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

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IOP PUBLISHING BIOMEDICAL MATERIALS

Biomed. Mater. 6 (2011) 015002 (18pp) doi:10.1088/1748-6041/6/1/015002

Fabrication and optimization of alginatehydrogel constructs for use in 3D neuralcell culture

J P Frampton1, M R Hynd1,2, M L Shuler3 and W Shain1,2

1 Department of Biomedical Sciences, School of Public Health, State University of New York at Albany,

Albany, NY 12210, USA2 NYS Department of Health, Biggs Laboratory, Wadsworth Center, Albany, NY 12210, USA3 Department of Biomedical Engineering, 270 Olin Hall, Cornell University, Ithaca, NY 14850, USA

E-mail: [email protected]

Received 30 August 2010Accepted 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 figures in this article are in colour only in the electronic version)

1. Introduction

Hydrogels are crosslinked polymers that, due to their

hydrophilic properties, retainhigh water content aftergelation.

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 theyexhibit lowimmunogenicityand low

cytotoxicity, and permit the exchange of gases and nutrients

between cells and the environment. It is also possible tomodify the mechanical and biochemical properties of many

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. Oncethe hydrogel has been formed, its porous nature allows

1748-6041/11/015002+18$33.00 1 2011 IOP Publishing Ltd Printed in the UK
http://dx.doi.org/10.1088/1748-6041/6/1/015002mailto:[email protected]://stacks.iop.org/BMM/6/015002http://stacks.iop.org/BMM/6/015002mailto:[email protected]://dx.doi.org/10.1088/1748-6041/6/1/015002


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Biomed. Mater. 6 (2011) 015002 J P Frampton et al

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 aroundsuspended cells, thus immobilizing the cells in 3D. Hydrogel-

based cell entrapment systems have been used with a variety

of cell types andhave been demonstrated to have no significant

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 fibronectin (RGD) and laminin (IKVAV and

YIGSR) have been incorporated into some types of hydrogel

matrices, providing attachment ligands that can be recognized

by specific cell types (Dhoot et al 2004, Rowley et al 1999,

Comisar et al 2007).Alginatehasbeenusefulfor theentrapmentof mammalian

cells because it satisfies 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 Ca

2+

and Ba2+, but not Mg2+ (Morch et al 2006). Crosslinking can

be reversed by exposure to the high concentrations of Na+ orto

Ca2+ chelating agents. By variation of alginate concentration,

composition, porosity andcell attachment factors, it is possible

to tailor alginate matrices for specific cell culture applications

(Rowley and Mooney 2002, Comisar et al 2006, Eiselt et al

2000).

Alginatematrices have beenusedextensivelyas substrates

for both 2D and 3D cell culture (Comisar et al 2007, Kong etal

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(Boisseauetal 1993, Li etal 2006b, Novikova etal

2006). This report describes an alginate construct system thathas been characterized in terms of its physical and biological

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% confluence

in T75 flasks prior to use. LRM55 cells were prepared

for 3D culture by washing monolayers of cells with HEPES

buffered Hanks saline (HBHS), followed by treatment withTrypLE (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

thenumbers required forentrapment in eitheralginate matrices

or cultured on the surface of poly-L-lysine (PLL)-treated glass

coverslips as a positive control. LRM55 cells were cultured

in Dulbeccos modified 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 postnatalday three Sprague-Dawleyratcortices(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 and50g 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 flasks or 150 mm2 Petri dishes. Glial cells were cultured

in DMEM containing 10% FBS. Medium was completely

replaced thedayafter plating andevery3 days thereafter. Cells

were passaged at 7090% confluence, not more than three

times. Pure astrocytecultures 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, thawingandreseeding,glialculturescontained

no microglia and were thus considered pure astrocyte cultures.

Microglia were collected from mixed glial culturesprior to freezing and subcultured in 150 mm2 Petri dishes.

