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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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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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Biomed. Mater. 6 (2011) 015002 J P Frampton et al
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
LRM 55 Cortical Astrocyte
1 3 7 14 1 3 7 14
Cells/Field
0
20
40
60
80
1 3 14 1 3 14
Days in Culture
LRM 55 Cortical Astrocyte
(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.)
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5
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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
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Biomed. Mater. 6 (2011) 015002 J P Frampton et al
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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