Autospecies and Post–Myocardial Infarction Sera Enhance the

Viability, Proliferation, and Maturation of 3D Cardiac Cell Culture

RAN SCHWARZKOPF, M.D, M.Sc.,1 MICHAL SHACHAR, M.Sc.,1 TAL DVIR, B.Sc.,1

YEHUDA DAYAN, M.Phil.,2 RADKA HOLBOVA, M.S.,3 JONATHAN LEOR, M.D.,3

and SMADAR COHEN, Ph.D.1

ABSTRACT

The limited ability of cardiac muscle to regenerate after an extensive myocardial infarction (MI) and thescarcity of cardiac donors have fueled the field of cardiac tissue engineering as a potential therapeuticapproach to enhance cardiac function in post-MI patients. We are exploring the ex vivo bioengineering ofcardiac muscle tissue by seeding isolated cardiac cells within alginate scaffolds and supplementing theculture with ‘‘smart’’ media. The hypothesis investigated herein is that sera derived from autospeciesand from post-MI animals contain agents that might induce cell proliferation, survival, and matura-tion in vitro. The results of the metabolic activity of the neonatal cardiac cell constructs (6.4–51�106

cells/cm3), as measured by MTT viability assay, indicated a significant advantage ( p< 0.05) to the con-structs supplemented with serum from normal and post-MI adult rats compared to fetal calf serum(FCS) supplementation. H&E staining and a-sarcomeric actin immunofluorescence staining revealedthick viable cardiac cell clusters (150–300lm), with abundant 3D architecture in the cardiac cell constructssupplemented with post-MI and normal adult rat serum. The number of cells positively immunostainedwith Ki-67, a cell proliferation marker, was significantly higher in post-MI adult rat serum-supplementedcultures compared to negative results in the FCS-supplemented culture. The results presented in thisstudy indicate that media supplemented with post-MI adult rat serum and normal adult rat serumcompared to FCS have a significant advantage in the regeneration of injured cardiac tissue.

INTRODUCTION

CARDIAC TISSUE ENGINEERING evolved to meet the ever

growing need to create a suitable biological tissue

replacement for the injured post–myocardial infarcted (MI)

heart. Today, when a patient is diagnosed with an acute MI

there are mainly 2 treatment options available to the medical

staff, a thrombolytic drug, or the more invasive option of

percutaneous angioplasty procedure. Still many of the pa-

tients who survive MI develop advanced heart failure, which

today remains mostly without a suitable treatment option.1

In both of these treatments, the best result we can expect is

to reopen the occluded vessel and save whatever was left of

the healthy cardiac tissue. Cardiac tissue engineering strives

to offer, in the future, a third better option for post-MI

patients. That is, to implant a tissue-engineered cardiac graft,

derived from culturing cardiac cells within 3D scaffolds

supplemented with the right assortment of growth factors.

A functioning cardiac tissue graft is still only yet a look into

the future. To achieve such a goal, a few challenges need to be

resolved; for example, developing advanced ways to harvest

cardiac progenitor cells, in sufficient amounts, and ways to

enhance the cardiac cell culture potential for proliferation,

viability, and maturation. These goals may be accomplished

1Department of Biotechnology Engineering, Ben-Gurion University of the Negev, Beer-Sheva, Israel.2Department of Statistics, London School of Economics, London, United Kingdom.3Neufeld Cardiac Research Institute, Tel-Aviv University, Sheba Medical Center, Tel-Hashomer, Israel.

TISSUE ENGINEERINGVolume 12, Number 12, 2006# Mary Ann Liebert, Inc.

3467

by means of culture medium (CM) growth factors and

bioreactors.2,3

Our group, by utilizing alginate scaffolds that are natural,

hydrophilic, and bear resemblance to the glycol-components

of the extracellular matrix,4 has been able to cultivate an as-

sortment of primary mammalian cells,4,5 among which are car-

diac cells, and enhance their ex vivo and in vivo performance.6

The present work addresses one of the main obstacles en-

countered during reconstruction of engineered cardiac tissue

from neonatal cardiac cells, namely cell viability and ma-

turation in long-term cultures. We challenge the offhand use

of the standard protocol culture media, fetal calf serum (FCS)

supplementation, in cardiac tissue engineering. In an attempt

to mimic the natural physiological and pathophysiological

processes that stimulate cardiac and progenitor cardiac cell

proliferation and maturation in vivo,7 we selected the optimal

‘‘smart’’ sera supplementation for cardiac tissue engineering,

the post-MI adult rat serum and as control, serum from normal

adult rat. Cardiac cell proliferation has been shown to occur in

response to unidentified cytokines and growth factors, which

are released during cardiac ischemic insult.8–11

Herein, we examine the hypothesis that supplementing

cardiac cell culture media with post-MI adult rat serum will

increase viability and maturation and induce proliferation

among the cardiac cells cultivated in vitro.

MATERIALS AND METHODS

Alginate scaffolds

Alginate scaffolds, 5 mm in diameter and 1 mm thick,

were prepared from sodium alginate with high guluronic

acid (G) content (Pronatal LF 5/60, 65% G; FMC Biopo-

lymers, Drammen, Norway) by a freeze dry technique as pre-

viously described.12 The scaffolds were over 90% in porosity

and had pore size in the range of 50–75 mm in diameter,

according to scanning electron microscopy.

