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Cardiac Tissue Engineering

Optimization of Cardiac Cell Seeding andDistribution in 3D Porous Alginate Scaffolds

Ayelet Dar,1 Michal Shachar,1 Jonathan Leor,2 Smadar Cohen1

1Department of Biotechnology Engineering and The Institute for AppliedBiosciences, Ben-Gurion University of the Negev, Beer-Sheva 84105 Israel;telephone: 972-8-6461798; fax: 972-8-6472915; e-mail:[email protected] Cardiac Research Institute, Sheba Medical Center, Tel-Hashomer,52621, Israel

Received 28 September 2001; accepted 16 April 2002

DOI: 10.1002/bit.10372

Abstract: Cardiac tissue engineering has evolved as apotential therapeutic approach to assist in cardiac re-

generation. We have recently shown that tissue-engi-neered cardiac graft, constructed from cardiomyocytesseeded within an alginate scaffold, is capable of pre-venting the deterioration in cardiac function after myo-cardial infarction in rats. The present article addressescell seeding within porous alginate scaffolds in an at-tempt to achieve 3D high-density cardiac constructs witha uniform cell distribution. Due to the hydrophilic natureof the alginate scaffold, its >90% porosity and intercon-nected pore structure, cell seeding onto the scaffold wasefcient and short, up to 30 min. Application of a mod-erate centrifugal force during cell seeding resulted in auniform cell distribution throughout the alginate scaf-folds, consequently enabling the loading of a largenumber of cells onto the 3D scaffolds. The percent cell

yield in the alginate scaffolds ranged between 6090%,depending on cell density at seeding; it was 90% atseeding densities of up to 1 108 cells/cm3 scaffold anddecreased to 60% at higher densities. The highly densecardiac constructs maintained high metabolic activity inculture. Scanning electron microscopy revealed that thecells aggregated within the scaffold pores. Some ofthe aggregates were contracting spontaneously withinthe matrix pores. Throughout the culture there was noindication of cardiomyocyte proliferation within thescaffolds, nor was it found in 3D cultures of cardio-broblasts. This may enable the development of cardiaccocultures, without domination of cardiobroblasts withtime. 2002 Wiley Periodicals, Inc. Biotechnol Bioeng80: 305312, 2002.

Keywords: alginate scaffolds; cardiac cells; cell seed-ing; tissue engineering

INTRODUCTION

The limited ability of a cardiac muscle to regenerateafter an extensive myocardial infarction and the scarcity

of cardiac donors indicate an urgent need for new

methods to regenerate myocardium. One approach that

has received recent attention pursues the in vitro for-

mation of cardiac muscle tissue by seeding dissociated

cardiac cells within three-dimensional (3D) polymeric

scaolds (Eschenhagen et al., 1997; Akins et al., 1999;

Carrier et al., 1999; Leor et al., 2000; Li et al., 2000;

Papadaki et al., 2001). The polymeric scaold provides a

temporary support for the seeded cells in culture until

they produce their own extracellular matrix and guides

their reorganization into a 3D tissue.Due to the critical role of the polymer scaolding in

tissue regeneration, material selection and design have

become a major area of research. Our group has fo-

cused its attention on the development of polysaccha-

ride scaolds that are natural, hydrophilic, and bear

resemblance to glyco-components of the extracellular

matrix. Alginate scaolds were fabricated using an all-

aqueous method based on a freeze-dry technique to

have 90% porosity and pore sizes of 50150 lm

(Shapiro and Cohen, 1997). The scaolds supported

the prolonged culture of various primary mammalian

cells and facilitated the performance of rat hepatocytes

in culture (Shapiro and Cohen, 1997; Glicklis et al.,

2000).

