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Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture

This article has been downloaded from IOPscience. Please scroll down to see the full text article. 2011 Biomed. Mater. 6 015002 (http://iopscience.iop.org/1748-605X/6/1/015002) View the table of contents for this issue, or go to the journal homepage for more

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IOP PUBLISHING Biomed. Mater. 6 (2011) 015002 (18pp)

BIOMEDICAL MATERIALS

doi:10.1088/1748-6041/6/1/015002

Fabrication and optimization of alginate hydrogel constructs for use in 3D neural cell culture
J P Frampton1 , M R Hynd1,2 , M L Shuler3 and W Shain1,2
Department of Biomedical Sciences, School of Public Health, State University of New York at Albany, Albany, NY 12210, USA 2 NYS Department of Health, Biggs Laboratory, Wadsworth Center, Albany, NY 12210, USA 3 Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850, USA E-mail: jf7674@albany.edu
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Received 30 August 2010 Accepted for publication 6 December 2010 Published 5 January 2011 Online at stacks.iop.org/BMM/6/015002 Abstract Two-dimensional (2D) culture systems provide useful information about many biological processes. However, some applications including tissue engineering, drug transport studies, and analysis of cell growth and dynamics are better studied using three-dimensional (3D) culture systems. 3D culture systems can potentially offer higher degrees of organization and control of cell growth environments, more physiologically relevant diffusion characteristics, and permit the formation of more extensive 3D networks of cellcell interactions. A 3D culture system has been developed using alginate as a cell scaffold, capable of maintaining the viability and function of a variety of neural cell types. Alginate was functionalized by the covalent attachment of a variety of whole proteins and peptide epitopes selected to provide sites for cell attachment. Alginate constructs were used to entrap a variety of neural cell types including astroglioma cells, astrocytes, microglia and neurons. Neural cells displayed process outgrowth over time in culture. Cell-seeded scaffolds were characterized in terms of their biochemical and biomechanical properties, effects on seeded neural cells, and suitability for use as 3D neural cell culture models. (Some gures in this article are in colour only in the electronic version)

1. Introduction
Hydrogels are crosslinked polymers that, due to their hydrophilic properties, retain high water content after gelation. Hydrogel matrices have been used in a variety of chemical, pharmaceutical and biomedical applications ranging from emulsifying agents, to drug elution systems, scaffolds for cell entrapment and extracellular matrix (ECM) analogs (Lee et al 2008, Li et al 2006a, Wheeler et al 1996, Hynd et al 2007a). Hydrogels are commonly used for tissue engineering applications because they exhibit low immunogenicity and low cytotoxicity, and permit the exchange of gases and nutrients between cells and the environment. It is also possible to modify the mechanical and biochemical properties of many
1748-6041/11/015002+18$33.00

hydrogel polymers (Ma 2008, Chan and Mooney 2008, Drury et al 2004, Rowley and Mooney 2002). Hydrogels can be formed from both synthetic and natural polymers. Many synthetic hydrogels are formed from acrylamide-based polymers as in the case of poly(ethylene glycol) diacrylate (PEGDA) and poly(hydroxyethyl methacylate) (HEMA) (Hynd et al 2007b). Natural polymers capable of forming hydrogels include agarose, chitosan, collagen, hyaluronan and alginate (Nair and Laurencin 2006). Many of these compounds can be prepared as solutions or as colloidal suspensions in aqueous buffers. Gelation can be achieved by a variety of mechanisms, including UV photopolymerization, redox initiation, ionic crosslinking, temperature change, or pH change. Once the hydrogel has been formed, its porous nature allows
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2011 IOP Publishing Ltd Printed in the UK

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most proteins, molecules, and nutrients to enter, as well as metabolites and waste products to diffuse out. However, pore sizes are often not large enough to permit cell migration without cell remodeling of the hydrogel matrix (George and Abraham 2006, Karageorgiou and Kaplan 2005). Cell entrapment within hydrogel matrices is achieved by polymerizing or crosslinking hydrogel polymers around suspended cells, thus immobilizing the cells in 3D. Hydrogelbased cell entrapment systems have been used with a variety of cell types and have been demonstrated to have no signicant effects on cell viability (Roberts et al 1996, Mann et al 2001, Hisano et al 1998, Elisseeff et al 2000, Desai et al 2006, Burdick and Anseth 2002, Kreeger et al 2006). However, selection of an appropriate hydrogel matrix for cell entrapment ultimately depends upon the chemical properties of the polymer and the sensitivity of the cells. Neural cell types, in particular neurons, are sensitive to reactive oxygen species due to low levels of endogenous antioxidant expression (Raps et al 1989). Therefore polymers that do not rely on free radical initiation for polymerization and do not produce reactive species as degradation products are desirable substrates for neural tissue engineering. Hydrogel matrices must also be able to provide attachment sites for anchorage-dependent cell types. Many hydrogel matrices can be functionalized to promote cell survival and process growth by the covalent attachment of peptide sequences or proteins. Peptide sequences from a variety of extracellular matrix proteins including bronectin (RGD) and laminin (IKVAV and YIGSR) have been incorporated into some types of hydrogel matrices, providing attachment ligands that can be recognized by specic cell types (Dhoot et al 2004, Rowley et al 1999, Comisar et al 2007). Alginate has been useful for the entrapment of mammalian cells because it satises the criteria described above and in addition, displays mechanical properties that are similar to tissue (Drury et al 2004, Kong et al 2003, 2004). Alginate is a polysaccharide derived from the cell walls of brown algae. It is composed of (14) -D-mannuronic acid and -L-guluronic acid residues linked either randomly or as homopolymeric blocks (Johnson et al 1997). The carboxylate groups present on the polysaccharide chains provide sites for the covalent attachment of peptides and proteins that promote cell attachment (Rowley et al 1999). Alginate is readily crosslinked in the presence of divalent cations including Ca2+ and Ba2+ , but not Mg2+ (Morch et al 2006). Crosslinking can be reversed by exposure to the high concentrations of Na+ or to Ca2+ chelating agents. By variation of alginate concentration, composition, porosity and cell attachment factors, it is possible to tailor alginate matrices for specic cell culture applications (Rowley and Mooney 2002, Comisar et al 2006, Eiselt et al 2000). Alginate matrices have been used extensively as substrates for both 2D and 3D cell culture (Comisar et al 2007, Kong et al 2003, Novikova et al 2006). However, only a few studies have addressed the use of alginate as a material for the culture of neural cells (Boisseau et al 1993, Li et al 2006b, Novikova et al 2006). This report describes an alginate construct system that has been characterized in terms of its physical and biological
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properties. A novel culture system is presented that has been rigorously characterized and optimized in order to sustain the growth of neural cells in 3D. This culture system provides a valuable in vitro tool for the fabrication of neural co-culture systems and the investigation of neural cellcell and cell substrate interaction.

