Structural and molecular micropatterning of dual hydrogel constructs for neural growth models using photochemical strategies

Abstract

Chemotactic and haptotactic cues guide neurite growth toward appropriate targets by eliciting attractive or repulsive responses from the neurite growth cones. Here we present an integrated system allowing both structural and molecular micropatterning in dual hydrogel 3D tissue culture constructs for directing in vitro neuronal growth via structural, immobilized, and soluble guidance cues. These tissue culture constructs were fabricated into specifiable geometries using UV light reflected from a digital micromirror device acting as a dynamic photomask, resulting in dual hydrogel constructs consisting of a cell growth-restrictive polyethylene glycol (PEG) boundary with a cell growth-permissive interior of photolabile α-carboxy-2-nitrobenzyl cysteine agarose (CNBC-A). This CNBC-A was irradiated in discrete areas and subsequently tagged with maleimide-conjugated biomolecules. Fluorescent microscopy showed biomolecule binding only at the sites of irradiation in CNBC-A, and confocal microscopy confirmed 3D binding through the depth of the construct. Neurite outgrowth studies showed contained growth throughout CNBC-A. The diffusion rate of soluble fluorescein-bovine serum albumin through the dual hydrogel construct was controlled by PEG concentration and the distance between the protein source and the agarose interior; the timescale for a transient protein gradient changed with these parameters. These findings suggest the dual hydrogel system is a usefulplatform for manipulating a 3D in vitro microenvironment with patterned structural and molecular guidance cues for modeling neural growth and guidance.

Photoreactive interpenetrating network of hyaluronic acid and Puramatrix as a selectively tunable scaffold for neurite growth

“…The simplicity of the lithography method gives the opportunity of micropatterning hydrogels with any kind of configuration in order to create hybrid scaffolds. This property also makes it possible to introduce spatial characteristics into regenerative therapies [17,20,25,26].…”

“…1 [17]. Briefly, photoreactive hydrogel solutions contained in permeable cell culture inserts with 0.4 lm pore size (Corning Inc., Corning, NY) were irradiated using an apparatus consisting of a collimated UV light source (OmniCure 1000 with 320-500 nm filter, EXFO, Quebec, Canada), a digital micromirror device (DMD) (Discovery™ 3000, Texas Instruments, Dallas, TX) as a dynamic photomask, and a 2Â Plan Fluor objective lens (Nikon Instruments, Tokyo, Japan) [17,20,25,26]. A solution of 10% (w/v) PEG-diacrylate (M n 1000; Polysciences Inc., Warrington, PA) and 0.5% (w/v) Irgacure 2959 in PBS was irradiated with 181 mW/cm 2 UV light, as measured by a radiometer (306 UV Powermeter, Fig.…”

“…Our model consisted of a photocrosslinkable polyethylene glycol (PEG) that surrounded an IPN hydrogel of methacrylated-HA and PM. By projecting UV light in defined geometries onto the photocrosslinkable substrates, the DMD enabled us to create a dynamic mask that could irradiate PEG and the IPN throughout the depth of the gel [17,[20][21][22][23]. We took advantage of this method in order to create a dual hydrogel system with a 3-D microenvironment to create an ideal in vitro environment for investigating neurite growth in the presence of varying mechanical properties.…”

Microscale Architecture in Biomaterial Scaffolds for Spatial Control of Neural Cell Behavior

“…The biological activity of immobilized proteins depends on the immobilization process and the specific protein tolerances to the processing environment(s). Photolithography was used to test if the activity of two known axon inhibitor proteins, semaphorin 6A and ephrin-B3, was affected by immobilizing them onto an agarose-based hydrogel Horn-ranney et al, 2014). The technique involved creating a mold from poly(ethylene glycol) (PEG) designed in the shape of a well attached to a channel that splits into two separate channels.…”

A Functional High-Throughput Assay of Myelination in Vitro

“…To demonstrate the feasibility of detecting action potential propagation in 3D hydrogel cultures with voltage-sensitive dye imaging, we have begun to use rat embryonic dorsal root ganglion (DRG) explants suspended in a Puramatrix gel contained within a PEG micro-mold (Curley, 2011;Horn-Ranney, 2013). We have obtained a newly-described voltage-sensitive dye as a generous gift from the lab of Roger Tsien.…”