In a more recent study, we demonstrated the feasi-

bility of bioengineering a cardiac tissue within alginate

scaolds (Leor et al., 2000). After implantation onto rat

infarcted myocardium, the cardiac biografts stimulated

intense neovascularization from the neighboring coro-

naries and attenuated left ventricular dilatation and

failure in an experimental rat model. Histology revealed

a small number of well-striated myobers scattered be-

Correspondence to: Prof. Smadar Cohen

Contract grant sponsor: The Israel Science Foundation

Contract grant number: 52/99-1

2002 Wiley Periodicals, Inc.


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tween collagen bers (Leor et al., 2000). The present

work addresses optimizing cell seeding and distributing

onto alginate scaolds in an attempt to achieve 3D

cardiac constructs with high and uniform initial cell

density. We than characterized the metabolic activity of

the constructs and followed their morphology over time

by scanning electron microscopy and immunohisto-

chemistry.

MATERIALS AND METHODS

Alginate Scaffolds

Alginate scaolds, diameter 5 mm, thickness 1 mm, were

prepared from alginate with high guluronic acid (G)

content (Pronatal LF 5/60, 65% G; Pronova Biopoly-

mer, Drammen, Norway) by the freeze-dry technique as

previously described (Shapiro and Cohen, 1997). The

scaolds were over 90% in porosity and had pore sizes in

the range of 5075 lm in diameter according to SEM.

Cell Isolation

The study was performed with the approval and ac-

cording to the guidelines of the Institutional Animal

Care and Use Committee. Cardiomyocytes were iso-

lated from the ventricles of 23-day-old neonatal or

fetal (1416 weeks) Sprague-Dawley rats using 10 cycles

of enzyme digestion (Leor et al., 2000). A total of 20

litters of neonates were used in the study; for the con-

tractility studies with embryonic cells, two litters were

used. Unless specied otherwise, the experiments were

conducted with neonatal cardiac cells. The dissociated

cells were subjected to preplating on 60-mm polystyrene

dishes for 15 min at 37C and the supernatants were

collected and analyzed for cell viability by Trypan blue

exclusion assay and for cardiomyocyte purity by im-

munouorescent staining for a-sarcomeric actinin (Leor

et al., 2000). Cardiomyocyte purity, determined by

calculating the percentage of immunostained cells in

four random elds from at least two rat litters, was

shown to be 70% when subjecting the cells to 15-min

preplating.

Cardiobroblasts constituted most of the adherent

cells following 15-min preplating on polystyrene dishes.

Highly enriched cardiobroblast cultures were obtainedwithin 2 weeks, when the adherent cells were further

cultured on polystyrene culture dishes (5% CO2, 95%

humidied air at 37C) in Dulbecco's modied Eagles

medium (DMEM, Biological Industries, Kibutz Beit

Ha'Emek, Israel) supplemented with 10% (v/v) fetal calf

serum (FCS), 1% (v/v) L-glutamine, 1% (v/v) pen-strep,

and 0.1% (v/v) gentamycin. Repeated splitting of the

plated cells every 23 days for 2 weeks resulted in cul-

tures that did not stain with the cardiomyocyte markera-sarcomeric actinin.

Cell Seeding and Cultivation

The cardiac cells were seeded onto the alginate scaolds

at dierent initial cell densities ranging from 3.2 106 to

2.8 108 cells/cm3 scaold. The cells were seeded onto

the scaolds and placed in 96-well plates by dropping up

to 10 ll of the cell suspension on top of the dry scaold.

After a short incubation for 5 min at 37C, the well

plates were placed in a plate holder-type rotor (Labo-

fuge GL, Heraus, Germany) and centrifuged for 6 min(1,000g, 4C). At the end of seeding stage (within 15

min), the volume of the culture media was adjusted ac-

cording to the initial seeded cell density and maintained

at 1 ml medium/106 cells. In the highly dense cell cul-

tures (>2.6 107 cells/cm3 scaold) the scaolds were

transferred into a 24-well plate. Cardiomyocytes were

cultivated in M-199 Earl's Salt Base medium with L-

glutamine (Biological Industries) supplemented with 5%

FCS, 0.6 mM CuSO45H2O, 0.5 mM ZnSO47H2O,

500U/ml penicillin and 100 lg/ml streptomycin. The

cardiobroblasts were cultured in DMEM supplement-

ed as described above. The dierent cell cultures were

incubated for up to 8 days in a humidied atmosphere of5%CO2 and 95% air at 37C, with medium change every

2 days.

