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Biomaterials 67 (2015) 264e273

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Biomaterials

journal homepage: www.elsevier .com/locate/biomateria ls

3D printing of layered brain-like structures using peptide modifiedgellan gum substrates

Rodrigo Lozano a, Leo Stevens a, Brianna C. Thompson a, Kerry J. Gilmore a,Robert Gorkin III a, Elise M. Stewart a, Marc in het Panhuis a, b, Mario Romero-Ortega c,Gordon G. Wallace a, *

a Intelligent Polymer Research Institute, ARC Centre of Excellence for Electromaterials Science, AIIM Facility, University of Wollongong, Wollongong,NSW 2522, Australiab Soft Materials Group, School of Chemistry, University of Wollongong, Wollongong, NSW 2522, Australiac Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080, USA

a r t i c l e i n f o

Article history:Received 29 June 2015Accepted 11 July 2015Available online 14 July 2015

Keywords:3D printingGellan gum (GG)GG modificationBrain-like structureDiffusion

* Corresponding author.E-mail address: gwallace@uow.edu.au (G.G. Walla

http://dx.doi.org/10.1016/j.biomaterials.2015.07.0220142-9612/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

The brain is an enormously complex organ structured into various regions of layered tissue. Researchershave attempted to study the brain by modeling the architecture using two dimensional (2D) in vitro cellculturing methods. While those platforms attempt to mimic the in vivo environment, they do not trulyresemble the three dimensional (3D) microstructure of neuronal tissues. Development of an accuratein vitro model of the brain remains a significant obstacle to our understanding of the functioning of thebrain at the tissue or organ level. To address these obstacles, we demonstrate a new method to bioprint3D brain-like structures consisting of discrete layers of primary neural cells encapsulated in hydrogels.Brain-like structures were constructed using a bio-ink consisting of a novel peptide-modifiedbiopolymer, gellan gum-RGD (RGD-GG), combined with primary cortical neurons. The ink was opti-mized for a modified reactive printing process and developed for use in traditional cell culturing facilitieswithout the need for extensive bioprinting equipment. Furthermore the peptide modification of thegellan gum hydrogel was found to have a profound positive effect on primary cell proliferation andnetwork formation. The neural cell viability combined with the support of neural network formationdemonstrated the cell supportive nature of the matrix. The facile ability to form discrete cell-containinglayers validates the application of this novel printing technique to form complex, layered and viable 3Dcell structures. These brain-like structures offer the opportunity to reproduce more accurate 3D in vitromicrostructures with applications ranging from cell behavior studies to improving our understanding ofbrain injuries and neurodegenerative diseases.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

The development of an appropriate in vitro model of the brainremains a big challenge, due in part to the difficulties associatedwith creating detailed three dimensional (3D) cell loaded regions ina layered architecture [1e8]. Beyond simple casting techniques[9,10], researchers have traditionally attempted to replicate 3Dtissue constructs of brain layers using silk scaffolds [7], PDMSmicro-chambers [5] and microfluidics [11,12]. These techniques arelimited in replicating the actual brain structure, as the resulting

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tissue model does not represent the complex in vivo organization.For example, many of these methods build along a transverse oraxial plane (Fig. 1AeC) contrary to the natural brain architecturewhich consists of layers in a medial or sagittal plane (Fig. 1D, E).Stand-alone artificial brain-like models would enable an improvedunderstanding of the complex electrophysiology of the brain aswell as neuronal regeneration and repair (neuroplasticity). Such amodel could also be used to perform preliminary evaluation of newtherapeutic treatments. Furthermore, a functional 3D layeredmodel could be used to probe multi-layered neural circuits,enabling a better understanding of traumatic brain injuries (TBI) aswell as the basis of physiological learning and memory [9e15].

The ability to create true 3D in vitro representations of the brainrequires the intersection of two related areas of research:

Fig. 1. Schematic representations of in vivo and in vitro brain layer structures. AeD) Bioengineered representation of brain-like structures using different methods. A) Microfluidiccasting can produce 6 layers of differing contents, however is restricted to horizontal configurations in PDMS chips (modified from ref. 2). B, C) Other casting methods haveattempted to form brain-like structures in 3D cylindrical shapes (modified from ref. 1). D) Proposed design for ideal stand-alone free-formed artificial brain-like structures. E) Arepresentation of the 6 layered brain architecture found in the human cortex. Image modified from http://www.imagequiz.co.uk/quizzes/10309002.