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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-specific marker ionized calcium-

binding adaptor molecule-1(Iba-1). Microglia cultures weregrown 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 fine 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 humidified

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 (figure 3(B)). Alginatewas 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 purified 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 fibrous product was stored at 20 C for

later use. Alginate functionalization was performed using

aqueous carbodiimide chemistry (figure 1) (Rowley et al

1999). Alginate was dissolved at 1.0% w/v in 0.1 M2-morpholinoethanesulfonic acid (MES) containing 0.3 M

O O

OH

OO

OOH

OHO

OH

OOH

n

N

NH

R1

Na

R2

Stabilized Intermediate

O O

OH

OO

O OH

OHO

OH

OOH

n

Na

Sodium Alginate 1% wt/v

EDC

Sulfo-NHS

O O

OH

OO

OOH

OHO

NH-Peptide

OOH

n

Na

Peptide-Alginate

Peptide

Figure 1. Alginate can be functionalized by the covalent attachmentof peptide sequences and proteins. Aqueous carbodiimide chemistrywas performed to functionalize alginate. First, alginate wasdissolved 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 alginatecarboxylate functional groups. Sulfo-NHS was addedsimultaneously to stabilize the reaction product in the form of anamine-reactive O-acylisourea ester. Peptide conjugation occurredduring 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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Deliver cells

in suspension

to substrate.

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca2+

Ca+

a+ Ca

2+

Ca2++

Ca2+

Ca2+

Ca2+

Shape alginate/cell suspension

with modified Millipore tissue

culture plate insert.

Deliver buffered

CaCl2solution to

upper chamber

of insert.

Remove insert

and add culture

medium.

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

molded using modified Millipore tissue plate inserts (figure 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 modified Millipore tissue plate insert was immediately

placed on top of the alginate scaffold to define the shape

of the scaffold. Scaffold dimensions were determined by the

viscosityof thealginate solution, volume of liquidalginate 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

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 SP5confocalmicroscope (Leica, Wetzlar

Germany) under differential interference contrast mode and

a LEO 1550VP scanning electron microscope (Zeiss SMT,

Peabody, MA)to evaluatethe chain structureof thecrosslinkedalginate. 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 defined

concentrations. Measurements were normalized to the

unmodified alginate.

2.5. Live/dead viability assay

Cell viability was determined using a live/dead dye exclusion

assay (Invitrogen). Syto40 green fluorescent 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 cellswith 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 fixable fluorescent probe Mitotracker

CMTMRos (Invitrogen). Mitotracker (200 ng mL1) was

applied in culture medium for 30 min and then fixed 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. Cellswere counted to determine the fraction of metabolically

functional cells. A total of five 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 final centrifugation and entrapment within alginate. Cell

seeding densities were varied between 1000 and 100000 cells

L1. Neural cell types, including astrocytes, microglia and

neurons, could be readily cultured within alginate matrices

and attachment was mediated through recognition of specific

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 weeksbeforefixation and immunohistochemicalprocessing.

Control over cell seeding conditions was confirmed 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

andmicroglia prior to entrapment. Cells of each cell type were

pre-counted and appropriate numbers centrifuged to produce

a pellet with the final density and the cell-to-cell ratio required

for construct formation. Glial co-culture constructs contained

100000 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 andpermit conditioningof theculture mediumby glia

cells, endothelial cells were seeded onto the construct surface.

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 Profiler plug-in for ImageJ was used to compute

fluorescence intensityas a functionof distance from thesurface

of the cultures. Z Profiler was capable of analyzing image

areas projected through the z dimension, beginning at the

alginate construct surface (the first 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 (figures 3 and 2(B)).

Astrocytes were labeled with a monoclonal-anti-GFAP

primary antibodyandan Alexa594-goat-anti-mousesecondary

antibody (red trace). Careful choice of fluorophores and

confocal imaging in combination with sequential scansminimized the amount of spectral overlap between detectors.

LRM55 cells and BAEC were transfected separately using

plasmid vectors containing DNA sequences encoding for

fluorescent proteins. Cells were transfected by nucleofection

using an Amaxa Nucleofector II Device and cell type-specific

kits designed to enhance transfection efficiency and vector

expression (Amaxa, Walkersville, MD). A rat astrocyte kit

was used to transfect LRM55 astroglial cells with an enhanced

green fluorescent 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 amplifier using

a gain of 10, 0.35 kHz 40 dB/decade filter 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 (Gaffield and Betz 2006). Samples were mounted

in live imaging chambers affixed 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

removedbywashingrepeatedly with HBHS.Thesampleswere

imaged by multi-photon microscopy, stimulated once more

with a high K+ loading solution to release loaded dye, andimaged again. Labeling of synaptic vesicles was confirmed

