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Facile micropatterning of dual hydrogel systems for 3D models ofneurite outgrowth

J. Lowry Curley, Michael J. Moore

Department of Biomedical Engineering, Tulane University, New Orleans, Louisiana 70118

Received 1 February 2011; revised 4 May 2011; accepted 10 June 2011

Published online 20 September 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.33195

Abstract: Understanding how microenvironmental factors

influence neurite growth is important to inform studies in

nerve regeneration, plasticity, development, and neurophysi-

ology. In vitro models attempting to more accurately mimic

the physiological environment by provision of a 3D growth

matrix may provide useful foundations. Some limitations of

thick 3D culture models include hampered solute transport,

less-robust neurite growth than on 2D substrates, and diffi-

culty in achieving spatial control of growth. To this end, we

describe a 3D dual hydrogel model for embryonic rat day 15

dorsal root ganglion tissue explant growth using a digital

micromirror device for dynamic mask projection photolithog-

raphy. The photolithography method developed allowed sim-

ple, reproducible, one-step fabrication of thick hydrogel

constructs on a variety of substrates, including permeable

cell culture inserts. The relationships between projected mask

size, crosslinked hydrogel resolution, and gel thickness were

characterized, and resolution was found generally to

decrease with increasing gel thickness. Cell viability in thick

(481 lm) hydrogel constructs was significantly greater on

permeable supports than glass, suggesting transport limita-

tions were somewhat alleviated. The observed neurite

growth was abundant and occurred in a spatially controlled

manner throughout the 3D environment, a crucial step in the

quest for a more effective biomimetic model of neurite out-

growth. VC 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A:

99A: 532–543, 2011.

Key Words: photolithography, tissue engineering, cell cul-

ture, nerve guidance, poly(ethylene glycol)

How to cite this article: Curley JL, Moore MJ. 2011. Facile micropatterning of dual hydrogel systems for 3D models of neuriteoutgrowth. J Biomed Mater Res Part A 2011:99A:532–543.

INTRODUCTION

Axonal pathfinding during development and repair occursby integration of a multitude of both chemotactic and hapto-tactic signals. Exploring the factors mitigating neuritegrowth and guidance may lead to better understanding oftargeting during development and may enable pathologiesto be better recognized and addressed. Thus, there is a criti-cal need for tunable culture environments in which struc-tural and molecular variables can be studied for advances inunderstanding axonal responses to environmental factors. Inthis report, we describe a quick and simple approach ena-bling spatial control of neurite growth within a 3D matrixas a constrained growth model that may serve as a basis forstudying effects of the microenvironment on neurite growth.

Tissue culture neurite outgrowth experiments have beencrucial tools for studying neurite growth responses to theirmicroenvironment. Culture models with 2D growth configu-rations have shown the influence of chemotaxis,1–4 proteingradients,1,2 topography,3–6 and preferences for specific celladhesion ligands over others.7 More recently, researchershave increasingly been turning toward 3D models that maymore accurately mimic the physiological environment.8,9

Indeed, cells cultured in 3D matrices have shown distinctdifferences in cellular characteristics such as gene expres-sion10 and morphology11 compared with identical cellsexamined in 2D cultures. Neuronal cells cultured in 3D havealso exhibited more biomimetic behaviors in terms of bothcytoarchitecture and electrophysiological characteristics,such as resting membrane potentials, calcium dynamics,action potential propagation, and voltage-gated ion channelfunctionality.12,13

Although there is a distinct advantage in studying neu-rite growth in 3D models, suitable materials and reproduci-ble methods for tuning them as neurite growth matrices arestill being developed.14 Several naturally derived hydrogels,including agarose15 and hyaluronic acid,16 have been foundto be effective for the culture of neurons and glia and pro-motion of neurite growth, as have some synthetic polymers,such as self-assembling peptide gels,17 and ionically chargedhydrogels.18 A number of methods to tailor the structuralmicroenvironment of hydrogels as culture environmentswith distinct geometries are being developed and have beenrecently reviewed.19,20 Such techniques include the use ofmicroprinted photomasks,21 etched silicon masters,22

No benefit of any kind will be received either directly or indirectly by the author(s).

Correspondence to: M. J. Moore; e-mail: [email protected]

Contract grant sponsor: Louisiana Board of Regents; contract grant number: LEQSF[2009-10]-RD-A-18

Contract grant sponsor: NIH; contract grant number: NS065374

532 VC 2011 WILEY PERIODICALS, INC.

elastomeric stamps,23 laser ablation,24 and dynamic maskmicrostereolithography.25 The ability to functionalize hydro-gels with biomolecules is another important facet of themicroenvironment to control. Functionalized 3D matriceshave shown that axons respond differently to soluble versusimmobilized factors and growth tends to depend more heav-ily on the magnitude of the gradient than on absolute concen-tration.26,27 Highly specific, spatial control of chemical modifi-cation has been demonstrated using multiphotonexcitation28,29 and photolabile deprotection for biomoleculecoupling.30

Despite the potential of 3D neural culture systems forstudying neurite growth, they are not without limitations.First, nutrient and waste transport can become problematicbecause of diffusion limitations in thick 3D gels. Also, uni-form 3D environments do not allow for spatial control ofgrowth, which can lead to random neurite outgrowth andmake assessment difficult due to variability. Some 3D hydro-gels could also hinder growth cone extension because oflack of attachment ligands or an inability of cells to remodelthe matrix. Some of these limitations may be responsible foran observed deficiency in neurite extension within hydro-gels, which is not as extensive as on 2D substrates.31 Addi-tionally, complications arising from imaging spatiallyunbound neurite growth through thick gels often make visu-alization of neurites difficult.

We sought a neurite outgrowth culture model thatwould combine a biomimetic 3D microenvironment with thespatial control of growth seen in 2D models. Toward thisend, we adapted a ‘‘dynamic mask’’ projection photolithogra-phy technique for micropatterning photocrosslinkablehydrogels in thick configurations that can contain self-assembling hydrogels in specifiable geometric configura-tions. We use poly(ethylene glycol) dimethacrylate (PEG), aphotocrosslinkable hydrogel that inherently inhibits cell ad-hesion and infiltration,32,33 together with Puramatrix, a syn-thetic peptide hydrogel that has shown a propensity for cellmigration and extensive neurite outgrowth.17,34 Using thedual hydrogel approach, we demonstrated simple andrepeatable fabrication of thick micropatterned constructsthat promoted robust neurite growth from tissue explantsin a spatially defined manner that also minimized some lim-itations previously observed in 3D culture models.