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biomaterials and biofabrication. The choice of biomaterial is acritical element to enable cell survival, proliferation and function ofthe neural network. In addition, the biomaterial must be amenableto fabrication of 3D structures in a manner that does not harmliving cells [16,17]. It is widely known that when dissociated cellsare encapsulated into biomaterial scaffolds, these structures func-tion as a synthetic extracellular matrix (ECM) [18,19]. Both the grossscaffold structure and its internal architecture affect the behaviorand viability of the cells seeded inside. These properties facilitatecell attachment, proliferation and differentiation within the entirescaffold to organize the cells in a tissue-like manner [20]. In the lastfew decades, hydrogels have been adopted as standard ECM sub-stitutes due to their biocompatibility, degradability, exceptionaloxygen and nutrient permeability [21e24] and structural stability[25]. However not all hydrogels fit the criteria needed to fabricate3D structures.

In recent years, natural gum polymers such as gellan gum (GG)have gained interest in the tissue engineering arena due to theirunique properties including gelling temperature, texture, gelstrength, clarity, the rate of gelation and ability to dissolve in water[26]. GG is a polysaccharide obtained as a result of a fermentationprocess by the microorganism Pseudomonas elodea [27e29], andwas first introduced in 1978 as an alternative to agar and gelatin inthe food industry. Applications in the pharmaceutical industry, gelelectrophoresis, and more recently in tissue engineering have fol-lowed. GG as a bio-ink has many benefits over other hydrogels asoutlined in Ferris et al. 2013 [27,28], including low production cost,high gelling efficiency at body temperature, good processability, awide spectrum of achievable mechanical properties, suitablerheological properties, and biocompatibility [29,30]. Additionally,GG achieves similar levels of mechanical strength as gelatin or agarat lower concentrations [31,32], thus maximizing the use of thematerial. The combination of these material properties facilitatesthe handling and deposition of GG using bioprinters.

GG matrices have been primarily used as substrates for bone[29e32] and fibroblast tissue engineering [33], however, only a fewstudies have addressed the use of GG as a biomaterial for neural cellcultures [34]. Furthermore, studies have shown that peptidemodification of hydrogels can be improved as ECM by incorporatingpeptide sequences to influence and stimulate cell survival as well asgrowth and differentiation [30e34]. For example, alginate has beenmodified with peptide sequences GRGDS and RGD and GG withGRGDS peptides [35e40]. Very recently GG has been modified withRGD peptide, showing an increase in cell adhesion and proliferationcompared to the same materials without peptide modification [41].

Although many novel materials allow for the encapsulation ofdissociated cells, they have not yet accurately replicated tissue-likestructural organization. Much of this is due to the difficulties ofprocessing bio-inks and their use in building the complex layeredstructures required to replicate tissue. Recently, bioprinting orbiofabrication (a subset of additive manufacturing (AM) commonlytermed 3D printing), has emerged as a critical tool in tissue engi-neering [16,42e45]. This method allows the accurate positioning ofcells and biocompatible materials with the aim of forming func-tional 3D tissues [42,46]. Several different approaches have beenutilized to produce 3D bioprinted structures; with each specifictechnique tailored to produce a certain geometry and application[47e50]. Even within the range of techniques available, there is alimit to which biomaterials can be utilized, based upon their me-chanical properties and crosslinking methods.

In this study we utilized a newly-developed handheld reactivebathless printing process (3D printing) to easily create free-formed3D structures with layer architecture. Simultaneously, we prepareda suitable bio-ink for dissociated primary cortical neural cells basedon peptide-modified GG that was compatible with this printingprocess. Scanning electron microscopy was used as previously re-ported to probe the internal structure of the printed hydrogels[51,52]. Fluorescence staining and Confocal microscopy wereemployed to determine the cell viability, phenotype andmorphology of neural networks. A comparison of crosslinker so-lutions (either Dulbecco's Modified Eagle's Medium (DMEM) orCaCl2) was made to determine the best conditions to form the self-supporting 3D structure. This 3D printed in vitro model provides atool for the fabrication and investigation of multi-layered neuralcircuit formation, relevant for understanding traumatic brain injury(TBI) as well as the basis of physiological learning and memory.

2. Material and methods

2.1. Gellan gum (GG) purification

Commercial low-acyl gellan gum (GG) obtained from CP Kelco(traded as Gelzan CM) was purified to remove divalent cationsusing an established method [41,53]. Briefly, a 1% (w/v) solution ofGG was dissolved in Milli-Q water (18.2 MU) at 60 �C was mixedwith 1.25 g of pre-rinsed Dowex 50W-X8 cation exchange resin andstirred for 30 min. The supernatant was then passed through acoarse filter along with two rinses of the resin with 60 �C ultrapurewater. 4 M NaOHwas added dropwise until the pH stabilized at 7.5.This solution was then frozen and lyophilized over 2 days using an

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Alpha 2-4 LD plus freeze dryer (Christ).