2. Methods
2.1. Cell culture The LRM55 rat astroglioma cell line was used for rapid screening and selection of polymers for use with primary cultures (Martin and Shain 1979). LRM55 cells proliferate rapidly (22 h doubling time) and display a number of astrocytic phenotypes (Shain et al 1987, Madelian et al 1985). Cells were cultured to between 70% and 90% conuence in T75 asks prior to use. LRM55 cells were prepared for 3D culture by washing monolayers of cells with HEPES buffered Hanks saline (HBHS), followed by treatment with TrypLE (Invitrogen, Carlsbad, CA) to dissociate cells from their substrate. Cells were collected and serum-containing medium was added to quench the activity of the TrypLE. Cells were counted and centrifuged at 45 RCF to collect the cells at the numbers required for entrapment in either alginate matrices or cultured on the surface of poly-L-lysine (PLL)-treated glass coverslips as a positive control. LRM55 cells were cultured in Dulbeccos modied Eagles medium (DMEM) containing 10% fetal bovine serum (FBS). The medium was completely replenished every 3 days. Mixed glial cultures (astrocytes and microglia) were obtained from postnatal day three Sprague-Dawley rat cortices (Taconic Farms, Germantown, NY) (Banker 1998). Rats were decapitated and the cranial skin and bone were removed to expose the cortex. Cortices were removed using a metal spatula and placed in a balanced saline solution (BSS). Meninges and surface vasculature were removed using hooked forceps. The cortices were then dissected from the midbrain, cerebellum and hippocampi, and minced into 1 mm3 pieces using microdissection scissors. Minced cortices were dissociated into a single cell suspension by enzymatic digestion in 0.25% trypsin and 50 g mL1 DNaseI in BSS, with stirring at 37 C for 30 min. The cell suspension was then triturated and strained through an 80 m pore-size, sterile nylon mesh (Wildlife Supply Company, Buffalo, NY) to remove undissociated tissue. Cells were cultured in either T75 asks or 150 mm2 Petri dishes. Glial cells were cultured in DMEM containing 10% FBS. Medium was completely replaced the day after plating and every 3 days thereafter. Cells were passaged at 7090% conuence, not more than three times. Pure astrocyte cultures were produced by cryogenically freezing mixed glial cell preparations in liquid nitrogen. Freezing medium consisted of complete growth medium containing 10% dimethyl sulfoxide. Following storage in liquid nitrogen, thawing and reseeding, glial cultures contained no microglia and were thus considered pure astrocyte cultures. Microglia were collected from mixed glial cultures prior to freezing and subcultured in 150 mm2 Petri dishes.

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OH OH O O O HO OH OH O

Microglia subcultures were generated by pipetting the culture medium across the surface of mixed glial cultures 1015 times to remove cells that were loosely attached to the surface of astrocytes. Microglial lineage was determined through examination of cell morphology, growth pattern, and expression of the microglia-specic marker ionized calciumbinding adaptor molecule-1(Iba-1). Microglia cultures were grown in DMEM containing 10% FBS, with medium being changed every 3 days. Hippocampal neurons were obtained from embryonic day 18 Sprague-Dawley rats (Taconic Farms) (Banker 1998). Embryos were removed from the timed pregnant dams following anesthesia with CO2 and euthanasia by bilateral thorectomy. Embryos were surgically removed from the uterine horn, decapitated, and brains removed using microdissection scissors. Meninges and surface vasculature were removed using hooked forceps, midbrain and hindbrain regions were dissected away, and hippocampi were carefully dissected using ne tipped jewelers forceps and microdissection scissors. Hippocampi were placed in ice-cold BSS, and then transferred to BSS containing 0.25% trypsin and placed in a 37 C incubator for 15 min. Single cell suspensions were obtained by triturating with a 1 mL pipette. Cells were counted and centrifuged at 45 RCF to obtain a concentrated pellet for use in culture. Neurons were plated in medium consisting of Eagles minimum essential medium (EMEM) with 10% horse serum. After 24 h, plating medium was replaced with Brewers medium consisting of Neurobasal medium (Invitrogen) supplemented with B27 (Stemcell Technologies, Vancouver, BC) and Glutamax (Invitrogen) as per the manufacturers instructions. Brewers medium was replenished by removal and replacement of 50% of the culture medium every 3 days. All cell cultures were maintained in a humidied incubator at 37 C, 5% CO2 . All animal procedures were approved by the Wadsworth Center Institutional Animal Care and Use Committee. 2.2. Alginate chemistry Alginic acid sodium salt was obtained from Sigma (Sigma, St Louis, MO). Sigma alginate is composed primarily of D-mannuronic acid residues containing minimal impurities from the manufacturing process (gure 3(B)). Alginate was dissolved in Ca2+ -free HBSS at concentrations ranging between 0.5% and 4.0%. To remove residual impurities, alginate was dissolved in HBHS at 1.0% w/v and dialyzed in a 3500 molecular weight cutoff (MWC) snakeskin dialysis membrane (Pierce, Rockford, IL) for 24 h against Milli-Q water (18.2 M cm2 ). Water was replaced four times, 1 L each time. The puried alginate was collected and placed in 100 mL glass vessels, frozen using a dry-ice/ethanol slurry and placed on a freeze dryer over night or until completely desiccated. The brous product was stored at 20 C for later use. Alginate functionalization was performed using aqueous carbodiimide chemistry (gure 1) (Rowley et al 1999). Alginate was dissolved at 1.0% w/v in 0.1 M 2-morpholinoethanesulfonic acid (MES) containing 0.3 M
3

O O Na O

n Sodium Alginate 1% wt/v

EDC Sulfo-NHS

N R2 N H OH O O O HO OH

R1 OH OH O

O NaO

n Stabilized Intermediate

Peptide

NH-Peptide OH O O O HO OH OH O

O O Na O

n Peptide-Alginate

Figure 1. Alginate can be functionalized by the covalent attachment of peptide sequences and proteins. Aqueous carbodiimide chemistry was performed to functionalize alginate. First, alginate was dissolved at 1.0% w/v in 0.3M NaCl MES buffer, pH 6.5. EDC was added to react with alginate by nucleophillic attack on the alginate carboxylate functional groups. Sulfo-NHS was added simultaneously to stabilize the reaction product in the form of an amine-reactive O-acylisourea ester. Peptide conjugation occurred during a 24 h long incubation.

NaCl, pH 6.5. Sulfo-N-hydroxysuccinimide (sulfo-NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) (both from Pierce) were added to the solution to produce a stable, amine reactive, O-acylisourea ester on the alginate carboxylate groups. N-terminal glycine amine acid sequences were synthesized by the Wadsworth Center Peptide Synthesis Core (Wadsworth Center, Albany, NY) and conjugated to the alginate during a 24 h long incubation at room temperature. The molar ratio of EDC to alginate was 0.005:1, sulfo-NHS to

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Ca2+ a2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+

Deliver cells in suspension to substrate.

Shape alginate/cell suspension with modified Millipore tissue culture plate insert.

Deliver buffered CaCl2 solution to upper chamber of insert.

Remove insert and add culture medium.

Figure 2. Alginate hydrogels were micromolded into 3D constructs of dened shapes and sizes. Cells were suspended in an alginate solution at the desired seeding density and applied to surfaces pretreated with poly-L-lysine. Modied Millipore tissue plate inserts were used to shape the constructs and facilitate alginate crosslinking using HEPES buffered CaCl2 . The entire process could be performed in less than 1 min and resulted in constructs that were on average 85 m thick.

