confounding factors, the study of cellular response to gradient-based stimuli has necessitated the development of in vitro experimental platforms capable of generating concentration gradients in a reproducible and controllable manner. Developments in microfluidic-based technology have sought to overcome the shortfalls associated with these traditional platforms by providing precise, controllable concentration gradients through manipulation of convective flow or molecular diffusion (Lin and Levchenko, 2015). There is a current dearth and necessity of simple platforms capable of generating biologically applicable concentration gradients to study cellular response

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