MATERIALS AND METHODS

Dynamic mask projection photolithographyHydrogel micropatterns were formed via projection photoli-thography in a manner similar to previous reports that useda digital micromirror device (DMD) for layer-by-layer micro-stereolithography.25,35 A DMD development kit (DiscoveryTM

3000, Texas Instruments, Dallas, TX) with USB computerinterface (ALP3Basic) served as a dynamic mask by convert-ing digital black and white images to micromirror patternson the DMD array, in which individual mirrors may beturned ‘‘on’’ or ‘‘off’’ by rotating the angle of reflection fromþ12� to �12�, respectively. Ultraviolet (UV) light (filtered at320–500 nm) from an OmniCure 1000 (EXFO, Quebec, Can-ada) Hg vapor light source was collimated with an adjusta-

ble collimating adapter (EXFO) and projected onto the DMDarray. The reflected light was projected through a 4� PlanFluor objective lens (Nikon Instruments, Melville, NY) withnumerical aperture 0.13 and focused directly onto a photo-crosslinkable hydrogel solution [Fig. 1(A)]. The iris of theUV light source was adjusted to maintain an irradiance out-put of 5.0 watts/cm2 as measured with a radiometer(EXFO). Hydrogel solutions were cured for �55 s, inducingcrosslinking through free radical chain reaction. Unlike thereports cited earlier, our method initiated crosslinkingthroughout the bulk with a single irradiation, negating theneed for a layer-by-layer approach.

Formation of dual hydrogel constructsHydrogel polymerization was performed as previouslydescribed for dynamic mask projection photolithography.36

The photocrosslinkable solution was made by diluting poly(ethylene glycol) dimethacrylate (PEG) with average molec-ular weight (MW) 1000 Da (Polysciences, Warrington, PA)to 10% (w/v) in either PBS or growth medium with 0.5%(w/v) Irgacure 2959 (I-2959) (Ciba Specialty Chemicals,Basel, Switzerland) as a photoinitiator. The concentrationand molecular weight of PEG was chosen, based on previ-ously published data, to both minimize cell adhesion andmaximize hydrogel adherence to the polymerization sur-face.33 Micropatterned PEG constructs were crosslinkeddirectly onto one of three types permeable cell cultureinserts: polyester, polycarbonate, and collagen-coated PTFETranswellV

R

Permeable Supports (Corning, Corning, NY) with24-mm diameter membranes and 0.4-lm pores. Inner wallsof the culture inserts, not the membranes themselves, weretreated with Rain-XV

R

(SOPUS Products, Houston, TX) toreduce meniscus effect of PEG solution. Each support wasplaced on the stage of an inverted microscope positioneddirectly below the lithography projection lens. After cross-linking, supports were rinsed, removing excess uncros-slinked PEG solution, and the micropatterned PEG remainedattached to the surface. Hydration of PEG gel was main-tained in buffered saline solution (4�C) if not usedimmediately.

A self-assembling peptide gel, Puramatrix (BD Bioscien-ces, Bedford, MA), was diluted to 0.15% (w/v) in deionizedH2O prior to use and was supplemented pregelation with1 lg/mL soluble laminin (Invitrogen, Carlsbad, CA) whenused for neurite outgrowth experiments. Both the concen-tration of Puramatrix and the addition of laminin wereaccording to manufacturer’s instructions for neural applica-tion. Using a pipette, this solution was carefully added tovoids within the micropatterned PEG hydrogels. Contactwith salt solution hydrating the PEG gel induced self-assem-bly of the Puramatrix, which remained confined within thePEG geometry. Puramatrix gelation was maintained by incu-bating at 37�C and 5% CO2.

Hydrogel adherence and stabilityAdherence of PEG hydrogels to cell culture inserts wasassessed daily. PEG constructs were polymerized asdescribed previously, and any PEG rectangles which

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | 15 DEC 2011 VOL 99A, ISSUE 4 533

detached from the substrate were counted as nonadherent.To assess the stability of Puramatrix gel formed within PEG,nine PEG constructs were crosslinked on each of four poly-ester inserts, and 1.75 � 107 beads/mL of fluospheres (Mo-lecular Probes, Eugene, OR) were added to Puramatrix priorto gelation within PEG voids. Inserts were placed in wellplates, half of which were filled with 2 mL of PBS, sufficientto hydrate but not submerge the dual hydrogel constructs,while the other half were filled with 3 mL, fully submergingthe constructs. Fluorescence microscopy was used to deter-mine whether the Puramatrix remained inside the PEG con-structs after 48 h (Fig. 2).

Hydrogel thickness and feature sizeVolumes of 350, 400, 450, 500, and 550 lL of 10% PEGsolution were added to polyester culture inserts, and four

rectangular patterns at each volume were used to deter-mine the relationship between volume and crosslinked PEGheight. Samples were stained with Texas Red (Invitrogen),and thickness was measured using confocal imaging. Todetermine the minimum PEG feature size achievable, a se-ries of both circular and rectangular photomasks [Fig.3(B,C)] were used to form patterned voids in PEG solu-tions over a range of thicknesses. The smallest size voidachievable at each thickness was determined by decreasingthe size of the mask until the void was not visible aftercrosslinking. Six samples of each shape were used to deter-mine the average minimum void size at which point 100%of voids had formed. Circular void diameters were meas-ured in the x and y directions, with rectangular voidwidths taken at the center and alternating left or rightedges.

FIGURE 1. Micropatterning of PEG constructs with dynamic mask projection photolithography. (A) Schematic of DMD dynamic-mask photoli-

thography method. (B) Macro view of PEG constructs inside six-well cell culture insert. (C) Close-up of PEG constructs inside cell culture insert.