EDC 0.5:1 and peptide to alginate 0.002:1. After conjugation, alginate was dialyzed again against Milli-Q water in a 3500 MWC dialysis cassette (Pierce) to remove the unconjugated peptide and residual EDC and sulfo-NHS. The solution was freeze-dried as before and the desiccated product stored at 20 C for cell culture use. Peptides and proteins used included GGGGRGDY (cell attachment), GGGGIKVAVY (neuron attachment and neurite outgrowth) and whole laminin (Sigma). 2.3. Scaffold fabrication Alginate scaffolds of dened shapes and dimensions were molded using modied Millipore tissue plate inserts (gure 2) (Millipore, Billerica, MD). First, alginate was dissolved in HBHS at a concentration of 1.0% wt/v. Glass, silicon or polystyrene surfaces were treated with PLL. These were used as stable support structures for mounting alginate tissue constructs. PLL coverslips were prepared by rinsing in 70% ethanol and Milli-Q water, and then drying under a stream of nitrogen gas. Substrates were vacuum plasma treated for 30 s and incubated for 1 h in a sterile PLL solution prepared in borate buffer. Substrates were rinsed three times, 1 min each in Milli-Q water and dried on porcelain drying racks. Once dry, alginate tissue constructs were fabricated by applying 20 L of liquid alginate to the surface of the substrates. A modied Millipore tissue plate insert was immediately placed on top of the alginate scaffold to dene the shape of the scaffold. Scaffold dimensions were determined by the viscosity of the alginate solution, volume of liquid alginate and weight of the Millipore insert. Spacer legs were removed from inserts prior to use. To crosslink the alginate, buffered CaCl2 (200 mM) was placed in the insert chamber. Ca2+ diffused
4

across the permeable membrane of the insert to crosslink the alginate. Support structures with attached alginate constructs were placed in well plates or Petri dishes, washed twice with culture medium and then cultured as described previously. 2.4. Characterization of physical and biochemical properties Scaffold dimensions were measured using the total z-depth obtained during confocal evaluation of cell-seeded scaffolds. Water contact angle was measured using a contact angle stage goniometer. Elastic modulus was measured by using a DMA Q800 dynamic mechanical analyzer in compression mode (TA Instruments, New Castle, DE). Stress/strain force measurements were used to determine Youngs modulus as a function of alginate concentration. Alginate constructs were imaged using a Leica SP5 confocal microscope (Leica, Wetzlar Germany) under differential interference contrast mode and a LEO 1550VP scanning electron microscope (Zeiss SMT, Peabody, MA) to evaluate the chain structure of the crosslinked alginate. Peptide attachment was determined using UVvis spectroscopy at 270 nm. UVvis absorption was determined for each functionalized alginate sample and compared to standard curves determined from peptide solutions of dened concentrations. Measurements were normalized to the unmodied alginate. 2.5. Live/dead viability assay Cell viability was determined using a live/dead dye exclusion assay (Invitrogen). Syto40 green uorescent nuclear stain (5 g mL1 ) was used to label cell nuclei of all cells. Sytox Orange nuclear stain (0.5 g mL1 ) was used to label cells with compromised plasma membranes. Sytox was applied for

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15 min and then thoroughly washed using growth medium three times for 1 min each time. Live samples were then imaged by confocal microscopy to determine the total number of cells and the fraction of dead cells. Leica automated dye separation software was used to separate overlapping dye spectra. Cells were then counted from the Syto and Sytox images to determine the fraction of viable cells per sample. Five images were collected per condition at 1, 3, 7 and 10 days after plating. A density of 1000 cell L1 was used to facilitate counting of individual cells. 2.6. Mitochondrial function assay Actively respiring mitochondria of entrapped cells were labeled using the xable uorescent probe Mitotracker CMTMRos (Invitrogen). Mitotracker (200 ng mL1 ) was applied in culture medium for 30 min and then xed with 4% buffered paraformaldehyde (PFA), pH 7.4, for 15 min. Samples were counterstained using Syto40 nuclear stain, mounted on slides and imaged by confocal microscopy. Cells were counted to determine the fraction of metabolically functional cells. A total of ve images were collected per condition at 1, 3, 7 and 14 days after plating. A density of 1000 cells L1 was used to facilitate counting of individual cells. 2.7. Variation of cell seeding conditions Cell seeding conditions could be easily varied by controlling the cell type, total cell number, and ratio of cell types prior to nal centrifugation and entrapment within alginate. Cell seeding densities were varied between 1000 and 100 000 cells L1 . Neural cell types, including astrocytes, microglia and neurons, could be readily cultured within alginate matrices and attachment was mediated through recognition of specic attachment ligands. IKVAValginate and lamininalginate were used for cultures of neurons. RGDalginate was used for glial cell types (LRM55, astrocytes and microglia). Alginate cell constructs were generally maintained in culture for 2 weeks before xation and immunohistochemical processing. Control over cell seeding conditions was conrmed using confocal microscopy of labeled cells. 2.8. Generation of co-culture constructs Co-culture systems were created in order to model astrocyte microglia interactions and neuralvascular interactions. Mixed glial co-cultures were produced by mixing astrocytes and microglia prior to entrapment. Cells of each cell type were pre-counted and appropriate numbers centrifuged to produce a pellet with the nal density and the cell-to-cell ratio required for construct formation. Glial co-culture constructs contained 100 000 cells L1 , a density approaching what has been observed in vivo (Bjornsson et al 2008). Bilayer cultures were produced in a stepwise fashion. First, glial cells were entrapped in the alginate matrix. After an equilibration period of 24 h, designed to accommodate small amounts of hydrogel swelling and permit conditioning of the culture medium by glia cells, endothelial cells were seeded onto the construct surface.
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Glial cells were seeded at 60 000 cells L1 to provide 1:1 contact with endothelial cells growing on the surface of the alginate constructs. Bovine aortic endothelial cells (BAEC) were plated at 2500 cells mm2 . Co-cultures were maintained for 2 weeks before immunohistochemical and microscopic evaluation of cell morphology and localization was performed. The Z Proler plug-in for ImageJ was used to compute uorescence intensity as a function of distance from the surface of the cultures. Z Proler was capable of analyzing image areas projected through the z dimension, beginning at the alginate construct surface (the rst optical section in the image z-series). Analysis was performed on individual channels and plotted together as a function of distance from the construct surface (gures 3 and 2(B)). Astrocytes were labeled with a monoclonal-anti-GFAP primary antibody and an Alexa594-goat-anti-mouse secondary antibody (red trace). Careful choice of uorophores and confocal imaging in combination with sequential scans minimized the amount of spectral overlap between detectors. LRM55 cells and BAEC were transfected separately using plasmid vectors containing DNA sequences encoding for uorescent proteins. Cells were transfected by nucleofection using an Amaxa Nucleofector II Device and cell type-specic kits designed to enhance transfection efciency and vector expression (Amaxa, Walkersville, MD). A rat astrocyte kit was used to transfect LRM55 astroglial cells with an enhanced green uorescent protein (eGFP) plasmid construct. An endothelial cell transfection kit was used to transfect BAEC cells with a mCherry plasmid construct. 2.9. Electrophysiology Cultures of neurons were constructed around microfabricated acute neural probes (NeuroNexus Technologies, Ann Arbor, MI). Neuronal cultures were established at a density of 20 000 cells L1 and remained in culture for 2 weeks before measurement. Recordings of spontaneous electrical activity were acquired using a differential ac amplier using a gain of 10, 0.35 kHz 40 dB/decade lter and a 20 kHz sampling frequency. Recordings were digitized using a data acquisition device and displayed using LabView software. Electrophysiological recordings were made in a HBHS recording solution (288 mosm kg1 ) at 37 C. 2.10. Labeling of functional synapses using FM 143 dye FM 143 labeling was performed using a protocol adapted from Betz (Gafeld and Betz 2006). Samples were mounted in live imaging chambers afxed to a temperature-controlled microscope stage. FM 143 uptake into synaptic vesicles occurred following exposure of neuron cultures to a high K+ (15 mM KCl) loading solution containing FM 143 dye (10 M) (Hynd et al 2007a, Jun et al 2008). Excess dye was removed by washing repeatedly with HBHS. The samples were imaged by multi-photon microscopy, stimulated once more with a high K+ loading solution to release loaded dye, and imaged again. Labeling of synaptic vesicles was conrmed

