Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Abstract

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities [1][2][3] . Studies in fields such as cancer research , and cell-matrix interaction 7,8 have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization [9][10][11][12] . Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning . Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cellrestrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (<200 μm) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast. Video LinkThe video component of this article can be found at https://www.jove.com/video/2636/ Protocol 1. DMD Setup 1. The DMD board, UV light guide (with collimator) and 4x objective lens are all mounted vertically on a vibration isolation table. 2. The UV light guide sh...

Sensory axon guidance with semaphorin 6A and nerve growth factor in a biomimetic choice point model

“…Additionally, neurites were observed to extend first at the interface between PEG and agarose (Fig. 4), as described previously (Curley and Moore, 2011). With increased incubation time, some neurites then infiltrated the gel peripherally from this border, potentially exaggerating this effect in low NGF conditions.…”

“…Consistent with our previous reports, confocal micrographs revealed neurite growth throughout the 3D depth provided by the gels (data not shown). Growth was prominent at the interface of PEG and agarose (Curley and Moore, 2011), and some neurite growth into agarose could be seen infiltrating from this area.…”

“…The dual hydrogel environment used for tissue explant culture has been previously described (Curley et al, 2011; Curley and Moore, 2011). The fabrication process is summarized in Fig.…”

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.…”