The eect of cell density at seeding on cell yield in

alginate scaolds was studied in three separate experi-

ments. Twelve hours postcell-seeding, triplicate scaolds

per data point were removed from the seeding well and

transferred into a new well and the number of entrapped

cells was evaluated by the MTT viability assay.

Metabolic Activity

Metabolic activity of the cell constructs (n = 3 per data

point) was determined using the MTT (3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)

assay, which measures the ability of mitochondrial de-

hydrogenase enzymes to convert the soluble yellow

MTT salt into insoluble purple formazan salt. MTT

sterile stock solution (5 mg/ml PBS) was added to the

cell constructs placed in the well plates. The volume

added to each well depended on cell seeding density in

the scaolds; for constructs seeded with less than 1 107

cells/cm3 scaold, 20 ll of MTT were added and to the

more cell-dense constructs 40 ll were added to each

construct. The plates were incubated overnight at 37Cand then the medium was aspirated from the wells. The

formazan crystals were dissolved in a 100-ll mixture of

20% aqueous SDS/formamide (1:1 volume ratio), over-

night at 37C. Aliquots of the dissolved formazan (100

ll/well) were then transferred to 96-well plate and the

absorbance was read on a microtiter plate spectropho-

tometer at 570 nm. Empty scaolds in 100 ll of SDS/

formamide solution served as a blank. MTT assay re-

sults were expressed as cell numbers using a standard

curve, constructed from scaolds seeded with known

306 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 80, NO. 3, NOVEMBER 5, 2002


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cell concentrations. The cell concentration in the dif-

ferent scaolds was corrected for cell loss during seeding

by determining the number of free cells in the medium

(not entrapped in the scaolds) using a hemocytometer

and their viability was ascertained by Trypan blue ex-

clusion assay.

Immunohistochemistry and Scanning ElectronMicroscopy

At day 1 and 3 in culture, cell-seeded scaolds were

retrieved for SEM analysis (n = 2 per time point). The

samples were xed in 2% (v/v) aqueous glutaraldehyde

(Sigma, St Louis, MO) and dehydrated in a graded se-

ries of waterethanol solutions for 10 min each and then

critical point-dried. Thin sections of the dried samples

were gold-sputtered (100 A ) and examined by SEM

(Model JSM 35 CF, Jeol, Japan) at 25 kV electron beam

radiation.

Samples for histology were xed in a 2% glutaralde-

hyde in PBS, then in 10% neutral buered formalin,

embedded in paran, and serial cross-sectioned (5 lm

thick). The sections (three per sample, two samples pertime point) were stained with hematoxylin and eosin

(H&E).

Immunohistochemistry was performed on thin frozen

sections (12 lm thick) of the cell-seeded scaolds (n =

3). The frozen sections were placed on treated micro-

scope slides, air-dried, then blocked with 2% BSA (w/v)

in PBS. After washing twice with PBS for 15 min, the

samples were incubated with either anti a-sarcomeric

actinin (Sigma, Israel) to detect cardiomyocytes, or

sheep antihuman bronectin (Serotec, Oxford, UK).

After incubation for 60 min at 37C with the primary

antibody, the samples were washed twice with PBS. Theslides were incubated with FITC-conjugated goat anti-

mouse IgG or FITC-conjugated donkey antisheep IgG

(Jackson Laboratories, Bar Harbor, ME) for 60 min at

37C, rinsed, and viewed under an inverted uorescence

microscope equipped with a 490 nm bandpass lter with

a 510 nm cuto lter for uorescence emission. A native

neonatal heart tissue served as a positive control; an

empty scaold and cell-seeded scaolds that reacted

with the secondary FITC-conjugated antibody served as

negative controls.