Biomed. Mater. 6 (2011) 015002

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(A)

(B)

14 mm 3 1 2 poly-Lys + +

Medium Ca2+-Alginate 85 m 6.4

O HO OH O O O HO OH

OH OH O O

- + - + - + - + - + - + - + - + - + - + - +

n n

SiO2

(1-4) D-Mannuronate Alginate

(C )
6

(D)

90 85 80 75 70

65

60

55

Young's Modulus (kPa)

50 45 40 35 30 25 20 15 10 5 0

as Gl

nate Algi
Peptide Modified Alginate

0 0 1 2 3 4

Water Contact Angle ()

Alginate Concentration (%wt/v)

(F ) (E)

Figure 3. Physical properties of alginate constructs. (A) Alginate constructs were 14 mm in diameter and 85 6.4 m in thickness (actual diagram not to scale). Support structures (glass, silicon or polystyrene) were pretreated with PLL to promote electrostatic interaction with the alginate carboxylate groups. (B) Sigma alginate consisted primarily of (14) D-mannuronic acid residues. Mannuronic acid chains form more elastic constructs than do guluronic acid chains (rigid constructs). (C) Alginate Youngs modulus was measured over a range of alginate concentrations. Youngs modulus increased with increasing concentrations of alginate. At 1.0% w/v alginate Youngs modulus was 480 12 Pa, within the range of values reported for brain tissue (500 Pa). (D) Water contact angle was measured to provide a relative index of the hydrophilic/phobic properties of the crosslinked alginate. The mean contact angle for PLL-treated glass was 32.5 0.76 . The mean contact angle for alginate was 14.1 1.6 . The carboxylate groups on the mannuronic acid chains are responsible for the hydrophilic properties of alginate constructs, and also mediate attachment to peptide sequences and electrostatic bonding to PLL-treated glass. (E) Scanning electron microscopy was used to observe the surface structure of critically point dried, gold sputter-coated alginate constructs. Filamentous structures were observed on the surface of alginate constructs as well as within surface cracks. Scale bar = 10 m. (F) Differential interference contrast confocal microscopy was used to verify the presence of such structures in 3D. Fillamentous structures appear gray and white contrasted against a dark background in confocal z-projections. Image displayed as a maximum intensity z-projection, scale bar = 100 m. All values are reported as mean standard error. Error bars represent SEM.

Biomed. Mater. 6 (2011) 015002

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by repetition of FM 143 dye loading and release. Solutions and cultures were maintained at 37 C over the course of the experiment. 2.11. Immunochemistry Antibodies raised against glial brillary acidic protein (GFAP), Iba-1(Wako, Richmond, VA), (III)-tubulin and connexin43 (GJA1), and the actin-binding toxin phalloidin were used to label cell structures. Unless otherwise noted, samples were xed in 4% PFA buffered in 25 mM PIPES, 10 mM HEPES, 2.5 mM CaCl2 , pH 7.4 for 15 min. It was necessary to maintain at least 2 mM Ca2+ in all wash solutions and media to prevent degradation of the alginate constructs. Following xation, the samples were permeabilized using 0.1% Triton X-100, washed once in HBHS, and blocked for 30 min in 5% bovine serum albumin (BSA), all at room temperature. Primary antibody and secondary antibody incubations occurred for 24 h at room temperature. The samples were washed three times for 5 min in HBHS between steps unless otherwise noted. After labeling, the samples were mounted for microscopy using spacer shims attached to glass slides with superglue. Mounting medium consisted of 95% glycerol, 5% HBHS and N-propyl gallate. Coverslips with alginate constructs attached were sealed and xed in place by applying nail polish around the edge of the coverslip. 2.12. Microscopy, analysis and statistics Confocal microscopy was used for imaging of both live and xed samples. Zeiss 510 Meta and Leica SP5 microscopes equipped with multi-photon and single-photon laser systems were used for multichannel imaging. Image analysis was performed using Fluorescence Association Rules for Imagebased Insight (FARSIGHT) software (RPI, Troy, NY) and ImageJ. Statistical presentation and Students t-test analysis were performed using Sigmaplot 10.0 and Systat 12 software (Systat Software, Chicago, IL).

3. Results
3.1. Characterization of alginate properties Alginate cell constructs were created with biochemical and physical properties favorable for long-term (up to 1 month) stability of the culture environment and viability of neural cells. Modied Millipore inserts were used to control the shape and size of scaffolds (gure 2). Alginate scaffolds were 14 mm in diameter, as determined by the diameter of the Millipore inserts, and were on average 85 m thick (gure 3(A)). While these scaffolds are orders of magnitude thinner than brain tissue, they provide an environment in which multiple layers of cells (5 or more layers) can interact in a 3D environment. By treating the supporting substrate with PLL, scaffolds remained xed in place during experiments and were not subject to breakage during medium replacement, immunohistochemical processing and microscopic analysis. The entire process of cell entrapment, including alginatecell mixing, crosslinking and washing could be accomplished in less than 1 min, minimizing
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the amount of time added to conventional 2D cell culture routines. Sodium alginate purchased from Sigma was used for all experiments. Sigma alginate is composed primarily of D-mannuronic acid residues, contributing to the elasticity of the alginate scaffolds (gures 3(B),(C)). The mechanical properties of alginate varied as a function of alginate concentration. The average Youngs modulus for the functionalized alginate ranged between 200 Pa for 0.5% wt/v alginate and 3500 Pa for 4.0% wt/v alginate. Alginate constructs were routinely used at 0.5% or 1.0% for cell entrapment experiments because Youngs modulus values corresponded well with values reported for neural tissue (Cheng et al 2008, Discher et al 2005). Water contact angle was used as a measure of the hydrophilic properties of alginate constructs. As expected, the water contact angle indicated that alginate (14.1 ) was more hydrophilic than for glass (35.5 ) (gure 3(D)). The hydrophilic properties of alginate are determined by the presence of carboxylate groups on the alginate sugar chains, which mediate attachment to peptide sequences as well as electrostatic bonding to PLL-treated glass (gures 1, 3(A),(B)). The attachment of cells to alginate constructs was determined by the presence of both specic attachment ligands and the structural properties of the cross-linked alginate chains. The macrostructure of alginate could be observed using scanning electron microcopy of the surface of the gold sputter-coated alginate. Cracks in the surface revealed the 3D lamentous macrostructure (gure 3(E)). Alginate structure could also be inferred from the differential interference contrast confocal images of alginate, where structural elements consistent with those observed by electron microscopy were observed to extend throughout the entire volume of the alginate constructs (gure 3(F)). UVvis spectroscopy provided verication of peptide attachment independent of cell behavior (gure 4). Cterminal tyrosine amino acids permitted UV detection. Peptide attachment was determined by extrapolating from standard curves measured using the known concentrations of unconjugated peptides. Per-batch alginate-bound peptide concentrations routinely ranged from several hundred nanograms for GGGGRGDY to nearly 2 g for laminin per mL of alginate (1.0% w/v). These values corresponded to ligand densities of 0.3 nmol cm3 (GGGGRGDY), 1.0 nmol cm3 (GGGGIKVAVY) and 1.2 pmol cm3 (laminin). The peptide functionalized alginate provided sites for cell attachment and process outgrowth. Ligand densities were roughly equivalent between peptidealginate batches. Laminin attachment may have been less efcient due to the absence of free amine groups on the native protein for carboxylate attachment. Ligand densities were well in excess of the density requirements reported for other cell types (Rowley and Mooney 2002). In contrast to other alginate systems, construct degradation, shrinking or cell migration out of the constructs were not observed. Although the PLL support structure provides a permissive environment, cell escape, growth and proliferation at the construct edges or at the glassconstruct