2.2. RGD- modification of purified gellan gum (RGD-GG)

Peptides were coupled to purified GG using a previously re-ported method [41,53]. Briefly, a 1% (w/v) solution of NaGG wasprepared in a 50 mM solution of 2-(N-morpholino) ethanesulfonicacid (MES) buffer (Sigma) adjusted to pH 6.5. A 0.3 M solution ofEDC (Sigma) was formed in the same MES buffer and immediatelytransferred to the NaGG solution, followed by 50 mL of a 0.15 Msolution of Sulfo-NHS (Sigma) in MES buffer. The solution wasvortexed for 15 min at 37 �C. A short peptide (GGGGRGDSY, 95%,Auspep) was added to convert 1% of available carboxylate groupsand the mixture was kept overnight at 37 �C. The solution waspurified by dialysis against ultrapure water over 5 days using Slide-a-Lyzer mini dialysis devices (0.1 mL, MWCO 10 kDa, Thermo-Scientific). The dialyzed product was frozen and lyophilized for 2days using an Alpha 2-4 LDplus freeze dryer (Christ), sealed andstored at 4 �C. Prior to cell encapsulation, the freeze dried RGD-GGwas dissolved overnight at 60 �C in Milli-Q water (18.2 MU) to forma 1% (w/v) solution.

2.3. Scanning Electron Microscope (SEM) of RGD-GG

SEM studies of the samples were carried out using the JSM-6490LV Scanning Electron Microscope installed at the Electron Micro-scopy Centre (EMC, University of Wollongong). The gels (with andwithout cells) were fixed with 3.7% paraformaldehyde (PFA) for30 min prior to freeze-fracturing by immersion in liquid nitrogenfor 60 s and fracturing using a cold razor blade. Fractured gels wereexamined with magnifications of 500, 800 and 1500�.

2.4. Diffusion studies

Diffusion of solute into and out of the printed gels was charac-terized by using a similar method to that applied by Li et al. [54].Three replicates of 1 cm � 0.5 cm diameter cylindrical hydrogels(GG and RGD-GG) were soaked in solutions containing fluo-rescently labeled bovine serum albumin (FITC-BSA, Sigma) and theuptake of the protein was calculated after collecting the solutionand measuring the loss of solute over time until equilibrium wasreached. FITC-BSA concentration was determined using a micro-plate reader (Fluostar Omega, BMG Labtech). Concentrations ofBSAwere calculated from a standard curve. The vials containing thehydrogel cylinders were maintained at constant temperature of37 �C in a shaking water bath. The subsequent release was calcu-lated after by soaking the gels in fresh PBS, and taking fluorescencemeasurements over time. Diffusion coefficients of FITC-BSA in theprinted GG and RGD-GG cylindrical gels were calculated using anonlinear regression method, reported by Li [54], Carman and Haul[55] and Crank [56].

2.5. Cell culture

All animal work was approved and performed in accordancewith the Animal Ethics Committee (AEC, #13/04), University ofWollongong. Primary cortical neurons were harvested from E18embryos of BALB/cArcAusb mice obtained from Australian Bio-Resources (New South Wales). After the brain was exposed, themeninges of the frontal cortex region of the lateral ventricles wereremoved and the tissue was collected and digested with collage-nase (Sigma) for 10 min at 37 �C. The reaction was inhibited by theaddition of fetal bovine serum (FBS), and the digested tissue wasmechanically dissociated with a pipette and then filtered through a70 mm cell strainer (BD Falcon, USA). After centrifugation at 375 g

for 10 min, cells were resuspended at a density of 2 � 106 cells/mLin neurobasal media (Gibco), with 1% L-glutamine (Sigma), 2% B27neural supplement (Gibco) and 1% penicillin/streptomycin (Sigma)(Complete Neurobasal). The 2D control cells were seeded on poly Dlysine (PDL)/laminin-coated glass coverslips at 50,000 cell/cm2 andincubated in a humidified atmosphere at 37 �C with 5% CO2. A 50%media change was performed on all cultured cells every 48 h.