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SiO2

Ca2+-Alginate

poly-Lys +++ + + + +++++ ++ -- ----- -----

Medium

12

385 m

6.4

14 mm

Alginate Concentration (%wt/v)

0 1 2 3 4

Young'sModulus(kPa)

0

1

2

3

4

5

6

Peptide Modified Alginate

0

5

10

15

20

25

30

35

40

4550

5560

6570

75808590

O O

OH

HO

O

OOH

OHO

OH

OOH

O

n

n

(1-4) D-MannuronateAlginate

Water Contact Angle ()

G

lass

Alginat

e

(B)(A)

(D)(C)

(E)

(F)

Figure 3. Physical properties of alginate constructs. (A) Alginate constructs were 14 mm in diameter and 85 6.4 m in thickness (actualdiagram not to scale). Support structures (glass, silicon or polystyrene) were pretreated with PLL to promote electrostatic interaction withthe alginate carboxylate groups. (B) Sigma alginate consisted primarily of(14) D-mannuronic acid residues. Mannuronic acid chainsform more elastic constructs than do guluronic acid chains (rigid constructs). (C) Alginate Youngs modulus was measured over a range ofalginate concentrations. Youngs modulus increased with increasing concentrations of alginate. At 1.0% w/v alginate Youngs modulus was480 12 Pa, within the range of values reported for brain tissue (500 Pa). (D) Water contact angle was measured to provide a relativeindex of the hydrophilic/phobic properties of the crosslinked alginate. The mean contact angle for PLL-treated glass was 32.5 0.76. Themean contact angle for alginate was 14.1 1.6. The carboxylate groups on the mannuronic acid chains are responsible for the hydrophilicproperties 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 intensityz-projection, scale bar = 100 m. All values are reported as mean standard error. Error bars represent SEM.

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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

Antibodiesraised againstglialfibrillaryacidic 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

fixed in 4% PFA buffered in 25 mM PIPES, 10 mM HEPES,

2.5 mMCaCl2, pH7.4 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 fixation, 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 sampleswere washed three timesfor5 minin

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

fixed 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

fixed 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 Image-

based 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. Modified Milliporeinsertswere used to control theshape

andsize of scaffolds (figure2). Alginatescaffoldswere 14 mm

in diameter, as determined by the diameter of the Millipore

inserts, and were on average 85m thick (figure 3(A)). While

these scaffolds are orders of magnitude thinner than brain

tissue, they provide an environment in which multiple layers

of cells (5or more layers) can interact in a 3Denvironment. By

treating the supporting substrate with PLL, scaffolds remained

fixed in place during experiments and were not subject to

breakage during medium replacement, immunohistochemical

processing andmicroscopicanalysis. Theentire processof cell

entrapment, including alginatecell mixing, crosslinking andwashing could be accomplished in less than 1 min, minimizing

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 (figures 3(B),(C)). The mechanical

properties of alginate varied as a function of alginateconcentration. 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) (figure 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

(figures 1, 3(A),(B)).

The attachment of cells to alginate constructs was

determined by thepresence of both specific 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

filamentous macrostructure (figure 3(E)). Alginate structure

could also be inferred from the differential interference

contrastconfocal images of alginate, wherestructuralelements

consistent with those observed by electron microscopy wereobserved to extend throughout theentirevolume of thealginate

constructs (figure 3(F)).

UVvis spectroscopy provided verification of peptide

attachment independent of cell behavior (figure 4). C-

terminal 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 cm

3

(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 efficient 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 andproliferation at the construct edges or at the glassconstruct

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Peptide Concentration (g/mL)

Absorbtionat270nm(A.U.)