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(A)

3.2. Glial cells remain viable and retain metabolic function following cell entrapment LRM 55 astroglioma cells and primary astrocytes were used to assess the effects of cell entrapment on cell viability. Syto/Sytox staining demonstrated that more than 95% of LRM55 cells and primary astrocytes were viable after 7 days of culture in alginate constructs (gure 5(A)). After 14 days of culture, cell viability decreased slightly but not signicantly for both cell types, indicating that the cell entrapment process does not cause deleterious effects on cells, and that alginate is not cytotoxic to cells over time in culture. The slight decrease in cell viability is likely a result of cell death that occurs independently of culture conditions over the course of 2 weeks. Confocal images conrmed that both Syto and Sytox clearly labeled nuclear structures (gure 5(B)). Nonviable cells were sometimes observed to have fragmented nuclei, indicating that apoptosis was one mechanism by which infrequent cell death occurred. LRM 55 astroglioma and primary astrocytes remained metabolically active following cell entrapment (gure 5(C)). The percentage of metabolically active cells was found to be slightly lower at 1 day post entrapment than at 3 days, indicating that cells may undergo a recovery phase following dissociation and entrapment. There was a slight but signicant decrease in the fraction of metabolically active cells between 7 and 14 days in culture. This could reect a transition into senescence following attachment to the scaffold and adaptation to the 3D environment. Confocal images revealed that Mitotracker dye was localized to mitochondria indicating oxidative metabolism (gure 5(D)). Mitochondria were found in high numbers in cell somas as well as in the processes of primary astrocytes. Cell number increased over time in culture indicating that mitosis and cell proliferation were occurring in populations of both LRM55 cells and primary astrocytes (gure 5(E)). Signicant increases in cell number were observed at 3 and 14 days post entrapment for LRM55 cells and at 14 days for primary astrocytes. This behavior is consistent with the more rapid proliferation rate of LRM55 cells than primary astrocytes in monolayer cultures; however, both cell types showed smaller than expected increases in cell number over time as compared to cultures grown on 2D glass substrates. The average cell number for both cell types at 1 day indicated that cell seeding density was similar between samples (LRM55s: 12.7 cells/eld, primary astrocytes: 9.3 cells/eld). 3.3. Neural cells extend processes in 3D within alginate constructs Several different cell types entrapped within alginate scaffolds were observed to extend processes in 3D over the course of 14 days in culture. GFAP labeling of astrocyte cultures (red) demonstrated that cells formed clusters from which processes extended outwards for up to 100 m (gure 6(A)). In separate cultures, Iba-1 labeling (red) demonstrated that microglia were amoeboid in morphology, existing as either single cells or small clusters (gure 6(B)). Neurons were observed to extend processes several hundred m in length
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Figure 4. Alginate was biochemically functionalized through the covalent attachment of either peptide sequences or whole proteins. (A) UVvis spectroscopy was used to quantify peptide/protein attachment to alginate. Standard curves were generated for GGGGRGDY, GGGGIKVAVY and laminin protein at concentrations ranging from 10 ng to 100 g mL1 . The amount of peptide attached to alginate was determined by extrapolating from the standard curves. Per-batch alginate-bound peptide concentrations ranged from several hundred nanograms for GGGGRGDY to nearly 2 g for laminin per mL of alginate (1.0% w/v). These values corresponded to ligand densities of 0.3 nmol cm3 (GGGGRGDY), 1.0 nmol cm3 (GGGGIKVAVY) and 1.2 pmol cm3 (laminin). (B) UVvis spectrum for the unmodied alginate. (C) UVv is spectrum for the peptide-modied alginate. A peak at 270 nm was detected indicating the presence of the attached peptide.

interface were not usually observed, indicating that the constructs can be used for long-term experiments in which cell localization and construct integrity are important.

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Figure 5. Cells entrapped in alginate remained viable and functional. (A) Greater than 90% of glial cells remained viable after 14 days in culture. Sytox Orange exclusion demonstrated that cell membrane integrity was not affected by cell entrapped. No signicant loss in cell viability was observed between 1 and 14 days post-entrapment. (B) An example of live dead staining at 7 days in primary astrocyte cultures. Syto40 (green) labeled the nuclei of all cells within the alginate constructs. Sytox Orange staining was observed in cells with the compromised plasma membrane (red, karyorrhexic nuclei, as indicated by arrows). Scale bar = 100 m. (C) Mitotracker Orange CMTMros staining conrmed that entrapped cells remained metabolically active. Slight but signicant decreases in Mitotracker staining were observed between 7 and 14 days ( ). (D) An example of Mitotracker staining at 7 days in primary astrocyte cultures. Actively respiring mitochondria (red) were observed in both cell somas and processes. Cells were counterstained with Syto40 (green). Arrows indicate individual mitochondria in cell processes and near cell somas. Scale bar = 20 m. (E) Total cell number in alginate constructs increased over time in culture. Signicant increases were observed between 1 and 3 days ( ), and 3 and 14 days (#) for LRM55 cells. Signicant increases in the cell number were observed between 1 and 14 days for primary astrocytes ( ). Images are displayed as maximum intensity z-projections. Students t-test was used as a test of signicance between conditions. P-values of <0.01 were considered to be signicant. Bars denote SEM.

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Figure 6. Primary neural cell cultures exhibited process outgrowth in 3D. (A) Astrocytes were labeled for GFAP (red) and nuclei were counterstained with Hoechst 33342 (blue). Astrocytes displayed stellate morphologies and extensive process outgrowth over the course of 2 weeks in 3D culture. Astrocytes were observed as single cells and as small clusters of cells. (B) Microglia were labeled with Iba-1 (red) and counterstained with Hoechst 33342 (blue). Over the course of 2 weeks in 3D culture microglia exhibited amoeboid (activated) morphologies with outgrowth of short processes. Astrocytes and microglia were cultured within RGDfunctionalized alginate constructs. (C) Neurons cultured for 2 weeks in lamininfunctionalized alginate constructs were labeled with (III)-Tubulin (red), and counterstained with phalloidin (green) and Hoechst 33342 (blue). Neurons exhibited neurite outgrowth and formation of growth cones as demonstrated by (III)-Tubulin labeling and actin-phalloidin staining. (D) Neurons were not able to form stable attachments to RGDfunctionalized alginate. Virtually all neurons were observed to die within RGDalginate constructs by 1 month in culture. Neurons were observed to attach to and extend processes within both IKVAValginate (E) and lamininalginate (F). Process baring cells could be observed after 1 month in IKVAV- and lamininalginate constructs. All images are displayed as maximum intensity z-projections. Scale bar in (A) = 100 m. All other scale bars = 50 m.