2.6. Cell encapsulation

The primary neural cells were encapsulated in RGD-GG solu-tions before the gels were crosslinked. A gellan gum crosslinkingprotocol based on Ferris et al. [41] was used to crosslink the gelscontaining the primary cortical neurons. In this study, the protocolwas modified to test the use of an alternative ionic crosslinker, (5�)Dulbecco's Modified Eagle's Medium (DMEM, containing 9 mMCaCl2 and 4.05 mMMgSO4), which is widely used in the culture of avariety of cells. The 1% (w/v) RGD-GG solution and the crosslinkerwere sterilized by passing the solutions separately through 0.2 mmfilters (Millipore) prior to use, and the RGD-GG solution waswarmed to 37 �C in a water bath. Cells were suspended in a 1:1dilution with bio-ink (RGD-GG) to obtain a final cell concentrationof 1 � 106 cells/mL and a final RGD-GG concentration of 0.5% (w/v).Themixture was carefully triturated with a 1mL pipette tip until aneven cell distribution was observed. For 3D non-printed controls,50 ml of cells in RGD-GG were directly pipetted into a 6 well plate.To crosslink the deposited bio-ink, a 5 ml drop of the crosslinkersolution (5� DMEM or 1 M CaCl2) was added onto the samples. Thebio-ink was allowed to crosslink at 37 �C in a humidified 5% CO2atmosphere for 3e5 min prior to the addition of 1 mL of CompleteNeurobasal media. Samples were then maintained in CompleteNeurobasal media in a humidified atmosphere at 37 �C with 5% ofCO2 with regular media changes.

2.7. Bio printing

Printing of cortical neurons was achieved using a simple, hand-held printing process using two different ionic crosslinkers, the 1 MCaCl2 or (5�) DMEM. Prior to printing, the equipment was sterilizedby rinsing with 70% ethanol and transferred to a biosafety cabinet.A Luer-locking coaxial needle tip (Rame Hart) with inner diameter0.2mm and outer diameter 1mm (Fig. 2BeC) as well as a 45 cm lineof silicon tubing were rinsed with sterile H2O and dried under N2.The bio-ink/cell suspension and crosslinker solutions were trans-ferred aseptically into separate 10 mL Harpool Luer-lock syringes.The cross-linker syringe was connected to the outer ring input ofthe printing needle via silicon tubing and then loaded into a KDScientific computer-controlled screw syringe pump. Thebiopolymer-loaded syringe was attached directly to the innerdiameter input of the coaxial syringe tip. Lines were then manuallyprimed in preparation for printing. The fully assembled system(Fig. 2A) was programed to deliver crosslinker solution at a rate of0.1 mL/min and manual depression of the bio-ink/cell syringeyielded solid hydrogel structures, captured in a receiving well plate(Fig. 2).

2.8. Live/dead staining of encapsulated cortical neurons

Standard calcein/propodium iodide assay was used estimate thepercentage of live/dead cells. Calcein AM solution was added tosamples at a final concentration of 5 mM and incubated at 37 �C for10 min. Propidium iodide (PI) was added at 1 mg/mL and incubatedfor a further 5 min before rinsing and imaging using Confocal mi-croscopy. Using this protocol, living cells are stained with the greenfluorescent marker calcein and dead cells with red PI. Living and

Fig. 2. Hand-held reactive bathless printing. A) Cell printing system inside a biosafety cabinet. B) 3D printed structure. B) Schematic diagram of the extrusion tip.

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dead cells were counted using Fiji (Image J) software [57]. Briefly, Z-stacks of 50e90 mm in height were flattened, and the green and redchannels analyzed separately to determine numbers of live anddead cells. Each experiment was repeated four times, with twointernal replicates, so each point represents the average of 8samples.

2.9. Immunofluorescence staining of cortical neurons and Confocalmicroscopy

The encapsulated and printed neurons were fixed with 4% (w/v)paraformaldehyde (PFA) for 30 min, followed by permeabilizationand blocking with 0.3% TritonX-100 with 10% donkey serum in PBSfor 2 h. After blocking, cells were washed three times for 15 min inPBS followed by incubation in rabbit anti-GFAP (Millipore) andmouse anti-b-III-tubulin (Covance) antibodies in PBS with 10%donkey serum/PBS. After overnight incubation of the primaryantibody at 4 �C, 3 washes with PBS (15 min each) were performed,followed by addition of the secondary antibodies (Alexa Fluor 488-conjugated donkey anti-mouse, Alexa Fluor 594-conjugated goatanti-rabbit, Invitrogen) and 10% donkey serum in PBS. After 3 hincubationwith secondary antibodies, DAPI (Molecular probes)wasadded to the solution, and incubated for a further 30 min. Finally,the printed cells were washed three times in PBS. All PBS used forthe cell staining process was supplemented with 5 mM CaCl2 toprevent gel dissolution. Confocal microscopy was used for imagingof both live and fixed samples. Confocal imaging using Leica TSCSP5 II Confocal microscope was used for multichannel imaging.Image analysis was performed using Leica 3D projection tool,ImageJ and Student's t-test analysis for determination of statisticalsignificance.