Statistical Analysis

Statistical analysis of data was performed by analysis of

variance (ANOVA) single factor using Excel Software

2000.

RESULTS

Cardiac Cell Seeding Within Alginate Scaffolds

Alginate scaolds were seeded with neonatal rat cardiac

cell suspensions of dierent cell concentrations. Once

the cell suspensions were overlayered on top of the dry

alginate scaold, the polysaccharide matrix readily ab-

sorbed the culture medium, while pulling the cells into

the matrix-interconnecting pores. To achieve a uniform

cell distribution within the alginate scaolds, a moderate

centrifugal force (1,000g, 6 min) was applied during cell

seeding, immediately after overlayering the cardiac cells

on top of the scaold. Figure 1 is a micrograph of serial

horizontal cross sections in three representative alginate

scaolds, initially seeded with three dierent cell densi-ties, i.e., 0.25 (right column), 0.5 (middle column), and

1.0 108 cardiac cells/cm3 scaold (left column). The

upper cross section in each column represents the sur-

face of the cell-seeded scaold, the middle is a cross

section at 0.5 mm from the surface, and the lower cross

section represents the scaold bottom. For a given cell

density, the serial cross sections along the cell-seeded

scaold reveal a similar picture of cell distribution and

occupation, indicating to a uniform cell distribution in

the alginate matrix. Uniformity in cell distribution was

maintained with the 4-fold increase in the initial cell

seeding concentration. Thus, it appears that centrifu-gation during cell seeding contributes to a uniform cell

distribution throughout the alginate matrix, conse-

quently enabling an ecient cell seeding onto the algi-

nate scaolds.

Increasing cell density at seeding from 3.2 106 to 2.8

108 cell/cm3 scaold resulted in a concomitant increase

in the number of cardiac cells entrapped within the

alginate scaolds (Figs. 1, 2). Cell loading eciency was

8090% and decreased to 60% at seeding densities equal

or higher than 1.0 108 cells/ cm3 scaold. The signif-

icant decrease in cell yield is probably due to cell ag-

gregation, which occurs at highly concentrated cell

suspensions. The cell density achieved herein, 1.6 108

cells/cm3 scaold, is among the highest cell densities ever

reached when cardiac cells were seeded onto 3D polymer

scaolds.

Cardiac Cell Cultivation

Cardiac cells (70% myocytes) seeded within alginate

scaolds at cell densities of 5 106, 1.25 107, 2.5 107,

or 5 107 cell/cm3 scaold were maintained in a static

culture for 12 weeks. At dierent days in culture, the

cell-seeded scaolds were removed from the wells in

order to determine the metabolic activity of the en-trapped cells by the MTT uptake assay, as well as the

number of leaked cells from the scaolds. The extent of

MTT conversion to formazan crystals by the entrapped

cardiac cells, 12 h postcell-seeding, corresponded to the

initial cell number seeded within the scaold (Fig. 3). In

addition, the number of leaked cells from the dierent

cell density constructs was negligible.

The cardiac cells maintained their metabolic activity

within the alginate scaolds in a static culture (Fig. 3).

The extent of MTT conversion by the seeded cells

DAR ET AL.: CARDIAC TISSUE ENGINEERING 307


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revealed no signicant change in the number of living

cardiac cells within the scaold with time, regardless of

the initial cell seeding density within the scaold. The

relatively constant number of viable cardiac cells within

the alginate scaolds and the negligible cell leakage from

the scaold indicate that the neonatal rat cardiac cells

do not proliferate to a signicant extent in the 3D

constructs.

Figure 1. Cardiac cells distribution within alginate scaolds. The scaolds were seeded with 0.25 (right column), 0.5 (middle column) and 1.0

108 cardiac cells/cm3 scaold (left column). Twelve hours postcell-seeding, the scaolds were xed, paran-embedded, serial cross-sectioned, and

stained with H&E.