((III)-tubulin labeling, red) (gure 6(C)). Phalloidin-labeled actin-rich growth cones (green) were observed in neurons, demonstrating that neurite outgrowth continued to occur even after 14 days in culture. Cell processes extended throughout the alginate scaffolds in both x, y and z dimensions. Single cells and small clusters of cell were distributed evenly in the z dimension.
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Glial cells demonstrated process outgrowth within RGDand laminin-modied alginate. RGDfunctionalized alginate was used to promote attachment and process outgrowth for astrocytes and microglia. In contrast, neurons did not attach to RGDfunctionalized alginate (gure 6(D)). Neurons were found to extend processes within IKVAV- and laminin functionalized alginate constructs (gures 6(E),(F)).

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Figure 7. Cell constructs could be created with densities as high as 100 000 cells L1 . Primary astrocytes were precounted and resuspended within alginate to produce constructs with dened cell densities. Constructs were stained with Hoechst 33342. (A) 25 000 cells L1 . (B) 50 000 cells L1 . (C) 75 000 cells L1 . (D) 100 000 cells L1 . All images are displayed as maximum intensity z-projections. Scale bars = 100 m.

3.4. Modulation of cell density within 3D alginate constructs Cell density could be varied within alginate matrices. Alginate cell scaffolds could be produced with cell densities as high as 100 000 cells L1 , a cell density approximating what has been observed in vivo. To demonstrate control over experimental cell density, scaffolds were produced with cell densities of 25 000, 50 000, 75 000, and 100 000 cells L1 (gures 7(A)(D)). Differences in cell density can be easily observed in the confocal images of cell nuclei collected from cultures of primary glial cells. At high cell densities, cells were more likely to form small clusters, possibly because of increased cellcell proximity during the cell entrapment process. By varying cell density it was possible to produce alginate scaffolds that could be used to study a variety of neural cell functions such as gap junction formation. Gap junction formation was assessed using alginate cell cultures with the densities described above. Glial cell gap junction interactions were assessed by immunohistochemical localization of the GJA1 antibody to connexin43 (gures 8(A)(D)). At 25 000 cells L1 little immunoreactivity was observed indicating relatively few gap
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junction complexes formed (gure 8(A)). Most GJA1 labeling was localized to the boundaries between cells growing in small clusters. As cell density increased, the incidence of gap junction formation between cells increased, because cell processes were more likely to encounter other cell processes and somas. At 75 000 and 100 000 cells L1 extensive networks of glial cells were observed to contain gap junction complexes (gures 8(C), (D)). 3.5. Functional activity of neurons within alginate constructs FM-143 labeling of neurons demonstrated the formation of functional synaptic elements within alginate constructs (gure 9(A)). Neurons showed release of FM143-loaded vesicles from presynaptic elements after stimulation with High K+ solution (gure 9(B)). Vesicular activity was conrmed by repeated loading and release, and repeated imaging of the cultures using multiphoton microscopy. To further verify neuronal function, primary hippocampal cultures were constructed within alginate matrices surrounding acute recording devices. Neurons were frequently observed in close

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Figure 8. Entrapped primary glial cells form more extensive gap junction networks at high cell densities. Glial cell constructs were labeled using the GJA-1 antibody to connexin43 (green) and counterstained with Hoechst 33342 (red). (A) and (B) At low cell densities (25 000 and 50 000 cells L1 ) relatively few gap junction plaques were observed to form between cells. (C) and (D) At higher cell densities (75 000 and 100 000 cells L1 ) increases in gap junction labeling were observed. Glial cell interconnectivity appeared to be more extensive at higher cell densities, as demonstrated by increases in the amount of GJA-1 labeling. All images are displayed as maximum intensity z-projections. Scale bars = 100 m.

proximity to recording electrodes as well as growing directly on the devices and device electrodes (gure 9(C)). After 14 days in culture, eld potentials could be recorded from cultures of primary hippocampal neurons surrounding the electrodes at a density of 20 000 cells L1 (gure 9(D)). Single units were also observed with amplitudes of 6080 V and duration of less than 10 ms, presumably originating from neurons entrapped in regions close to the electrodes. 3.6. Generation of co-culture systems for monitoring cellcell interactions For preliminary testing, cells requiring identical medium formulation were used to construct co-culture systems. These included cultures of microglia and astrocytes and LRM55 and BAEC, all requiring standard DMEM 10% FBS medium. It was possible to construct several types of co-culture systems. Glial cells were cultured within RGDfunctionalized alginate using both astrocytes and microglia. The second conguration tested was a bilayer co-culture system. Bilayer
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cultures were made by plating BAEC onto the surface of preformed alginate constructs containing LRM55 cells. It was necessary to allow at least 1 day between construct formation and cell seeding for the alginate construct to equilibrate. Glial cell/endothelial cell bilayer cultures required alginate constructs functionalized with laminin protein to promote growth of conuent endothelial cell monolayers. As described for single cell type 3D cultures, microglia displayed amoeboid morphology, although outgrowth of small processes was observed. Astrocytes exhibited stellate morphologies as observed in single cell type 3D cultures. It was visually apparent from inspecting mixed 3D cultures that cells were evenly distributed through the volume of the alginate construct although small clusters of cells were sometimes observed (gure 10(A)). Z-dimension prole plots revealed that signal was uniform through the z dimension of the cultures (gure 10(B)). Spectral plots demonstrated that there was no difference in localization or organization of glial cells in 3D cultures, suggesting that both cell types were randomly distributed.

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Figure 9. Neurons retain synaptic and spontaneous electrical activity within 3D alginate constructs. Neurons were entrapped in lamininfunctionalized alginate at a density of 20 000 cells L1 . (A)-(B) FM 143 dye labeling demonstrated that neurons retain synaptic activity in 3D cultures. (A) Following a second round of FM 143 labeling and three consecutive washes with HBHS, punctuate labeling could be observed in cultured neurons (arrows). (B) Synaptic labeling was extinguished by depolarization of neurons using a hyperkalemic solution. (C) For recording of local eld potentials, neurons were entrapped around NeuronNexus acute probes. Neurons (labeled with (III)-Tubulin (green)) were observed in close proximity to the probes (red signal). (D) Spontaneous electrical activity was recorded from populations of neurons surrounding acute neural probes. The blue trace represents a control electrode (no cells) from which no signals were recorded. The green trace shows small bursts of spontaneous activity with amplitudes of 6080 V and durations of less than 10 ms (enlarged in the red trace). Recorded units are indicated by arrows. All images are displayed as maximum intensity z-projections. Scale bars = 100 m.