3. Results and discussion

3.1. RGD-GG pore structure

The RGD-GG gels described in this study (0.075%, 0.15% and0.5%) were porous enough to facilitate nutrients, oxygen and wasteexchange between cells and culture medium. This is demonstratedby the viability and growth of neural cells throughout the entire gelafter 7 days of culture. An indication of the pore-structure of thebio-ink (RGD-GG) was obtained by using freeze fracturing and lowvacuum scanning electron microscopy (SEM). The SEM images ofRGD-GG showed that the three concentrations of bio-ink (0.075%,0.15% and 0.5%) form porous networks. The gels showed a widedistribution of pore diameters ranging from 10 to 250 mm(Fig. 3AeB, DeE, GeH). Furthermore, in addition to the inter-connected large macropores, smaller sized pores were observed inthewall regions connecting thosemacropores (1.5e5 mm) as shownin Fig. 3C, F, I.

3.2. Analysis of encapsulated primary cells

To test cell viability and behavior of cortical neurons in the bio-ink (RGD-GG), cells were suspended in RGD-GG at 1 � 106 cells perml, and the gels cross-linked using 5� DMEM. After 5 days the cellsencapsulated in the gel were immunostained and the neural andglial populations were analyzed using Confocal microscopy in orderto determine the optimal RGD-GG concentration for primary cellsurvival, attachment and differentiation. The results of this studyconfirmed that the RGD peptide supported and enhanced primarycortical adhesion and differentiation compared to that of unmodi-fied GG (Supplementary Fig. S1). Confocal microscopy of immu-nostained cells revealed that after 5 days, cells exhibitedattachment and viability at three different concentrations of RGD-GG as shown in Fig. 4. Also, Fig. 4 DeF demonstrate that the cellsremained viable and suspended in 3D over the 5 days of cells cul-ture, extending filipodia in the X,Y and Z planes. This demonstratesthat the gels exhibit sufficient mechanical support to maintain thecells in 3D, but also sufficient porosity for nutrient and wastediffusion, and for cell growth and neurite extension. Fig. 4 A, B, Cshow b-III tubulin-stained neurons (red) which at all RGD-GGconcentrations demonstrated formation of neural projections andnetworks. Neuronal cells not only remained viable within thedifferent concentrations of RGD-GG gel, but also exhibited cellmorphologies typical of differentiated neurons, extending pro-cesses several hundredmicrometers in length throughout the RGD-GG gel in the x, y and z planes. The 0.15% (w/v) RGD-GG gel sup-ported fewer clustered neurons, as showed in Fig. 4B.

While all tested concentrations of RGD-GG supported neuronaldevelopment and differentiation to varying degrees, the survivaland growth of glial cells in the encapsulating gel is vital for theformation of brain-like constructs in 3D [58]. It is widely knownthat glial cells are a key factor in the survival and differentiation ofneurons [59,60]; therefore, glial cell survival in the three concen-trations of RGD-GG was also considered. Glial cell attachment andviability was supported in all three tested concentrations of RGD-GG (Fig. 5). Encapsulated glial were evenly distributedthroughout the gels and demonstrated typical stellate morphol-ogies and widespread process outgrowths within the three RGD-GG gel concentrations. Also, they were observed as single cells aswell as forming small clusters, a typical behavior, which supportsthe suitability of the model. Furthermore, visual inspectiondemonstrated differences in cell density and process length be-tween different RGD concentrations. Cells in 0.15% (w/v) RGD-GGconcentration had longer processes and 0.5% (w/v) RGD-GG sup-ported a higher density of cells as observed in Fig. 5C.

It was demonstrated that with all tested concentrations of RGD-GG gel, a platform was established for the production of viable 3Dneuronal networks, in which acceptable survival, morphology anddifferentiation of neurons (Fig. 4) and excellent support for glialcells (Fig. 5) was obtained. However, the 0.5% (w/v) RGD-GG gel was

Fig. 3. SEMs of cross sections of fixed, freeze-fractured RGD-GG showing pore sizes. AeC) Cross-section of 0.075% RGD-GG. DeF) Cross-section of 0.15% RGD-GG. GeI) Cross-sectionof 0.5% RGD-GG. Scale bars in A, D, G represent 50 mm, in B,E,H represent 20 mm and C,F,I represent 10 mm. Arrows indicate the large range of pore sizes.