(D) DMD photomask. (E) PEG construct crosslinked around adhered DRG. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]

534 CURLEY AND MOORE FACILE MICROPATTERNING OF 3D NEURITE GROWTH

FIGURE 2. Stability of Puramatrix within PEG constructs relative to volume of PBS added. Representative images of fluorescent micrographs of

Fluosphere-labeled Puramatrix 48 h after gelation in PEG (broken white outline indicates PEG void) and schematic diagrams of dual hydrogel

constructs are shown above bar plot of Puramatrix stability with respect to volume of PBS added (n ¼ 18 for each of three experiments, bars

represent standard error of the mean). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIGURE 3. Minimum void size as a function of hydrogel thickness. (A) Plot of average minimum feature size versus average thickness of PEG (n

¼ 6). (B) DMD photomask for circular voids. (C) DMD photomask for rectangular voids. (D) Orthographic view showing confocal Z-stack of circu-

lar void with thickness of 481 lm. (E) Orthographic view showing confocal Z-stack of rectangular void with thickness of 481 lm. Each image

slice was manually adjusted for intensity and threshold before assembly to Z-stacks. Image slices were interpolated to account for distance

between slices. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Tissue harvesting and cultureNIH guidelines for the care and use of laboratory animals(NIH Publication #85-23 Rev. 1985) have been observed.Embryonic day 15 (E-15) pups were removed from timed-pregnant Long Evans rats (Charles River, Wilmington, MA)and placed in Hank’s Balanced Salt Solution. Spinal columnswere isolated from embryos, from which dorsal root ganglia(DRG) were harvested and placed in Neurobasal Mediumsupplemented with nerve growth factor (NGF), 10% fetalbovine serum (FBS), and penicillin/streptromycin (P/S)(Invitrogen) to promote adhesion. After adhesion, DRGswere placed on collagen-coated cell culture inserts andmaintained in an incubator at 37�C and 5% CO2 with B-27and L-glutamine replacing FBS for growth medium.

Primary DRG neurons were obtained through dissocia-tion of DRGs by tripsinization and trituration, followed bythe subsequent removal of supportive cells using fluoro-deoxyuridine and uridine (3-day treatment). Cells were thensuspended in Puramatrix according to the manufacturer’sprotocol at a concentration of 3 � 105 cells/mL. The cellsuspension was added, at a volume sufficient to obtain�480-lm thick hydrogels, to either 24-well cell cultureinserts or 24-well tissue culture plates (Corning), and self-assembly was initiated upon addition of growth medium (n¼ 4). The constructs were incubated for 48 h before testingcell viability.

Neurite outgrowth in dual hydrogelsCollagen-coated PTFE cell culture inserts were soaked over-night in adhesion medium to hydrate the membrane. FourDRGs were then placed on the surface of an insert andallowed to adhere for 2 h before the medium was replacedwith 500 lL of 10% PEG in growth medium as describedearlier, without FBS. This volume could be adjusted to varythe thickness of the PEG constructs. The DMD was illumi-nated with a visible light source to aid alignment of the pro-jected mask with each adhered DRG. The visible light sourcewas then replaced by the UV source and the PEG hydrogelcrosslinked around the tissue explant. DRG-containing PEGconstructs were washed three times with PBS to removeany uncrosslinked PEG solution. When applicable, modifiedPuramatrix was added to the void inside the PEG, and toinduce Puramatrix self-assembly, 1.5 mL of growth mediawas introduced beneath the insert. Constructs referred to aswithout Puramatrix were made as described earlier exceptwithout the addition of Puramatrix, thereby restrictingDRGs to the 2D environment of the collagen-coated PTFEmembrane. Constructs were maintained in an incubator at37�C and 5% CO2 for 7 days, and medium was changedafter the second and fifth days.

Constructs were prepared and visualized for morpho-logy, viability, neurite outgrowth, and containment. If noneurites were visualized growing on or outside the PEGvoid, the sample was considered to have contained growth.This was done in identical PEG constructs both with andwithout Puramatrix added, and twelve trials for each condi-tion were attempted for the five different PEG heights

described previously. Trials were thrown out in cases ofincomplete PEG polymerization or lack of DRG adhesion.

Specimen preparation and visualizationLive specimens were evaluated for viability with a Live/DeadV

R

assay (Invitrogen) per manufacturer’s instructions.For cell suspensions in Puramatrix, widefield fluorescentimages were captured at multiple focal planes throughoutthe depth of the gel in three different areas of each hydrogelspecimen. Standard deviation projections were then ana-lyzed for cell viability in both cell culture inserts and tissueculture plates by counting calcein AM (live marker) and eth-idium homodimer-1 (dead marker), giving a total of 12 sam-ples per condition. Specimens evaluated with immunohisto-chemistry were fixed in 4% paraformaldehyde for 2 h. Cellnuclei were stained with DAPI Nucleic Acid Stain (MolecularProbes) per manufacturer’s instructions. Neurites werestained using mouse monoclonal [2G10] to neuron specificb III tubulin primary antibody and goat-antimouse IgG-H &L (CY2) secondary antibody, and dendrite staining was car-ried out using rabbit polyclonal to MAP2 primary antibodyand donkey-antirabbit IgG Dylight 594 secondary antibody(AbCam, Cambridge, MA). Each step was carried out in PBSwith 0.1% Saponin and 2.0% BSA overnight followed bythree washes in PBS with 0.1% Saponin. Bright field andconventional fluorescent images were acquired with a NikonAZ100 stereo zoom microscope (Nikon, Melville, NY)equipped with florescence cubes, while confocal imageswere acquired using a Zeiss LSM 510 Meta microscope(Zeiss, Oberkocken, Germany). Average depths of b III la-beled structures were calculated from confocal images tomeasure the distance between the first and last focal planecontaining fluorescence (n ¼ 7). Image processing wasperformed with Image J (National Institutes of Health, Be-thesda, MD), and V3D software (Howard Hughes MedicalInstitute, Ashburn, VA) used to visualize confocal imagestacks in 3D.37 The proportion of neurite growth over thedepth of the gel was quantified from pixel counts of man-ually thresholded confocal slices. Confocal slices werebinned in 10% increments of total depth, and the measuredfluorescence of binned slices was compared with the totalmeasured for each Z-stack to give the proportion of neuritegrowth throughout the depth of the construct (n ¼ 3). TheVolumeJ plugin was used to create depth coded Z-stack pro-jection of neurite growth. Confocal z-stacks were acquiredthrough the maximum depth of visible neurite growth (186lm) with 3.0-lm thick slices (1024 � 1024 � 63) for bothPuramatrix and non-Puramatrix containing constructs. TheZ Code Stack function with spectrum depth coding LUT wasused to add color, and the stacks were merged using astandard deviation z projection. Last, Z-stacks were des-peckled to remove background noise. Cryogenic scanningelectron microscopy (Cryo-SEM) was performed by freezingspecimens in slushed liquid nitrogen and imaging with aHitachi S4800 Field Emission SEM (Hitachi, Krefeld, Ger-many) and Gatan Alto 2500 Cryo System (Gatan, Warren-dale, PA) at 3 kV and �130�C.


