Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment, Customizable Scaffolds for Tissue Engineering

Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego.
Journal of Visualized Experiments (Impact Factor: 1.33). 02/2013; DOI: 10.3791/50018
Source: PubMed


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.

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    ABSTRACT: The ability to independently assemble multiple cell types within a three-dimensional matrix would be a powerful enabling tool for modeling and engineering complex tissues. Here we introduce a strategy to dynamically pattern distinct subpopulations of cells through genetic regulation of cell motility. We first describe glioma cell lines that were genetically engineered to stably express constitutively active or dominant negative Rac1 GTPase mutants under the control of either a doxycycline-inducible or cumate-inducible promoter. We culture each population as multicellular spheroids and show that by adding or withdrawing the appropriate inducer at specific times, we can control the timing and extent of Rac1-dependent cell migration into three-dimensional collagen matrices. We then report results with mixed spheroids in which one subpopulation of cells expresses dominant negative Rac1 under a doxycycline-inducible promoter and the other expresses dominant negative Rac1 under a cumate-inducible promoter. Using this system, we demonstrate that doxycycline and cumate addition suppress Rac1-dependent motility in a subpopulation-specific and temporally-controlled manner. This allows us to orthogonally control the motility of each subpopulation and spatially assemble the cells into radially symmetric three-dimensional patterns through the synchronized addition and removal of doxycycline and cumate. This synthetic biology-inspired strategy offers a novel means of spatially organizing multiple cell populations in conventional matrix scaffolds and complements the emerging suite of technologies that seek to pattern cells by engineering extracellular matrix properties.
    Soft Matter 03/2014; 10(14):2372-80. DOI:10.1039/c3sm52265b · 4.03 Impact Factor


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