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chosen to investigate printing of encapsulated primary cells as itwas best candidate for production of brain-like printed 3D struc-tures. This was due to its ability to maintain cultured cells in aviable and differentiated state, and that this concentration providedthe optimal flow for printing coherent gel structures whileretaining shape.

Fig. 4. Cortical neurons encapsulated in RGD-GG at different gel concentrations (0.075%, 0tubulin (red) for cortical neurons and DAPI (blue) for nuclei. DeF) Confocal microscope imdecoding for the depth of the cells in the RGD-GG gel along the Z-axis is given (0e60 mm)50 mm.

As revealed by immunofluorescence staining, the cells werelabeled with specific cell markers such as b-III tubulin (red) forcortical neurons (Fig. 4) and GFAP (green) for glial cells (Fig. 5), bothneuronal and glial cells survived in the bio-ink when crosslinkedwith a solution known to be well-tolerated by cultured cells (5 �DMEM). However, parallel gel samples cast into thick films in 12

.15%, and 0.5% w/v respectively) after 5 days of culture. AeC) Cells stained with b-IIIages (depth decoding) of neuronal 3D culture models after 5 days of culture. Color

. Different colors represent the different planes along the Z-axis. Scale bars represent

Fig. 5. Glial cell differentiation in RGD-GG at different concentrations of gel (0.075%, 0.15%, and 0.5% w/v respectively) after 5 days of culture. AeC) Encapsulated cells were stainedwith GFAP (green) to specifically label glial cells. Scale bars represent 50 mm. (For interpretation of the references to colour in this figure legend, the reader is referred to the webversion of this article.)

Fig. 6. Viability of printed and non-printed primary cortical neurons encapsulated in gels using different crosslinkers. Cortical neurons encapsulated in 0.5% RGD-GG gels werelabeled with Calcein AM (green, live cells) and PI (red, dead cells) 2 h, 3 days and 5 days after the printing process. Scale bars represent 100 mm. Time course for cortical cell viabilityafter the bioprinting process with two crosslinkers: (A) 5 � DMEM and (B) 1 M CaCl2. Cell viability was assessed by staining with Calcein (live) and PI (dead), imaging andquantification using Fiji software. Each point represents the average of eight 50e90 mm gel slices imaged by Confocal microscopy (n ¼ 8). The error bars indicate one standarddeviation of the mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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well plates did not have a well-defined structure, indicating thatcrosslinking may have not been sufficient to build a free-standingmultilayered structure. As an alternative, a higher concentrationof CaCl2, (the divalent cation that is primarily responsible for cross-linking of the gel), than is present in DMEM was examined as across-linking agent. Both neurons and glial cells are known to bemore susceptible to chemical buffers and to printing processes thancell lines [61], requiring us to perform a viability study, to minimizethe duration of time over which neural cells were exposed to thehigh extracellular concentrations of CaCl2.

3.3. Viability analysis of printed primary cells

Comparison of the viability of encapsulated primary cultureswhen exposed to both 5� DMEM and CaCl2 as cross-linking agentsis presented in Fig. 6. Cell viability was determined by differentialstaining of live (Calcein AM, green) and dead (propidium iodide,red) cells, at 2 h, 3 days and 5 days after printing (Fig. 6).

An initial cell viability of 78e80% was determined immediatelyafter dissection and derivation of the primary culture. After the cellswere printed and incubated for 2 h, 74 ± 2% cell viability wascalculated for the DMEM crosslinker, 73 ± 8% for gels crosslinkedwith CaCl2 while non-printed, RGD-GG encapsulated controlsshowed 76 ± 7% viability with DMEM and 73 ± 2% with CaCl2(Fig. 6AeB). Similar results were obtained after 3 days in culture,when printed samples crosslinked with DMEM and CaCl2 showed73 ± 10% and 73 ± 6% viability respectively, compared with non-printed, RGD-GG encapsulated cells which were 75 ± 2% viablewith DMEM crosslinker and 74 ± 4% with CaCl2. At 5 days printedsamples remained at 73± 8% in both crosslinkers, whereas the non-printed RGD-GG encapsulated controls showed 75 ± 2% and 74 ± 1%viable cells, respectively. Printed samples crosslinked with CaCl2therefore showed no significant differences in viability from the