StatisticsStatistical analysis was performed using a student’s t-test ofequal variances. Statistically significant differences weredetermined using the requirement that p < 0.05. Equality ofvariance was calculated using Levene’s test (p < 0.05).Unless otherwise specified, data are presented as mean 6standard deviation.

RESULTS

Dual hydrogel construct formationDynamic mask projection photolithography reproduciblyproduced thick hydrogel micropatterns, �200–500 lm, withspecifiable geometries in a simple, one-step procedure. Toillustrate the versatility of this approach, a number of thickhydrogel constructs of varying sizes and shapes were cross-linked one after another directly on permeable cell cultureinserts in less than 1 min with 5.0 watts/cm2 incident onthe DMD, as shown in Figure 1(B,C). Crosslinked hydrogelsshowed some loss of fidelity as compared with the originalmask, which is evident in Figure 1(D,E), in which cornersbecame rounded, slightly changing proportions of the micro-patterns formed. This effect, as well as the micropatternresolution achievable, was found to depend heavily onhydrogel thickness. These relationships between photomasksize, hydrogel feature size, and thickness are summarized inTable I and further discussed in the ‘‘Results: HydrogelThickness and Feature Size’’ section.

Gelation of Puramatrix occurred upon injection of Pura-matrix solution into the voids of PEG hydrogels hydratedwith buffered salt solutions. This in situ gelation was con-firmed by addition of fluorescent microparticles (Invitrogen)to the Puramatrix solution before injection into the PEGvoids. When suspended in Puramatrix and injected into PEGhydrogel structures, both of which were devoid of salt toprevent peptide self-assembly, microparticles were observedto disperse. In contrast, as shown in Figure 2, microparticlesremained confined to PEG structures when injected in thepresence of salt solution, suggesting peptide self-assemblyand gel formation took place in situ upon contact with saltsolution, as reported for this type of gel.17 Figure 2 alsodemonstrates that the gel appeared to contract upon gela-tion, slightly pulling away from the borders of the PEG gel.This sometimes led to the complete detachment of thePuramatrix gel from the PEG gel, but as discussed in thefollowing section, Puramatrix remained inside of the PEGgiven correct conditions.

Hydrogel adherence and stabilityPEG hydrogel constructs remained adherent to both polyes-ter and collagen treated PTFE inserts throughout the lengthof our experiments. In fact, the hydrogel constructs adheredthroughout the length of a 3-week test (data not shown).These results are consistent with those seen by Moelleret al.33

Puramatrix containing fluorescent microspheres wasinjected into the micropatterned voids of PEG structurescrosslinked onto permeable culture inserts and allowed to gelvia self-assembly. When the inserts were placed in the wellsof a six-well plate, Puramatrix remained in 100% of the con-structs throughout the 48 h experiment when hydrated witha volume (2 mL) of PBS insufficient to completely submergethe constructs, as confirmed in Figure 2. However, Puramatrixfrequently detached from within the PEG structures whencompletely submerged in PBS (3 mL): 13 6 13% remainedafter the initial equilibrium, and only 10 6 11% of the con-structs still contained Puramatrix after 48 h (Fig. 2). A statis-tically significant difference in detachment existed betweenthe two volumes both immediately and after 48 h.

Hydrogel thickness and feature sizeThe minimum void size achievable over a range of PEGthicknesses was characterized by crosslinking both circularand rectangular voids within different volumes of PEG solu-tion added to culture inserts. PEG gels were stained andimaged with confocal microscopy to determine the relation-ship between the volume of PEG solution added and result-ing crosslinked gel thickness. Volumes of 350, 400, 450,500, and 550 lL of 10% PEG solution added to polyesterculture inserts resulted in crosslinked structures with thefollowing average thicknesses, respectively: 233 6 24, 3686 32, 433 6 19, 481 6 14, and 543 6 9 lm. The minimumvoid size achievable at each thickness was taken to be thesize for which 100% of specimens formed a measurablevoid. The average widths of these voids for each of the fiveexperimental thicknesses of PEG described earlier wereobtained for both circular and rectangular voids. As shownin Table I, the smallest features formed were circular voids62 6 6 lm in diameter and rectangular voids 85 6 4 lmwide, both at a thickness of 233 6 24 lm. For both circularand rectangular voids, as the thickness of PEG increased,the minimum feature size also increased, representing adecrease in resolution (Table I). The trend was similar forboth types of void features, but it may have been more lin-ear for rectangular channels than for circular wells [Fig.

TABLE I. Comparison Between Projected Mask Size and Minimum Void Size Formed at Thicknesses Tested

Projected UVSize (lm)

Minimum FeatureSize (lm) % of Projection

Volume (lL) Thickness (lm) Circle Channel Circle Channel Circle Channel

350 233 6 24 220 130 62 6 6 85 6 4 28% 6 3 65% 6 3400 368 6 32 290 160 92 6 5 118 6 5 32% 6 2 73% 6 3450 433 6 19 360 190 145 6 11 141 6 4 40% 6 3 74% 6 2500 481 6 14 400 230 174 6 10 159 6 5 44% 6 2 69% 6 2550 543 6 9 430 290 206 6 12 190 6 7 48% 6 2 65% 6 3

ORIGINAL ARTICLE


3(A)]. In all cases, a pronounced beveling effect was appa-rent because of the nature of the optics used. As seen inFigure 3(D,E), the void size was smallest at the membranesurface, where the light is focused and widened toward thetop of the gel.

Cell viabilityCell viability in �480-lm thick Puramatrix hydrogels wasevaluated in both cell culture inserts and tissue cultureplates. As shown in Figure 4, the average proportion of livecells was 89.4 6 4.0% when cultured in permeable inserts,but only 83.5 6 3.4% in rigid plates, representing a statisti-cally significant difference (p < 0.001).