Figure 2. Eciency of cell loading onto alginate scaolds at dierent

cell-loading densities. The cardiac cells were seeded onto the scaold

by overlayering the cell suspension on top of the dry scaold, followed

by a moderate centrifugation cycle. Twelve hours postseeding, the cell

yield in triplicate alginate scaolds was analyzed by the MTT viability

assay. Each data point is the average of a triplicate from three separate

experiments. The numbers in parentheses represent the percentage of

entrapped cells within the scaold. Asterisk = percent cell yield sig-

nicantly greater than the group seeded with 1.0 108 cells/ cm3

scaold (ANOVA, single factor P = 0.00013 for P < 0.01).

Figure 3. Metabolic activity of the dierent cell-density 3-D cardiac

constructs. Alginate scaolds were loaded with ( 106, (n) 1.25 107,

(m) 2.5 107, and () 5 107 cells/cm3 scaold. The MTT uptake

assay was performed daily on triplicate scaolds and the data were

converted to viable cell numbers, using the appropriate calibration

curve. Each data point on the curve is an average of 23 separate

experiments.



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Cardiac Cell Morphology

Because of the relative transparency of the alginatematrix after wetting with the medium, the behavior of

the cardiac cells within the 1-mm thick scaolds could

be observed via an inverted light microscope. To verify

cell viability, MTT uptake assay was performed on the

constructs. Figure 4A shows that small crystals of the

insoluble purple formazan salt are scattered in the

entire scaold 12 h postseeding. At day 3 in culture,

the size of the deposited formazan crystals appears to

be larger (Fig. 4B). SEM micrographs of the cell-

seeded scaolds, taken at the same time points, depict

the cardiac cell aggregation process, which occurs

within the alginate scaolds with time (Fig. 4C,D).

Cardiac cell aggregation was observed in all cell-seeded

alginate scaolds, regardless of the initial cell density atseeding.

Hematoxylin-eosin staining of thin sections (5 lm

thickness) of the cardiac constructs at day 8 postseeding

revealed the presence of cell aggregates wherein the

constituent cells maintain a spherical shape (Fig. 5).

Most of the cells, including those in the center of the

aggregates, had an intact nucleus, further supporting the

maintenance of cell viability in the 3D cultures.

The cell aggregates were a mixture of myocytes and

nonmyocytes in the same particle. Figure 6 compares

two micrographs of the same eld taken by a light mi-

croscope (Fig. 6A) vs. a uorescence microscope (Fig.

6B), after immunostaining thin sections of the cell ag-gregates with a specic marker for cardiomyocytes, anti-

a-sarcomeric actinin. As seen, most of the cells in the

aggregate are immunouorescently stained for actinin.

The nonstained cells are probably cardiobroblasts,

which constitutes 60% (by number) of the cells in neo-

natal rat myocardium (Brilla et al., 1991).

Contractility of the Cardiac Cell Aggregates

Microscopic inspection for 2 weeks of the neonatal

cardiac cell aggregates in the 3D constructs, seeded at

an initial cell density of 2.6 107

/cm3

scaold, revealedthat some of the aggregates were spontaneously con-

tracting within the alginate scaold pores. In compar-

ison, most of the cell aggregates formed from

embryonic cardiac cells, seeded at the same cell density

within the scaolds, were contracting at a higher rate

(23 10 beat/min). The embryonic constructs con-

tracted at a similar rate as observed when the cells are

plated on 2D polystyrene plates (cell density 7.18.8

106 cells/cm2), where they formed a conuent layer of

Figure 4. Cardiac cells morphology within alginate scaolds at day

1 (A,C) and 3 (B,D) postseeding. A,B: Light micrographs of the

3D cultures after incubation with MTT salt. C,D: SEMs of the

cultures.

Figure 5. H&E stained thin cross-section of cardiac cell-seeded

scaolds (1 106 cells/cm3 scaold) at day 8 postseeding (1,000).