Fig. 7. Development of 3D encapsulated and printed primary cortical cell networks 7 days aof all three. Cell nuclei (DAPI, blue, top left), glial cells (GFAP, green, top right), and corticshowing cortical neuron adhesion and proliferation on printed RGD-GG (0.5% w/v). Arrows inC) 3D projection of cortical neurons (b-III tubulin stained) obtained from Fiji (Image J) softwreferred to the web version of this article.)

non-printed RGD-GG encapsulated control samples, and no sig-nificant differences were observed between DMEM and CaCl2crosslinked samples (by Student's t-test). We conclude that theprinting process is compatible with primary cortical neurons andglial cells and that RGD-GG is sufficient to protect the encapsulatedcells from the shear pressure exerted during printing. Therefore,both DMEM and CaCl2 are suitable crosslinkers to produce free-standing structures while maintaining cell viability at acceptablelevels.

In addition to determining the viability of cells in the gels usinglive/dead staining, cells were immunostained 7 days after printingto determine if encapsulated, printed cells retained the ability toform neural networks. The results shown in Fig. 7 demonstrate thatneuronal networks were formed and that glial cells were welldistributed throughout the network (gel). In addition to immuno-staining the interaction between cells and RGD-GG bio-ink wasexamined by SEM (Fig. 7B).

3.4. Diffusion of protein BSA in gels

The diffusion of BSA into and out of the 3D printed hydrogels(0.5% GG and RGD-GG) was investigated using the non-steady statediffusion model (Li et al. [54]). The results show that BSA uptakereached equilibrium by 48 h as shown in Fig. 8A. The subsequentrelease appeared to be slower than the uptake (Fig. 8B); therewas arelease of ~10% of the solute by120 min, followed by a linear releaseprofile over the next 46 h. The diffusion coefficients were calculatedto be 5.78 � 10�7 for GG and 5.15 � 10�7 for RGD-GG. Confocalimaging was utilized to demonstrate that FITC-BSA diffusedthrough large printed structures (0.5 cm diameter by 1 cm inlength, Fig. 8C). Furthermore, we used finite element modeling(COMSOL) to demonstrate that BSA diffuses through the entirety ofprinted gels (Fig. 8D) The modeling shows that BSA reached

fter printing. A) Side-by-side comparison of three fluorescence channels and an overlayal neurons (b-III tubulin, red, bottom left). Scale bars represent 50 mm. B) SEM imagedicate cortical neuron nuclei and ellipse indicates the axon. Scale bar represents 10 mm.are. (For interpretation of the references to colour in this figure legend, the reader is

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equilibrium by 48 h (Supplementary Fig S2).

3.5. Printed 3D brain layer structure

Most culturing systems do not accurately replicate the layeredarchitecture that occurs in brain tissue. The printing methoddescribed here provides an approach through which this structurecan be more precisely replicated (Fig. 9A, E).

To validate that the printing technique could be used to createcomplex, layered and viable 3D cell structures, we first printed a 6-layered structure using 0.5% (w/v) RGD-GG with different dyecolors (blue, green and red) in each printed layer to ensure that theconfiguration could fuse together to form a solid structure whilemaintaining separated layers (Fig. 9BeE). Visual inspection showedthat the layers remained distinct after printing, and that the addi-tion of up to 5 subsequent layers did not compromise the structuresof the bottom section.

Secondly, cortical cell survival and axonal development wasstudied in the 3D brain layer structure after printing a three layeredstructure (Fig. 9D). The structure was made with a top layer con-sisting of 0.5% RGD-GG containing 1 � 106 cells/mL cortical cells, amiddle layer with no cells and a bottom layer identical to the toplayer. After 5 days the cells formed a neuronal network, and axonsbeginning to penetrate into the acellular middle layer as showed in

Fig. 8. Diffusion of FITC-BSA from solution into and out of printed gels. A) Uptake of BSA intsoaking solution. B) Released of BSA from large printed structures. C) FITC-BSA uptake intoelement model of BSA diffusion in the printed gels using COMSOL Multiphysics 5.0: 2D axisyand 48 h of soaking, data is normalized between concentration at specific time point and fi

Fig. 9FeG. Also, DAPI staining (Supplementary fig. S3) confirms thatcells remained distributed through the printed layers and thatneuronal cell bodies did not migrate to the lower layer. Neverthe-less, Fig. 9F and G demonstrate that the porosity of the gel is suf-ficient to allow nutrients and waste to diffuse through in order tokeep cells alive and differentiating over 5 days of culture, as well assufficient to allow neurite penetration through the gel across layers,while providing sufficient mechanical support to maintain the cellbodies in the initial printed layers. Overall results indicated that thebio-ink has the capability to contain and support growth andnetwork formation of cells in specific layered structures, and that itis possible to print a 3D multilayer brain-like structure.