Neurite outgrowth in dual hydrogelsThe PEG thickness necessary to constrain neurite growthwas investigated by culturing DRG explants in constructswith increasing thicknesses. Containment was measuredhere because it was crucial to the ability of our system tofunction reliably as an in vitro model. Impartial polymeriza-tion frequently occurred with 233-lm thick PEG, leading tounusable constructs. Additionally, throughout the polymer-ization process, some DRG detached from the surface of themembrane, leading to a lower than expected number of tri-als for analysis. For the constructs containing Puramatrix, adistinct increase in the containment of neurites was seen asgel thickness increased (Table II). At a thickness of 233 lm,no constructs limited the growth of neurites. The rates ofcontainment for the subsequent heights of 368, 433, 481,and 543 lm were: 10%, 22.2%, 63.6%, and 87.5%. Overall,higher percentages of containment were seen in constructslacking Puramatrix. Neurites appeared able to grow overthe sloping PEG walls at certain thicknesses in both groups[Fig. 5(A,B)], but in constructs without Puramatrix therewas more efficient containment at similar heights (otherthan 534 lm) and more effective containment by a lowerheight than in PEG with Puramatrix (Table II).

In an effort to balance void size resolution and patternfidelity with neurite containment, subsequent neuritegrowth experiments were carried out in constructs with anaverage PEG thickness of 481 lm (500 lL solution). Con-structs monitored for cell viability after 5 days showed anoverwhelming amount of live cells, with an extremely smallportion of dead cells located in the DRG itself [Fig. 6(A)]. Af-ter fixation and staining at 7 days, neurites and migratingcells were constrained by the geometry of the PEG hydrogel,as suggested by dual labeling with b III tubulin and DAPI[Fig. 6(B)]. Neurite outgrowth was consistently robust andall labeled structures were concentrated inside the Purama-trix portion of the dual hydrogel (Fig. 6). Additionally, MAP2antibody labeling suggests that a substantial portion of theneurite growth in the constructs appeared to be dendritic[Fig. 6(E)]. Growth appeared to occur first along the bound-ary between the two gels, as is evident in Figure 6(A), inagreement with reports of similar preferential growthbetween the interfaces of two materials.31,38 However,behind the neurites extending along the channel, growthwas seen filling in the inner space between the PEG

FIGURE 4. Cell viability in Puramatrix constructs on tissue culture

plates versus permeable supports. (A) Graph representing the average

percent live cells for both conditions (p < 0.001). Representative

images of Live/Dead assay on (B) 24-well permeable insert and (C)

24-well tissue culture plate with live cells represented in green and

dead cells in red. Median images are shown as representatives of

each experimental group (n ¼ 12). [Color figure can be viewed in the

online issue, which is available at wileyonlinelibrary.com.]


[Fig. 6(A)]. Images of leading neurite growth in the 3D bulkof the Puramatrix showed a tendency to grow in randomdirections [Fig. 6(D)]. In direct contrast, we found that neu-rites growing along the surface of the cell culture insert ori-ented themselves obliquely, apparently closely following thefibers of the insert membrane [Fig. 6(F)]. Outgrowth wasobserved to fan out at the bifurcation point with no appa-rent preference in direction. A considerable amount ofbranching and fasciculation was observed, which was espe-cially apparent at the leading edge of growth [Fig. 6(D)]. In�7 days, outgrowth was seen throughout the length of thechannels. Significantly more growth was observed in Pura-matrix filled constructs, as compared with the apparentlyabortive limited growth seen in constructs without Purama-trix [Fig. 7(B,C)]. Confocal imaging confirmed that neuritegrowth occurred in three dimensions, as shown in Figure 7.The average thickness of b III labeled structures was 159.86 23.9-lm thick in Puramatrix-containing constructs, whilethe average thickness in constructs without Puramatrix was85.4 6 38.6 lm, a difference which was found to be statisti-cally significant [Fig. 8(A), p < 0.005]. Figure 7(D) repre-sents an example of growth in a construct lacking Purama-trix, where growth appeared crowded and neurites grew toa maximum height of 54.0 lm. Neurite growth in constructs

without Puramatrix was visualized growing along the mem-brane of the collagen-coated PTFE, with no growth occuringin the PEG itself. Alternatively, Figure 7(E) demonstratesneurite growth in a dual hydrogel construct, with notablyless neurite crowding observed, and individual neuritesgrowing through Puramatrix in multiple focal planes, reach-ing a maximum height of 120.0 lm. Figure 8(B) furtherdemonstrates that neurite growth was not confined toeither the membrane or the top surface of the Puramatrix,as 7.3 6 2.9% and 4.9 6 1.3% of total growth was seen inthe bottom and top 10% of the slices, respectively. Unlikethe neurites, DAPI staining indicated migrating cells werenot influenced to migrate into Puramatrix, remaining con-fined near the support surface [Fig. 7(A)], although previousresearch suggests that glial cell migration and neuritegrowth often occur together.30,39

DISCUSSION

Understanding how microenvironmental factors influenceneurite growth is important to inform studies in nerveregeneration, plasticity, development, and neurophysiology.Neurite growth within 3D culture models may more accu-rately represent neural growth in vivo. While 3D cultureenvironments may represent a more biomimetic approach,implementation may be hampered by transport limitationsand a lack of spatial control of growth. The 3D dual hydro-gel system described in this article was found to be simple,reproducible, and highly effective for constraining and spa-tially controlling neurite outgrowth from embryonic tissueexplants. This approach could be useful as part of an overallstrategy to study microenvironmental effects on neuriteextension in vitro.

Our approach to photolithography proved beneficialbecause of the simplicity of the design and the versatility offabrication. The use of a DMD eliminated the need to fabri-cate masks, allowing instead for the use of simple, adaptableblack-and-white images. The UV exposure could be adjusted,if necessary, to influence crosslinking kinetics or address

TABLE II. Neurite Growth Containment as a Function of

Hydrogel Thickness

Volume(lL)

Thickness(lm) n % Contained

Puramatrix 350 233 6 24 6 0.0%400 368 6 32 10 10.0%450 433 6 19 9 22.2%500 481 6 14 11 63.6%550 543 6 9 8 87.5%

Non-Puramatrix 350 233 6 24 5 40.0%400 368 6 32 10 30.0%450 433 6 19 6 83.3%500 481 6 14 10 90.0%550 543 6 9 8 87.5%

FIGURE 5. Neurite growth outside of PEG void. (A) Cryo-SEM image of dual hydrogel construct showing neurite overgrowth. (B) Cryo-SEM

image of PEG only construct without Puramatrix showing neurite overgrowth. The average height of PEG in both (A) and (B) was 481 lm.