4.0

-0.8

Absorbtion

0.0570.091 Wavelength

4.0

-0.8

Absorbtion

0.0570.091 Wavelength

(A)

(B)

(C)

Figure 4. Alginate was biochemically functionalized through thecovalent attachment of either peptide sequences or whole proteins.(A) UVvis spectroscopy was used to quantify peptide/proteinattachment to alginate. Standard curves were generated forGGGGRGDY, GGGGIKVAVY and laminin protein atconcentrations ranging from 10 ng to 100 g mL1. The amount ofpeptide attached to alginate was determined by extrapolating fromthe standard curves. Per-batch alginate-bound peptideconcentrations ranged from several hundred nanograms forGGGGRGDY to nearly 2 g for laminin per mL of alginate(1.0% w/v). These values corresponded to ligand densities of0.3 nmol cm3 (GGGGRGDY), 1.0 nmol cm3 (GGGGIKVAVY)and 1.2 pmol cm3 (laminin). (B) UVvis spectrum for theunmodified alginate. (C) UVv is spectrum for the peptide-modifiedalginate. A peak at 270 nm was detected indicating the presence ofthe attached peptide.

interface were not usually observed, indicating that the

constructs can be used for long-term experiments in whichcell localization and construct integrity are important.

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 (figure 5(A)). After 14 daysof culture, cell viability decreased slightly but not significantly

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 thecourseof 2 weeks.

Confocal images confirmed that both Syto and Sytox clearly

labeled nuclear structures (figure 5(B)). Nonviable cells were

sometimesobserved to have fragmented nuclei, indicating that

apoptosis was one mechanism by which infrequent cell death

occurred.

LRM 55 astroglioma and primary astrocytes remainedmetabolically active following cell entrapment (figure 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

dissociationand entrapment. There wasa slight but significant

decrease in the fraction of metabolically active cells between

7 and 14 days in culture. This could reflect 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 (figure 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 (figure 5(E)).

Significant 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 LRM55cells 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/field, primary astrocytes: 9.3 cells/field).

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 (figure 6(A)).

In separate cultures, Iba-1 labeling (red) demonstrated that

microglia were amoeboid in morphology, existing as either

single cells or small clusters (figure 6(B)). Neurons wereobserved to extend processes several hundred m in length

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Days in Culture

%Respiring

Cells

0

10

20

30

40

50

60

70

80

90

100

LRM 55 Cortical Astrocyte

1 3 7 14 1 3 7 14

Days in Culture

%Viability

0

10

20

30

40

50

60

70

80

90

100


1 3 7 14 1 3 7 14

Cells/Field

0

20

40

60

80

1 3 14 1 3 14

Days in Culture


(A)

(C)

(E)

(D)

(B)

Figure 5. Cells entrapped in alginate remained viable and functional. (A) Greater than 90% of glial cells remained viable after 14 days inculture. Sytox Orange exclusion demonstrated that cell membrane integrity was not affected by cell entrapped. No significant loss in cellviability 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 thecompromised plasma membrane (red, karyorrhexic nuclei, as indicated by arrows). Scale bar = 100 m. (C) Mitotracker OrangeCMTMros staining confirmed that entrapped cells remained metabolically active. Slight but significant decreases in Mitotracker stainingwere observed between 7 and 14 days (). (D) An example of Mitotracker staining at 7 days in primary astrocyte cultures. Actively respiringmitochondria (red) were observed in both cell somas and processes. Cells were counterstained with Syto40 (green). Arrows indicateindividual mitochondria in cell processes and near cell somas. Scale bar = 20 m. (E) Total cell number in alginate constructs increasedover time in culture. Significant increases were observed between 1 and 3 days (), and 3 and 14 days (#) for LRM55 cells. Significantincreases in the cell number were observed between 1 and 14 days for primary astrocytes (). Images are displayed as maximum intensityz-projections. Students t-test was used as a test of significance between conditions. P-values of


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(A)

(C)

(F)(E)(D)

(B)

Figure 6. Primary neural cell cultures exhibited process outgrowth in 3D. (A) Astrocytes were labeled for GFAP (red) and nuclei werecounterstained with Hoechst 33342 (blue). Astrocytes displayed stellate morphologies and extensive process outgrowth over the course of2 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 counterstainedwith 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 RGDfunctionalizedalginate. Virtually all neurons were observed to die within RGDalginate constructs by 1 month in culture. Neurons were observed to attachto and extend processes within both IKVAValginate (E) and lamininalginate (F). Process baring cells could be observed after 1 month inIKVAV- and lamininalginate constructs. All images are displayed as maximum intensity z-projections. Scale bar in (A) = 100 m. Allother scale bars = 50 m.