Figure 6. Immunostaining for a-sarcomeric actinin of thin sections of

cardiac cell aggregates seeded within alginate scaolds 7 days post-

seeding onto the scaolds. A: Light microscope image. B: Fluorescence

microscope image.



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centrifugal force, which apparently helps to overcome

the tortuous path of the pores. Yang et al. (2001) simi-

larly used centrifugation to increase hepatocyte loading

onto 3D constructs. Thus, the combination of an e-

cient seeding method and the material properties of

alginate is probably the main reason for the excellent cell

loading that was achieved in this study, 1.6 108 cells/

cm3 scaold. A similar value was recently reported by

Papadaki et al. (2001) for seeding cardiac cells in con-

structs made of laminin-coated PGA bers using con-centrated cell suspensions. The investigators did not

report, however, on cell distribution.

The highly dense cardiac 3D cultures maintained high

metabolic activity for over a week in a static culture. The

static conditions were optimized to maximize cellular

viability, i.e., by adjusting the volume of media and the

frequency of medium exchange of nutrients. It may be

that when using thicker scaolds (larger than 1 mm),

mixing will be needed in order to enhance mass transfer

into the seeded cells. Carrier et al. (1999) found that

cardiac cells seeded within 2-mm thick PGA scaolds

lost viability in static cultures and cultivation at HARVimproved their cellularity.

Under static culture conditions, rat neonatal car-

diomyocytes and cardiobroblasts aggregated within

the alginate scaold pores, creating particles with an

average diameter of 50 lm, at the same magnitude as

the scaold pore size. We have previously shown that

rat hepatocytes undergo aggregation within the scaf-

folds (Glicklis et al., 2000). The cell aggregation noted

herein is attributed to the hydrophilicity of the alginate

matrix, which resists cell and protein adhesion; the

latter process usually mediates cellmatrix interactions

with nonbiological matrices. The nonadhesive nature

of the alginate scaolding promoted cellcell interac-

tions, which contributed to maintenance of cell via-

bility and enhanced the functional properties of the

cells, such as hepatocytes (this work; Glicklis et al.,

2000).

Cardiobroblasts may play an important role in

cardiac tissue regeneration in vitro. Thus, the nding

that their proliferation is diminished within the algi-

nate scaolds is signicant, as it implies that they can

be cocultivated with cardiomyocytes, without the rst

cells dominating the culture with time. In the native

myocardium, cardiobroblasts are responsible for the

production of bronectin, type I and III collagen, andother ECM components, as well as for the synthesis

and secretion of tissue remodeling involved growth

factors. In the present study, we showed that the

cardiobroblasts secrete bronectin within the alginate

scaolds in culture. Fibronectin is the most abundant

noncollagenous protein synthesized by these cells

(Bashey et al., 1992). It plays an important role in

development, cell adhesion, and wound healing and it

is upregulated in the heart upon mechanical overload

(Villarreal et al., 1998). Secretion of bronectin within

the nonadhesive alginate scaolds should promote

specic cellmatrix interaction via integrins and may

stimulate cardiac cell hypertrophy (Ogawa et al.,

2000). The outcome of these opposing forces, i.e., cell

cell vs. cellmatrix interactions, would probably de-

pend on the extent and strength of each interaction

(Ryan et al., 2001). It will be possible to manipulate

them, for example, by varying the proportions of co-

cultivated cells.In summary, the present study was aimed at devel-

oping highly dense 3D cardiac cultures within alginate

scaolds. These cultures were obtained primarily due to

the material properties of the alginate scaolds, i.e.,

hydrophilicity and porosity. We envision that the highly

dense cardiac cultures would further facilitate the per-

formance of tissue-engineered cardiac biografts in re-

pairing an infarcted myocardium.

The authors thank Lilia Shapiro for making the scaolds,

Israel Barbash and Sharon Etzion for help in isolating the

cardiac cells, and Parvin Zerin for histology.

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