ORIGINAL ARTICLE


phototoxicity issues. Through the use of dynamic photo-masks and projection optics, there was no need for a maskto be in direct contact with the polymerization medium,allowing production of thick hydrogel structures, fullyexposed to air, in a single step with flexibility in choosingthe polymerization surface. The use of cell culture insertsalso allowed us to overcome some of the transportation lim-itations for thick explant cultures (discussed further below),as well as permitting crosslinking around previouslyadhered DRG explants.

The feature sizes achieved by our setup were compara-ble with, or even showed improvement over, previouslypublished work, even with thicker hydrogels (>300lm).21,25 Additionally, with still thicker gel depths, >500lm, feature sizes were sufficient for our neurite growthmodels. As demonstrated by Lu et al.,25 employing a layer-by-layer approach produced intricate geometries of PEGstructures with feature sizes near the order of 50 lm.Although our feature sizes were slightly larger, by negatinga layer-by-layer approach, we decreased the time necessaryfor total polymerization, an important consideration whenincorporating cells or tissue explants. Beebe et al. describe aphotolithography method providing comparable featuresizes with spacing of �100 lm obtainable, given sequentialpolymerization.21 However, we improved upon these results,as we were able to simultaneously polymerize our shapes,where they saw partial polymerization between objects witha spacing of <250 lm. Finally, while the DMD developmentkit may be specialized and costly, our use of a standardmicroscope objective suggests that a commercially available

DMD-microscope could be employed, which may make thisapproach to micropatterning attractive and readilyadaptable.

The two commercial hydrogels we employed were effec-tive for neurite growth, and our model could be easilyadapted to include other hydrogels. Crosslinked PEG consis-tently adhered to the permeable inserts, likely because theprepolymer liquid seeped into pores of the transparentmembranes, with physical adhesion occurring upon poly-merization. At a 0.15% concentration, Puramatrix alone ismechanically unstable, and it is barely more viscous thanwater prior to self-assembly via beta sheet formation,17 soit requires carefully controlled conditions to be effective.The PEG hydrogel on a permeable support served as astructural barrier to contain and shape Puramatrix as wellas protect peptide beta sheets from breaking apart. Use ofpermeable cell culture inserts in our dual hydrogel modelalso served to improve cell viability as seen in the increasednumbers of live cells in Figure 4 and the proportion of via-ble cells in Figure 6(A), suggesting transport limitationsassociated with thick constructs required to contain neuritegrowth were likely alleviated because of oxygen diffusionthrough the gels from below as well as the sides and top.The viability experiment supports the advantage of usingcell culture inserts over tissue culture plates as the poly-merization surface for 3D constructs.

We believe that our methods represent a more biomi-metic manner in which to create 3D constructs for the studyof neurite growth. Comparison of Puramatrix functionalityas a 3D environment for cell encapsulation has been

FIGURE 6. DRG neurite growth and cell migration in dual hydrogel constructs. (A) Live/Dead stained construct (live cells and cellular structures

in green, dead cells in red, brightfield in blue) after 5 days in culture. (B–C) DRG explants cultured in dual hydrogel constructs for 7 days,

indicated by b III tubulin-positive neurites (green) and DAPI-stained nuclei (blue). (D) Close up view of leading growth inside channel (b III tubu-

lin) after 5 days. (E) DRG explants cultured for 7 days, stained for MAP2-positive dendrites (red) and b III tubulin-positive neurites (green).

(F) Bifurcating portion of the construct focused at the surface of the cell culture insert (b III tubulin). [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]


previously demonstrated with other popular hydrogels.40

Agarose has been frequently used as a 3D matrices to inves-tigate neurite growth, although modification of agarose isnecessary to obtain significant axon extension.15 Matrigelhas shown promise as a 3D matrix for neural growth andneuronal-astrocyte cocultures,13 but the presence of incom-pletely defined ECM proteins and cytokines in Matrigel maycomplicate studies of molecular neurite guidance. Purama-trix has previously been shown to support neurite growthand cocultures with or without additional components. Inone recent advance, Xu et al. describe another method inwhich to print 3D neural sheets up to �1-mm thick.41 How-ever, none of the previous examples of 3D neural culturesexhibit directed growth, thereby negating any sort of spatial

control and limiting study of axonal projections. We believethat our presentation of the peptide gel within a micropat-terned construct represents a novel way to promote growthin a 3D environment within a constrained geometry. Ourapproach incorporates ease of use, fabricated spatial controlof abundant neurite growth, along with the ability to pro-duce a ‘‘blank slate’’ scaffold, allowing for the selectiveincorporation of growth factors to inform future studies ofneurite growth and guidance.

The approach we describe, although effective for itsintended purposes, was not without some limitations. First,the minimum attainable feature size was limited by the re-solution of our system and was observed to decrease withincreasing PEG thickness. Similar to observations described

FIGURE 7. Confocal micrographs of b III tubulin (green) and DAPI (blue, A only) stained constructs. (A) 3D representation of growth near bifurca-

tion point, showing both an orthographic view, and a side view to demonstrate thickness. Image slices were interpolated to account for distance

between slices. (B) Merged Z-stack projection of neurite growth in dual hydrogel construct. (C) Merged Z-stack projection of neurite growth in

PEG construct without Puramatrix. (D) Depth-coded Z-stack projection of neurite growth in PEG construct without Puramatrix. (E) Depth-coded

Z-stack projection of neurite growth in dual hydrogel construct. In figures B–E, a standard deviation projection was used.