((III)-tubulin labeling, red) (figure 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.

Glial cells demonstrated process outgrowth within RGD-

and laminin-modified alginate. RGDfunctionalized alginate

was used to promote attachment and process outgrowth

for astrocytes and microglia. In contrast, neurons did not

attach to RGDfunctionalized alginate (figure 6(D)). Neurons

were found to extend processes within IKVAV- and lamininfunctionalized alginate constructs (figures 6(E),(F)).

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(A ) (B)

(C) (D)

Figure 7. Cell constructs could be created with densities as high as 100 000 cells L1. Primary astrocytes were precounted andresuspended within alginate to produce constructs with defined cell densities. Constructs were stained with Hoechst 33342. (A) 25 000 cellsL1. (B) 50 000 cells L1. (C) 75 000 cells L1. (D) 100000 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 withinalginate matrices. Alginatecell scaffolds could be produced with cell densities as highas 100 000 cells L1, a cell density approximating what

has been observed in vivo. To demonstrate control overexperimental cell density, scaffolds were produced with cell

densities of 25 000, 50 000, 75 000, and 100 000 cells L1

(figures 7(A)(D)). Differences in cell density can be easilyobserved in the confocal images of cell nuclei collected fromcultures of primary glial cells. At high cell densities, cells

were more likely to form small clusters, possibly becauseof increased cellcell proximity during the cell entrapmentprocess. By varying cell density it was possible to produce

alginate scaffolds that could be used to study a variety of neuralcell 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 byimmunohistochemical localization of the GJA1 antibody to

connexin43 (figures 8(A)(D)). At 25 000 cells L1 littleimmunoreactivity was observed indicating relatively few gap

junction complexes formed (figure 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 pro-

cesses were more likely to encounter other cell processes and

somas. At 75000 and 100000 cells L1 extensive networks

of glial cells were observed to contain gap junction complexes

(figures 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

(figure 9(A)). Neurons showed release of FM143-loaded

vesicles from presynaptic elements after stimulation with

High K+ solution (figure 9(B)). Vesicular activity was

confirmed by repeated loading and release, and repeated

imaging of the cultures using multiphoton microscopy. To

further verify neuronalfunction, primary hippocampal cultures

were constructed within alginate matrices surrounding acuterecording devices. Neurons were frequently observed in close

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(A) (B)

(C) (D)

Figure 8. Entrapped primary glial cells form more extensive gap junction networks at high cell densities. Glial cell constructs were labeledusing the GJA-1 antibody to connexin43 (green) and counterstained with Hoechst 33342 (red). (A) and (B) At low cell densities (25 000 and50 000 cellsL1) relatively few gap junction plaques were observed to form between cells. (C) and (D) At higher cell densities (75 000and 100000 cells L1) increases in gap junction labeling were observed. Glial cell interconnectivity appeared to be more extensive athigher cell densities, as demonstrated by increases in the amount of GJA-1 labeling. All images are displayed as maximum intensityz-projections. Scale bars = 100 m.

proximity to recording electrodes as well as growing directly

on the devices and device electrodes (figure 9(C)). After

14 days in culture, field potentials could be recorded from

cultures of primary hippocampal neurons surrounding the

electrodes at a density of 20 000 cells L1 (figure 9(D)).

Single units were also observed with amplitudes of 6080 Vand 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 cellswere cultured within RGDfunctionalized

alginate using both astrocytes and microglia. The secondconfiguration tested was a bilayer co-culture system. Bilayer

cultures were made by plating BAEC onto the surface ofpreformed 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 promotegrowth of confluent endothelial cell monolayers.

As described for single cell type 3D cultures, microglia

displayed amoeboid morphology, although outgrowth of

small processes was observed. Astrocytes exhibited stellatemorphologies as observed in single cell type 3D cultures. It

was visually apparent from inspecting mixed 3D cultures that

cellswere evenly distributedthrough thevolume of thealginate

construct although small clusters of cells were sometimes

observed (figure 10(A)). Z-dimension profile plots revealed

that signalwas uniformthrough thez dimensionof thecultures

(figure 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 randomlydistributed.