In contrast, bilayer-type cultures were observed to support the growth of cells in spatially separated zones (gure 10(C)). By using cells transfected with vectors coding for spectrally resolvable uorophores it was possible to observe and quantify the localization of both populations of cells. Visual inspection of 3D images demonstrated that mCherry-transfected endothelial (red trace) were distributed in 2D on the surface of alginate constructs, while eGFPtransfected LRM55 (green trace) were only found in 3D within the construct. Cell migration of BAEC into constructs and LRM55 cells out of constructs was not observed. Based on spectral analysis using Z Proler, endothelial cells displayed a sharp peak in uorescence intensity 020 m from the surface of the construct, while the signal from glia cells displayed a broad peak beginning at 15 m and persisting throughout the
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remaining volume of the construct (gure 10(D)). A region of overlap between the two cell types could be observed in the spectral prole consistent with cells making physical contact with one another. These data demonstrate that 3D alginate hydrogel coculture systems can relieve many of the constraints imposed by conventional co-culture systems, permitting control over cell organization and providing more realistic growth environments for co-cultured cells. Co-culture parameters can be varied to permit the culture of several types of neural cells either as mixed populations within a 3D matrix or as bilayer cultures with one or more cell types growing in 3D within the alginate construct and a second cell type growing on the surface of the alginate construct (gures 10(E),(F)). Coculture systems were designed using parameters obtained from

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Figure 10. Analysis of 3D co-cultures. (A) Astrocytes (labeled for GFAP, red) and microglia (labeled for Iba-1, green) were randomly distributed in 3D within the alginate constructs. An image of a 1:1 astrocyte:microglia condition is displayed as a set of maximum intensity xy and xz projections, scale bar = 100 m. (B) Cell distribution was quantied using the Z Proler plugin for ImageJ. Confocal images were proled from the construct surface through the entire z volume of optical image sections. Fluorescence intensity was plotted as a function of distance from the surface of the construct. Fluorescence proles were similar between cell type-specic labels indicating that microglia (red trace) and astrocytes (green trace) were randomly distributed within mixed alginate co-cultures. Bars denote SEM, n = 3 image series. (C) LRM55 astroglia (transfected with eGFP, green) and BAEC (transfected with mCherry, red) were organized within discrete regions of the alginate constructs. An image of a transfected bilayer co-culture is displayed as a set of maximum intensity xy and xz projections, scale bar = 50 m. BAEC were observed to grow as a 2D monolayer on the construct surface. (D) Cell distribution was quantied in bilayer cultures using the Z Proler plugin for ImageJ. Confocal images were proled from the construct surface through the entire z volume of optical image sections. Fluorescence intensity was plotted as a function of distance from the surface of the construct. A large peak in uorescence intensity for BAEC was observed near the surface of the constructs (red trace) and LRM55 astroglia (green trace) were distributed randomly in 3D within the alginate co-cultures. Fluorescence intensity plots revealed that BAEC and LRM55 cells might interact near the alginate construct surface. Bars denote SEM, n = 3 image series. (E) and (F) Tested (E) and hypothesized (F) co-culture congurations for alginate constructs. Alginate constructs can be congured to produce at least four types of co-cultures in addition to the single cell type constructs described in section 2. (i) Single cell type 3D alginate constructs. (ii) 3D mixed co-cultures. Separate populations of astrocytes and microglia can be combined to produce 3D mixed cultures of neural cells at dened densities and cell-to-cell ratios. (iii) Bilayer co-cultures can be produced by fabricating single cell type 3D alginate constructs and plating a second cell type on the construct surface 24 h later. (iv) Patterned bilayer co-cultures could be produced by using soft photolithography to pattern biomolecules on the surface of alginate constructs before plating additional cells. (v) Dual layer 3D bilayer constructs could be produced by sequentially fabricating two layers of single cell type 3D alginate constructs.

quantitative analysis of brain tissue and were observed using immunohistochemical and microscopic methods to identity and describe the distribution of cells in both types of culture congurations.

4. Discussion
Neural cell viability and function were examined within 3D alginate matrices dened in terms of geometry and
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biochemical/physical characteristics. This culture system possesses several signicant features that improve upon previous methods for alginate construct formation. Firstly, the fabrication method described here is rapid and generates highly reproducible multicellular constructs that incorporate neurons, glia and endothelial cells. The fabrication process can be performed in most cell culture facility and does not require specialized equipment. The fabrication method

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also minimizes the exposure of cells to excess Ca2+ , which can be detrimental to cell survival and function (particularly for neural cells). Secondly, the entire process can be carried out at physiological pH, temperature and osmolarity. Additionally, the size scale of the constructs (85 m maximum distances from the bulk medium) permits the analysis of cell morphology, process outgrowth, cellcell interaction and function independent of mass transport limitations that would affect thicker (5001000 m thick) constructs. The size of these constructs offers a true 3D environment for growth in which cells can interact with the alginate matrix and other cells up to 810 cell layers away. Although our scaffolds are smaller than tissues or entire organs, and therefore are limited in terms of the scale in which 3D cell and biochemical processes may occur, they do however provide a valid model for analyses of local cellcell interactions in 3D environments. Using this novel fabrication platform a comprehensive analysis of material properties, cell viability and function were performed over time in culture. In contrast to previous reports our constructs were seeded at physiologically relevant concentrations of cells (up to 100 000 cells L1 ), i.e. similar to cell densities found in brain tissue. Thus we present an improved alginate culture system for the in vitro analysis of neural cells that can be varied in terms of material properties and the properties of cells (type, density and conguration). We have demonstrated that this system can be used to enhance in vitro culture models by providing 3D growth environments, and by permitting the formation of co-culture systems consisting only of cells and alginate matrix material. Alginate tissue constructs were produced that provided many of the biochemical and biomechanical properties required for recapitulating the brain microenvironment. Alginate could be readily functionalized by covalently tethering integrin receptor ligands such as RGD and IKVAV, as well as with the laminin protein, thus providing cell attachment
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sites on the alginate scaffold for both glial cells and neurons. Neural cells not only remained viable within the alginate constructs, but also displayed cell type-specic functions as demonstrated by both the formation of gap junction complexes between glial cells, and the synaptic and electrical activity of neurons. Alginate constructs provided a platform for the production of viable 3D cell cultures using a variety of neural cell types for which morphology, behavior and functions could be described. Alginate culture systems have been described previously for a variety of mammalian cells including myoblasts, osteoblasts, chondrocytes and ovarian follicles (Rowley and Mooney 2002, Kong et al 2003, West et al 2007, Lin et al 2009, Comisar et al 2007). It was demonstrated with each cell type that alginate was non-cytotoxic and non-immunogenic. However, the ionic concentrations required for cross-linking alginate constructs were of concern for culturing neural cells. Both glial cells and neurons are sensitive to uctuations in Ca2+ concentrations, and it has been demonstrated that mM nM1 changes in the extracellular/intracellular concentration of Ca2+ can lead to abnormal cell signaling in neural cells and excitotoxicity in neurons (Lucke et al 1995, Higley and Sabatini 2008, Agulhon et al 2008). It was necessary to minimize the concentration of Ca2+ used to cross-link the alginate constructs and the duration of time over which neural cells were exposed to the high extracellular concentrations of Ca2+ . Here we demonstrate that alginate can be crosslinked in less than 30 s with 200 mM CaCl2 and that excess Ca2+ could be removed with successive washes in HBHS (1.3 mM CaCl2 ). Therefore, it is unlikely that Ca2+ -dependent physiological processes were perturbed for sustained periods of time following construct formation. The potential for neural cell attachment to alginate constructs is critical for long term cell viability. Glial cells recognized and attached to RGD and laminin, while neurons were found to attach to IKVAV and laminin. This was