ORIGINAL ARTICLE


by Beebe et al. with beveling in deep channels (150–300lm),21 we noticed that PEG voids narrowed throughout thetheir depth [Fig. 3(D,E)]. This phenomenon would not beproblematic except for the possibility that it enables neuritegrowth, presumably because of the adsorption of proteinsfrom the medium to the PEG surface, over the sloped sidesof the voids, limiting containment. If necessary, these effectscould perhaps be overcome by choosing optics with lowernumerical aperture or higher UV transmission, or by adjust-ing the focus to reverse the direction of the beveling. Sec-ond, while we hypothesized that Puramatrix gelation in situwould lead to physical entanglement of the two gels, thePuramatrix was frequently observed to detach from the PEGvoid, even while maintaining its shape (Fig. 2). Maintenanceof the height of the media combined with careful handlingavoided detachment, although this may not be ideal.Another possible solution could be choosing crosslinkedgels with larger mesh sizes to encourage entanglement orusing photocrosslinkable semi-interpenetrating net-works,42,43 rather than two different hydrogels. Finally,staining for b III tubulin revealed growth did not occurthroughout the entire height of the PEG constructs. It islikely that the depth of Puramatrix is considerably less thanthat of PEG because of the minimal volume (<1 lL) addedto prevent Puramatrix overflowing the PEG void, as well ascontraction during the self-assembly process. Production ofthicker PEG constructs could increase the depth of neuritegrowth, although this would inherently further limit theattainable feature size.

We chose a bifurcating pathway suggestive of nervegrowth, and future studies will take advantage of this designby allowing us to explore axon guidance molecules and ana-lyze the growth pattern at a divergent point in the growthprocess. The model was relatively simple in regards to pro-viding specific structural guidance cues, namely the presen-tation of a cell permissive region, surrounded by a cell re-strictive border, facilitating neurite containment; futurework will merge the ability to physically constrain growthwith molecular signaling. More specific to the findingsreported herein, we also hope to further investigate themigration of supportive cells, the fasciculation of neurites,and the phenomenon of preferential growth alongthe hydrogel borders. The ultimate informative value ofour dual hydrogel system may lie in maximizing and direct-ing neurite growth using combinations of biomimeticsignaling.

CONCLUSIONS

The dual hydrogel model laid out in this article was effec-tive for promoting neurite outgrowth in a spatially definedmanner within a 3D environment. Using a DMD for dynamicmask projection photolithography, we were able to fabricatethick hydrogel constructs quickly, easily, and on a variety ofsupport substrates. Photocrosslinked hydrogels consistentlyadhered to permeable supports, and the self-assemblingpeptide gel formed within the space provided. The twohydrogels remained stable and intact in culture conditionsfor the duration of the culture period, although care in han-dling was required to prevent separation of the gels. Therelationships between projected mask size, crosslinkedhydrogel feature size, and gel thickness were characterized,and resolution found generally to decrease with increasinggel thickness. Cell viability in thick hydrogel constructs wassignificantly greater on permeable supports than glass sup-ports, suggesting that solute transport limitations were atleast partially alleviated. The thick constructs were effectivefor containing neurite growth from tissue explants. Neuritegrowth was robust and occurred in a spatially controlledmanner throughout the 3D environment of the Puramatrix,a crucial observation in the quest for a more effective bio-mimetic model of neurite outgrowth.

ACKNOWLEDGMENTS

The authors thank Ms. Jewell Podratz and the laboratory ofProf. Anthony Windebank for sharing their expertise in DRGdissection and culture as well as Prof. Shaochen Chen for help-ful discussions regarding the DMD setup.

REFERENCES1. Dertinger SK, Jiang X, Li Z, Murthy VN, Whitesides GM. Gradients

of substrate-bound laminin orient axonal specification of neurons.

Proc Natl Acad Sci U S A 2002;99:12542–12547.

2. Cao X, Shoichet MS. Investigating the synergistic effect of com-

bined neurotrophic factor concentration gradients to guide axonal

growth. Neuroscience 2003;122:381–389.

FIGURE 8. Analysis of depth of neurite growth in constructs. (A) Aver-

age height of b III labeled neurites in constructs both with and with-

out Puramatrix (p < 0.005). (B) Neurite growth throughout depth of

Puramatrix as a percentage of total neurite growth.


3. Johansson F, Carlberg P, Danielsen N, Montelius L, Kanje M. Axo-

nal outgrowth on nano-imprinted patterns. Biomaterials 2006;27:

1251–1258.

4. Mahoney MJ, Chen RR, Tan J, Saltzman WM. The influence of

microchannels on neurite growth and architecture. Biomaterials

2005;26:771–778.

5. Gomez N, Lu Y, Chen S, Schmidt CE. Immobilized nerve growth

factor and microtopography have distinct effects on polarization

versus axon elongation in hippocampal cells in culture. Biomate-

rials 2007;28:271–284.

6. Miller C, Jeftinija S, Mallapragada S. Micropatterned Schwann

cell-seeded biodegradable polymer substrates significantly

enhance neurite alignment and outgrowth. Tissue Eng 2001;7:

705–715.

7. Gomez TM, Letourneau PC. Filopodia initiate choices made by

sensory neuron growth cones at laminin fibronectin borders in-

vitro. J Neurosci 1994;14:5959–5972.

8. Abbott A. Cell culture: Biology’s new dimension. Nature 2003;424:

870–872.

9. Cushing MC, Anseth KS. Materials science. Hydrogel cell cultures.

Science 2007;316:1133–1134.

10. Schindler M, Nur-E-Kamal A, Ahmed I, Kamal J, Liu HY, Amor N,

Ponery AS, Crockett DP, Grafe TH, Chung HY, Weik T, Jones E,

Meiners S. Living in three dimensions: 3D nanostructured envi-

ronments for cell culture and regenerative medicine. Cell Biochem

Biophys 2006;45:215–227.

11. Smalley KS, Lioni M, Herlyn M. Life isn’t flat: Taking cancer biol-

ogy to the next dimension. In Vitro Cell Dev Biol Anim 2006;42:

242–247.

12. Desai A, Kisaalita WS, Keith C, Wu ZZ. Human neuroblastoma

(SH-SY5Y) cell culture and differentiation in 3-D collagen hydro-

gels for cell-based biosensing. Biosens Bioelectron 2006;21:

1483–1492.

13. Irons HR, Cullen DK, Shapiro NP, Lambert NA, Lee RH, LaPlaca

MC. Three-dimensional neural constructs: A novel platform

for neurophysiological investigation. J Neural Eng 2008;5:333–

341.

14. Yu LMY, Leipzig ND, Shoichet MS. Promoting neuron adhesion

and growth. Mater Today 2008;11:36–43.