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0.1

-0.1

0.0

5.00. 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 4. 0 4. 2 4. 4 4. 6 4. 8

0.1

-0.1

0.0

1.0010 .8 00 0 .8 10 0 .8 20 0 .8 30 0 .8 40 0 .8 50 0. 86 0 0 .8 70 0 .8 80 0 .8 90 0 .9 00 0. 91 0 0 .9 20 0 .9 30 0 .9 40 0 .9 50 0 .9 60 0 .9 70 0 .9 80 0 .9 90

0.1

-0.1

0.0

5.00. 0 0. 2 0. 4 0. 6 0. 8 1. 0 1. 2 1. 4 1. 6 1. 8 2. 0 2. 2 2. 4 2. 6 2. 8 3. 0 3. 2 3. 4 3. 6 3. 8 4. 0 4. 2 4. 4 4. 6 4. 8

(D)

Amplitude(V)

Time (msec)

(A) (B)

(C)

Figure 9. Neurons retain synaptic and spontaneous electrical activity within 3D alginate constructs. Neurons were entrapped inlamininfunctionalized alginate at a density of 20 000 cells L1. (A)-(B) FM 143 dye labeling demonstrated that neurons retain synapticactivity in 3D cultures. (A) Following a second round of FM 143 labeling and three consecutive washes with HBHS, punctuate labelingcould be observed in cultured neurons (arrows). (B) Synaptic labeling was extinguished by depolarization of neurons using a hyperkalemicsolution. (C) For recording of local field 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 frompopulations of neurons surrounding acute neural probes. The blue trace represents a control electrode (no cells) from which no signals wererecorded. 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. Scalebars = 100 m.

In contrast, bilayer-type cultures were observed tosupport the growth of cells in spatially separated zones(figure 10(C)). By using cells transfected with vectors coding

for spectrally resolvable fluorophores it was possible toobserve and quantify the localization of both populationsof cells. Visual inspection of 3D images demonstrated thatmCherry-transfected endothelial (red trace) were distributedin 2D on the surface of alginate constructs, while eGFP-

transfected LRM55(greentrace)were only found in 3D withinthe construct. Cell migration of BAEC into constructs andLRM55 cells out of constructs was not observed. Based onspectral analysis using Z Profiler, endothelial cells displayed asharp peak in fluorescence intensity 020 m from the surface

of the construct, while the signal from glia cells displayed abroad peak beginning at 15 m and persisting throughout the

remaining volume of the construct (figure 10(D)). A region ofoverlap between the two cell types could be observed in thespectral profile consistent with cells making physical contact

with one another.These data demonstrate that 3D alginate hydrogel co-

culture systems can relieve many of the constraints imposedby conventional co-culture systems, permitting controlover cell organization and providing more realistic growth

environments for co-cultured cells. Co-culture parameterscan be varied to permit the culture of several types of neuralcells either as mixed populations within a 3D matrix or asbilayer cultures with one or more cell types growing in 3Dwithin the alginate construct and a second cell type growing

on thesurface of thealginate construct (figures 10(E),(F)). Co-culture systems were designed using parameters obtained from

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Distance from surface (m)

0 10 20 30 40

Iba-1Alexa-488FluorescenceIntensity(A.U.)

8

10

12

14

GFAPAlexa-594Fluores

cenceIntensity(A.U.)

8

10

12

14

Distance from Surface (m)

0 10 20 30 40 50 60

mC

herryFluorescenceIntensity(A.U.)

4

5

6

7

8

9

eGFPFluorescenceIntensity(A.U

.)

4

6

8

10

12

14

16

18

A (B)

(D)C

(A)

(C)