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surprising since other studies have demonstrated that RGD is capable of promoting neurite outgrowth in 2D cultures and since more than half of all known integrin receptor subtypes recognize the RGD sequence (Ruoslahti 1996, Shi and Ethell 2006). It is possible that neurons require higher ligand densities for 3D attachment. However, it is also possible that neurons either were not able to recognize the RGD epitopes because of the conformation in which they were presented on alginate chains or because the neurons did not express the integrin receptors required for mediating attachment to RGD. We suggest in future studies that scaffolds are designed in order to evaluate the effect that other integrin receptor subtypes have on neurons, thus providing a tool to study specic cellmatrix interaction in a 3D in vitro environment. The morphologies of neural cells, particularly astrocytes, were dramatically different from cells cultured on 2D substrates. Astrocytes cultured on 2D substrates displayed epithelial-like morphologies, whereas astrocytes cultured within 3D alginate constructs displayed stellate morphologies more similar to astrocytes growing within brain tissue. Cell morphology may also be inuenced by the mechanical properties of the alginate constructs and by the contactinhibited environment that provided more restrictive pathways for process outgrowth. The cell morphologies observed in 3D alginate constructs may appear similar to those observed in brain tissue, because in brain tissue neural cells are also surrounded by a complex environment consisting of many other cells and elastic matrix materials rich in carbohydrate chains (such as hyaluronan and proteoglycans). Glial cells were observed to proliferate within alginate constructs and extend processes; however, whole cell migration was rarely observed. While small clusters of cells were occasionally observed, their presence was particularly noted at higher densities. These clusters may have formed during construct formation, e.g. at the time of the nal cell suspension preparation, or by isolated occurrences of cell migration. Process growth may occur due to local porosity and architecture of alginate scaffolds, where openings between alginate chains may be large enough for cell processes to grow, yet not large enough to permit the movement of cell somas. Alternatively the alginate constructs may not have openings large enough to permit extension of cell processes, but rather process outgrowth could occur through localized remodeling of the tissue construct. Large-scale remodeling of alginate constructs was not observed. The mechanical properties of alginate constructs could be modied by varying alginate concentration. However, it is advantageous to vary mechanical properties independently of alginate concentration so as not to restrict the space through which cell processes or chemical signals might pass. It has been demonstrated that both the alginate composition and chain length have direct effects on the mechanical properties of alginate hydrogels (Rowley and Mooney 2002, Kong et al 2003, 2004). Thus, this culture system could be further modied through the optimization of such parameters, so as to more closely recapitulate the variety of biomechanical niches present in brain tissue. For instance, alginate was used at a concentration of 1.0% w/v which resulted in Youngs
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modulus values similar to cortical gray matter (alginate 480 Pa, gray matter500 Pa). However, white matter, vascular elements and scar tissue will impact measurements, and these elements have been shown to have larger Youngs modulus values (Cheng et al 2008). Most culture systems do not accurately recapitulate the cell types and densities that occur in brain tissue. The alginate construct system described here provides a platform by which multiple parameters can be modied for specic cell culture and tissue modeling applications. In vivo measurements of hippocampal and cortical cell densities have revealed that the average cell density in brain tissue exceeds 100 000 cells L1 (Bjornsson et al 2008). Alginate construct culture systems can approximate the cell densities observed in brain tissue, achieving a maximum cell density of 100 000 cells L1 . Such high cell densities can be achieved by mixing small amounts of concentrated alginate with loosely pelleted precounted cells. It is worthwhile to note that high density cultures required frequent medium replacement to replenish nutrients and remove waste products. LRM55 cultures required medium changes at least three times per day and primary cells required medium change at least once per day. In addition to providing near-physiological ranges of cell density this culture system also provides the ability to culture multiple cell types in mixed or spatially separated zones within the construct. The cell ratios used in the constructs were determined from in vivo FARSIGHT classication results and from other reported literature. For mixed 3D glial cocultures, appropriate cell ratios were determined based on the in vivo ratio of astrocytes to microglia, 3:1 to 4:1 depending upon brain region. Glial co-culture ratios were bracketed around 4:1 astrocytes to microglia, and therefore cultures of 8:1 and 1:1 astrocytes to microglia were tested. A number of reports have identied that intimate astrocyteendothelial cell communication is essential for the proper development of brain vasculature, where endothelial cells communicate with nearby neural cells with very minimum 1:1 associations. Therefore, a 1:1 ratio of astrocyte to endothelial cells was used as a benchmark for the establishment of bilayer co-cultures. Given the average process length for glial cells (30 m) it was possible to calculate and appropriate cell density for glial cells that would permit 1:1 contact with endothelial cells growing on the surface of constructs (Shain and Roysam, unpublished observation). The alginate construct systems described in this study were not so thick as to prohibit the efcient exchange of oxygen, nutrients and waste products between neural cells and the environment. Even at points furthest from the bulk medium, diffusion did not appear to limit the viability or sustained growth of neural cells. By using a micromolding technique it was possible to form 3D alginate constructs with volumes sufciently large to support neural cell outgrowth, yet thin enough to permit the efcient exchange of compounds between cells and culture medium. However, it could be possible to improve the long term viability and function of high density neural cell cultures and increase the size of culture systems through the incorporation of continuous medium perfusion or through the fabrication of microuidic channels

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within alginate constructs (Cullen et al 2007, Provin et al 2008). In conclusion, 3D cultures exhibited sustained viability and metabolic function, and in vivo like morphologies. This report provides a comprehensive evaluation of alginate constructs for use in neural cell culture. It was possible to study several neural cell functions in 3D including the formation of connexin43 gap junction formation, and neural communication through synaptic vesicle release and the measurement of local eld potentials. These results demonstrate that this culture system could be extended to study receptor-mediated processes such as cell responses to growth and trophic factors, as well as responses to other forms of environmental stimuli. Most importantly, alginate constructs can be adapted to support the co-culture of several cell types and to assess the interactions that occur in cell-tocell communication systems. Additional experiments have been undertaken to assess the utility of the alginate construct cell cultures for the development of co-culture systems capable of modeling the properties of the bloodbrain barrier and the brain response to neuroprosthetic devices.

Acknowledgments
The authors wish to thank the Wadsworth Center Advanced Light Microscopy, Biochemistry, Electron Microscopy and Peptide Synthesis core facilities for providing the equipment and technical training required for the analyses described in this report. The authors acknowledge the use of the Hudson Mesoscale Processing Facility at Cornell University, and in particular would like to thank Dr Yuanming Zhang for his assistance in performing measurements of alginate mechanical properties. We thank Roger Tsien for the mCherry vector. This work was supported in part by the Nanobiotechnology Center (NBTC) an STC program of the NSF under agreement number ECS-9876771, and by the NIBIB-supported Center for Neural Communication Technology (CNCT) P41-002030.

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