4. Conclusions

In this study, we have demonstrated a novel process to create a3D brain-like structure consisting of layered primary cortical cellsencapsulated in hydrogels representing cortical tissue. Wedemonstrated successful encapsulation, survival and networking ofprimary cortical neurons and glial cells in 3D printed RGD-GGmodified hydrogels, indicating that cortical neurons respondedbetter to the RGD peptide in RGD-coupled GG than to purified GG(supplementary information). Additionally, we investigated thediffusion of the model protein FITC-BSA, in our 3D printed

o a large printed structure is shown by the reduction in fluorescence of FITC-BSA in thea large printed RGD-GG gel at 0, 1 h and 3 h, scale bars represent 300 mm. D) Finite

mmetric; 2 domains: hydrogel (small rectangle) and solution (large rectangle) for 0, 3 hnal concentration (Ct/C).

Fig. 9. Printed brain-like layered structure. A) Solidworks representation of proposed brain-like layer structure. BeE) Printing process to create a brain-like structure, each colorrepresents a layer. F) Confocal microscope images of neurons in different layers after 5 days of culture. The image is colored for the distribution of the cells through the z-axis in thebio-ink RGD-GG gel as indicated. G) Expanded view of the area from the square of Fig. 8F, showing an axon penetrating into the adjacent layer. Scale bars represent 100 mm. (Forinterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

R. Lozano et al. / Biomaterials 67 (2015) 264e273272

hydrogels, to demonstrate the porosity of the gels as confirmed bythe used of low vacuum SEM to probe the internal structure. Tosupport those experiments we used finite element analysis models(COMSOL) to demonstrate that BSA is capable of diffusing throughthe entire printed gels. The modeling confirms that cell nutrientscan diffuse in and out of the printed gels, in support of the high cellviabilites observed experimentally. We explored the crosslinking ofthe RGD-GG gel with both DMEM and CaCl2, and determined thatwhile both crosslinkers supported similar cell viability by 5 days.CaCl2 produced crosslinked gels that better retained their printedstructures.

Cell viability of encapsulated cells after the printing processremained stable for 5 days regardless of the crosslinker used toproduce the RGD-GG gels. Importantly, no difference in viability orcell morphology was seen when comparing cells in printed struc-tures, compared with non-printed (cast) controls, demonstratingthat the shear forces generated during the printing process did notdamage the primary cortical cells. Also, Confocal microscopy im-ages (depth decoding) demonstrated that there was no differencein localization or organization of cells thought the entire 3Dconstruct, demonstrating that cell in the middle as well as at theperiphery of the scaffold received similar nutrients as a result ofinternal porosity which allowed nutrients to flow in and out of thescaffolds. Furthermore, given that cortical cells developed into 3Dneuronal networks in less than 5 days after the printing, constructsmay appear similar to those observed in brain tissue consisting ofdiscrete, although interdependent, brain regions. Furthermore, wevalidated that a low-cost, simple extrusion printing technique andbio-ink (RGD-GG) could be used to create contained, layered andviable 3D cell structures, with the potential to organize corticalneuron subtypes in layer structures, using a process that could beperformed in any cell culture laboratory. These brain-like structures

offer the opportunity to provide a more accurate representation of3D in vivo environments with applications ranging from cellbehavior studies, understanding brain injuries and neurodegener-ative diseases to drug testing.

Acknowledgments

The authors would like to thank the Australian Research CouncilCentre of Excellence Scheme (CE140100012) for their funding ofthis research. In addition, we are also grateful to the ARC for sup-port under the Australian Laureate Fellowship scheme(FL110100196) and the Consejo Nacional de Ciencia y Tecnologia(CONACYT) (292073) for financial support. Also, the authors thankthe Australian National Nanofabrication Facility e Materials nodefor equipment use. We also acknowledge the assistance of TonyRomeo at the UOWElectronMicroscopy Centre, Adam Taylor for hishelp with Solidworks, Aaron Waters for COMSOL modeling, Dr.Jennifer Seifert and Dr. Cameron Ferris, for their useful advice. Theauthors have no financial interests to disclose.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.biomaterials.2015.07.022.

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