Figure 10. Analysis of 3D co-cultures. (A) Astrocytes (labeled for GFAP, red) and microglia (labeled for Iba-1, green) were randomlydistributed in 3D within the alginate constructs. An image of a 1:1 astrocyte:microglia condition is displayed as a set of maximum intensityxy and xz projections, scale bar = 100 m. (B) Cell distribution was quantified using the Z Profiler plugin for ImageJ. Confocal imageswere profiled from the construct surface through the entire z volume of optical image sections. Fluorescence intensity was plotted as afunction of distance from the surface of the construct. Fluorescence profiles were similar between cell type-specific labels indicating thatmicroglia (red trace) and astrocytes (green trace) were randomly distributed within mixed alginate co-cultures. Bars denote SEM, n = 3image series. (C) LRM55 astroglia (transfected with eGFP, green) and BAEC (transfected with mCherry, red) were organized withindiscrete regions of the alginate constructs. An image of a transfected bilayer co-culture is displayed as a set of maximum intensity xy andxz projections, scale bar = 50 m. BAEC were observed to grow as a 2D monolayer on the construct surface. (D) Cell distribution wasquantified in bilayer cultures using the Z Profiler plugin for ImageJ. Confocal images were profiled from the construct surface through theentire z volume of optical image sections. Fluorescence intensity was plotted as a function of distance from the surface of the construct. Alarge peak in fluorescence 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 mightinteract near the alginate construct surface. Bars denote SEM, n = 3 image series. (E) and (F) Tested (E) and hypothesized (F) co-cultureconfigurations for alginate constructs. Alginate constructs can be configured 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 populationsof astrocytes and microglia can be combined to produce 3D mixed cultures of neural cells at defined 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 constructsurface 24 h later. (iv) Patterned bilayer co-cultures could be produced by using soft photolithography to pattern biomolecules on the surfaceof alginate constructs before plating additional cells. (v) Dual layer 3D bilayer constructs could be produced by sequentially fabricating twolayers 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

configurations.

4. Discussion

Neural cell viability and function were examined within3D alginate matrices defined in terms of geometry and

biochemical/physical characteristics. This culture system

possesses several significant 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 doesnot require specialized equipment. The fabrication method

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(E)

(F)

iiiiii

vvi

Figure 10. (Continued.)

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 3Denvironment 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

100000 cells L

1

), i.e. similar to cell densities found inbrain 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 configuration). 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 RGDandIKVAV, aswell as with the laminin protein, thus providingcell attachment

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-specific functions as

demonstratedby 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 andfunctionscould

be described.

Alginate culture systems have been described previouslyfor 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 fluctuations 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 andSabatini 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 cross-

linked 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 epitopesbecause 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 futurestudies that scaffoldsare designed in order

to evaluate the effect that other integrin receptor subtypes have

on neurons, thus providing a tool to study specific 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 influenced by the mechanical

properties of the alginate constructs and by the contact-

inhibited 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 cellswere 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 final cell

suspension preparation, or by isolated occurrences of cell

migration. Process growth may occur due to local porosity

and architecture of alginate scaffolds, whereopeningsbetween

alginate chainsmaybe 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 alginateconstructs was not observed.

The mechanical properties of alginate constructs could be

modified 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

modified 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 usedat a concentration of 1.0% w/v which resulted in Youngs

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 alginateconstruct system described here provides a platform by which

multiple parameters can be modified for specific cell culture

and tissue modeling applications. In vivo measurements of

hippocampal and cortical cell densities have revealed that the

average cell density in brain tissueexceeds 100000cellsL1

(Bjornsson et al 2008). Alginate construct culture systems

can approximate the cell densities observed in brain tissue,

achieving a maximum cell density of 100000cellsL1. 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

andremovewaste products. LRM55 cultures requiredmedium

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 spatiallyseparatedzones within

the construct. The cell ratios used in the constructs were

determined from in vivo FARSIGHT classification results

and from other reported literature. For mixed 3D glial co-

cultures, 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 of8:1 and 1:1 astrocytes to microglia were tested. A number

of reports have identified 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 efficient 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 sufficiently large to support neural cell outgrowth,

yet thin enough to permit the efficient exchange of compounds

between cells and culture medium. However, it could be

possible to improve the long term viability and function of

high density neuralcell cultures andincrease thesize of culture

systems through the incorporation of continuous mediumperfusion or through the fabrication of microfluidic channels

16


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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 includingthe formation of connexin43 gap junction formation, and

neural communication through synaptic vesicle release and

the measurement of local field 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-to-

cell communication systems. Additional experiments have

been undertaken to assess the utility of the alginate construct

cell cultures for thedevelopment of co-culture systems capable

of modeling the properties of the bloodbrain barrier and thebrain 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

assistancein performingmeasurements of alginate mechanicalproperties. We thankRoger Tsien for themCherry 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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