15. Yu X, Dillon GP, Bellamkonda RB. A laminin and nerve growth

factor-laden three-dimensional scaffold for enhanced neurite

extension. Tissue Eng 1999;5:291–304.

16. Suri S, Schmidt CE. Cell-laden hydrogel constructs of hyaluronic

acid, collagen, and laminin for neural tissue engineering. Tissue

Eng Part A 2010;16:1703–1716.

17. Holmes TC, de Lacalle S, Su X, Liu G, Rich A, Zhang S. Extensive

neurite outgrowth and active synapse formation on self-assem-

bling peptide scaffolds. Proc Natl Acad Sci U S A 2000;97:

6728–6733.

18. Dadsetan M, Knight AM, Lu L, Windebank AJ, Yaszemski MJ.

Stimulation of neurite outgrowth using positively charged hydro-

gels. Biomaterials 2009;30:3874–3881.

19. Tsang VL, Bhatia SN. Three-dimensional tissue fabrication. Adv

Drug Deliv Rev 2004;56:1635–1647.

20. Khademhosseini A, Langer R, Borenstein J, Vacanti JP. Micro-

scale technologies for tissue engineering and biology. Proc Natl

Acad Sci U S A 2006;103:2480–2487.

21. Beebe DJ, Moore JS, Yu Q, Liu RH, Kraft ML, Jo BH, Devadoss C.

Microfluidic tectonics: A comprehensive construction platform for

microfluidic systems. Proc Natl Acad Sci U S A 2000;97:

13488–13493.

22. Li CW, Cheung CN, Yang J, Tzang CH, Yang M. PDMS-based

microfluidic device with multi-height structures fabricated by sin-

gle-step photolithography using printed circuit board as masters.

Analyst 2003;128:1137–1142.

23. Khademhosseini A, Yeh J, Jon S, Eng G, Suh KY, Burdick JA,

Langer R. Molded polyethylene glycol microstructures for capturing

cells within microfluidic channels. Lab Chip 2004;4:425–430.

24. Sarig-Nadir O, Livnat N, Zajdman R, Shoham S, Seliktar D. Laser

photoablation of guidance microchannels into hydrogels di-

rects cell growth in three dimensions. Biophys J 2009;96:4743–

4752.

25. Lu Y, Mapili G, Suhali G, Chen SC, Roy K. A digital micro-mirror

device-based system for the microfabrication of complex, spa-

tially patterned tissue engineering scaffolds. J Biomed Mater Res

A 2006;77A:396–405.

26. Moore K, Macsween M, Shoichet M. Immobilized concentration

gradients of neurotrophic factors guide neurite outgrowth of pri-

mary neurons in macroporous scaffolds. Tissue Eng 2006;12:

267–278.

27. Kapur TA, Shoichet MS. Immobilized concentration gradients of

nerve growth factor guide neurite outgrowth. J Biomed Mater

Res A 2004;68:235–243.

28. Hahn MS, Miller JS, West JL. Three-dimensional biochemical and

biomechanical patterning of hydrogels for guiding cell behavior.

Adv Mater 2006;18:2679–2684.

29. Seidlits SK, Schmidt CE, Shear JB. High-resolution patterning of

hydrogels in three dimensions using direct-write photofabrication

for cell guidance. Adv Funct Mater 2009;19:3543–3551.

30. Luo Y, Shoichet MS. A photolabile hydrogel for guided three-

dimensional cell growth and migration. Nat Mater 2004;3:249–

253.

31. Bellamkonda RV. Peripheral nerve regeneration: an opinion on

channels, scaffolds and anisotropy. Biomaterials 2006;27:3515–

3518.

32. Gombotz WR, Wang GH, Horbett TA, Hoffman AS. Protein ad-

sorption to poly(ethylene oxide) surfaces. J Biomed Mater Res 1991;

25:1547–1562.

33. Moeller HC, Mian MK, Shrivastava S, Chung BG, KhademhosseiniA. A microwell array system for stem cell culture. Biomaterials2008;29:752–763.

34. Ellis-Behnke RG, Liang YX, You SW, Tay DK, Zhang S, So KF,Schneider GE. Nano neuro knitting: Peptide nanofiber scaffold forbrain repair and axon regeneration with functional return ofvision. Proc Natl Acad Sci U S A 2006;103:5054–5059.

35. Sun C, Fang N, Wu DM, Zhang X. Projection micro-stereolithogra-

phy using digital micro-mirror dynamic mask. Sensor Actuator A

Phys 2005;121:113–120.

36. Curley JL, Jennings SR, Moore MJ. Fabrication of micropatterned

hydrogels for neural culture systems using dynamic mask projec-

tion photolithography. J Vis Exp 2011;48:e2636.

37. Peng H, Ruan Z, Long F, Simpson JH, Myers EW. V3D enables

real-time 3D visualization and quantitative analysis of large-

scale biological image data sets. Nat Biotechnol 2010;28:

348–353.

38. Lemmon V, Burden SM, Payne HR, Elmslie GJ, Hlavin ML. Neurite

growth on different substrates—Permissive versus instructive

influences and the role of adhesive strength. J Neurosci 1992;12:

818–826.

39. Anton ES, Sandrock AW, Matthew WD. Merosin promotes neurite

growth and schwann-cell migration in-vitro and nerve regenera-

tion in-vivo—Evidence using an antibody to merosin. Arm-1. Dev

Biol 1994;164:133–146.

40. Gelain F, Lomander A, Vescovi AL, Zhang S. Systematic studies

of a self-assembling peptide nanofiber scaffold with other scaf-

folds. J Nanosci Nanotechnol 2007;7:424–434.

41. Xu T, Gregory CA, Molnar P, Cui X, Jalota S, Bhaduri SB, Boland T.

Viability and electrophysiology of neural cell structures generated by

the inkjet printingmethod. Biomaterials 2006;27:3580– 3588.

42. Brigham MD, Bick A, Lo E, Bendali A, Burdick JA, Khademhos-

seini A. Mechanically robust and bioadhesive collagen and photo-

crosslinkable hyaluronic acid semi-interpenetrating networks.

Tissue Eng Part A 2009;15:1645–1653.

43. Suri S, Schmidt CE. Photopatterned collagen-hyaluronic acid

interpenetrating polymer network hydrogels. Acta Biomaterialia

2009;5:2385–2397.

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