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Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer1. Current methodologies to achieve this include photopatterning2-3, lithography4, sequential functionalization5, freeze drying6, microfluidics7, or centrifugation8, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers9.
DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities10. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.
A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide11, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications10.
MALDI-TOF analysis confirms the conjugation of RGDS peptide to acryloyl-PEG (Figure 2). Gel imaging reveals alternating RGDS- 350 (blue) layers after photopolymerization (Figure 3A). As shown in Figure 3A, 2D DGMP gel size can be varied based on the diameter of the silicone molds (10 mm, left ; 8 mm, right), and therefore are easily customizable for use in multiple assays – in this case to fit a 48 well cell culture plate (Figure 3B). Epifluorescence and phase contrast microscopy of C2C12 myoblasts cultured on a DGMP gel shows selective attachment on RGDS-350-containing PEG layers (Figure 4), demonstrating compartmentalization of the cell adhesion peptide (RGDS).
Figure 1. Molecular weight analysis by MALDI-TOF comparing aPEG-SCM to aPEG-RGDS obtained after conjugation of RGDS peptide.
Figure 2. Schematic representation of DGMP gel fabrication. After the gradients are layered, they can be allowed to settle for varying periods of time (ts) to create graduated interfaces, followed by photopolymerization. Stratified DGMP gels can be easily extracted from the mold for further use. Click here to view larger figure.
Figure 3.A) 2D multilayered gels obtained after photopolymerization imaged using 350 nm and white light channels of VersaDoc gel documentation unit. The grayscale image reveals alternating layers containing RGDS in white. B) Insertion of DGMP gel into 48-well cell culture dishes.
Figure 4. Merged phase contrast and epifluorescence image of C2C12 myoblasts grown on DGMP gels (scale bar 50 μm).
Figure 5. Effect of iodixanol on gel surface elasticity. Atomic force microscopy measurements of static samples of crosslinked PEG substrates using previously established methods with a 2 nN force trigger 12. *p< 0.05 and **p<0.01.
DGMP is a simple strategy for preparing multilayered gels that does not rely on expensive instrumentation. This protocol can be adapted for creating scaffolds using other biocompatible materials, such as collagen and hyaluronic acid. Bioactive small molecules, for example cell adhesion-promoting RGDS peptide, can be tethered to the polymer matrix to prevent mixing of cues between layers. Proteins can be encapsulated in distinct layers without the need for chemical conjugation as they, depending on the matrix mesh size, are less prone to diffuse through hydrogels10. Here we used iodixanol (Nycoprep), an inert density modifier, which has previously been used for viable cell applications. Other density modifiers such as sucrose and dextrose can also be used. By varying the settling time (ts), one can fine-tune the interfaces between two layers to produce smooth or sharp transitions as needed (longer settling time gives smoother transitions)10. For example, smoother transitions between DGMP gel layers could be used to generate a continuous gradient of a biological cue to study cell processes such as chemotaxis.
The effect of density modifier on gel stiffness is shown in Figure 5 for a 15% aPEGda gel; a more complete characterization of stiffness and porosity as a function of PEGda and iodixanol concentrations is currently being evaluated. While the PEGda concentration in this example is relatively high, we observed a 60% greater elastic modulus in gels with 30% iodixanol compared to gels without. The change in gel stiffness can be adjusted for by modulating the macromer concentration or crosslinking density.
We have also applied the DGMP technique to create 3D multilayered gels using polyacrylamide and PEG precursors10. Varying the concentration or the degree of crosslinking of the prepolymer allows structural variation in the scaffolds, which can be used to explore cell behavior such as polarized growth and migration in 3D.
In summary, DGMP is an adaptable technique that can be applied to fabricate 2D and 3D scaffolds from a variety of biocompatible materials for a broad range of biomedical and basic research applications.
The authors have no conflicting interests to disclose.
The authors are grateful for support from NIH Director's New Innovator Awards (1DP2 OD006499-01 to A.A. and 1DP2 OD006460-01 to A.J.E.), and King Abdulaziz City for Science and Technology (UC San Diego Center of Excellence in Nanomedicine). We would like to thank Ms. Jessica Moore for her critical comments on the manuscript.