Post-MI adult rat serum isolation (S-serum)

The study was performed with the approval and accord-

ing to the guidelines of the Institutional Animal Care and

Use Committee of Ben-Gurion University. The left anterior

descending artery was ligated in Adult Sprague-Dawley rats

weighing250–300 g.13Theratswereanesthetizedwithacom-

bination of ketamine (40 mg/kg) and xylazine (10 mg/kg).

The chest was opened under sterile technique by left thor-

acotomy through the fourth intercostal space, the peri-

cardium was removed, and the left main coronary artery was

permanently occluded with an intramural stitch. The ani-

mals were euthanized 6 days after the procedure and 1–2 mL

of blood per animal was drawn from the heart in an attempt

to maximize capture of local paracrine and endocrine fac-

tors. The blood was centrifuged (600 g for 10 min) and the

serum above the pellet was drawn and filtered (0.2 mm).

Prepared serum was stored at �48C.

Cell isolation

The cardiac cells were isolated from the ventricles of 1-

to 4-day-old neonatal Sprague-Dawley rats using 6–7 cycles

of enzyme digestion.14 Briefly, neonatal rats (n ¼ 20–45)

were killed by decapitation, and their ventricles were

rapidly removed and placed in M-199 Earl’s Salt Base

medium with L-glutamine (Biological Industries, Beit

Haemek, Israel). After collecting the ventricles, and placing

them in cold buffer (in mM/L: NaCl 116.4, HEPES 20,

NaH2PO4 1, glucose 5.5, KCl 5.4, MgSO4 0.8; pH 7.4),

they were cut to 2 mm3 pieces and then incubated (at 378Cfor 30 min) repeatedly (6–7 times) in buffer supplemented

with collagenase type II (95 U/mL; Worthington, Lake-

wood, NJ) and pancreatin (0.6 mg/mL; Sigma, Rehovet,

Israel). After each round of digestion, the supernatant was

centrifuged (600 g for 5 min at room temperature) and the

resulting cell pellet was resuspended in cold M-199 sup-

plemented with 0.6 mM CuSO4.5H2O, 0.5 mM ZnSO4.

7H2O, 500 U/mL penicillin and 100 mg/mL streptomycin

(Biological Industries; CM) and 0.5% (v/v) FCS (Sigma,

Israel). The cardiac cells were pooled and suspended in an

ammonium chloride solution (0.83% NH4Cl, 0.1% KHCO3,

0.004% EDTA [w/v]; pH 7.4) for red blood cell detonation.

Afterward they were centrifuged (600g, 5 min) and washed

twice with CM supplemented with 5% FCS. After washing,

the cells were pooled and the number of viable cardiac cells

was counted by the use of Trypan blue exclusion assay

(Sigma, Israel). The cardiac cells harvested by this method

consisted of 70% cardiac myocytes and the remaining

nonmyocytes.4

Cell seeding and cultivation

The cardiac cells were seeded onto alginate scaffolds at

different cell densities, ranging from 6.4�106 to 51�106

cells/cm3 per scaffold. Before cell seeding, the cells were

washed from their previous culture media and suspended

with a fresh CM supplemented with either FCS, post-MI

adult rat serum, or normal adult rat serum (at a con-

centration of 5% V/V). The scaffolds were placed within a

96-well plate and the cells were seeded onto the scaffolds

by equally dropping 10 mL of the cell suspension on top of

the dry scaffold. After seeding, the plates were placed in a

plate holder-type rotor (Labofuge GL, Heraus, Germany)

and centrifuged (1000 g for 6 min at 408C) to enhance cell

distribution within the alginate scaffold. After a short (1–2

hours) incubation (at 378C, 5% CO2, 95% air) within the

96-well plate, supplemented with either 200 mL of

CMþFCS/CMþS-serum /CMþnormal rat serum, the sca-

ffolds were transferred to 12-well plates, supplemented with

1 mL of CMþFCS/CMþS-serum/CMþnormal rat serum,

to yield the ratio of 1 mL medium per 1�106 cells. Culti-

vation took place within a humidified atmosphere incubator

with medium exchange every 2 days. At different cultiva-

tion time points, samples were analyzed for metabolic ac-

tivity, viability, and culture morphology.

3468 SCHWARZKOPF ET AL.

Biochemical assays

Metabolic activity of the cell constructs (n ¼ 2–3 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 dehy-

drogenase enzymes to convert the soluble yellow MTT salt

into insoluble purple formazan salt. MTT sterile stock solu-

tion (Sigma, Israel; Biological Industries) (5 mg/mL phos-

phate-buffered saline) was added to the cell constructs after

transferring them from their cultivation vessels to a 96-well

plate. Forty microliters of MTT were added to constructs. The

plates were incubated for 24 hours and then transferred to an

additional period of 24-hour incubation in a 96-well plate,

containing 100mL mixture of 20% aqueous SDS/formamide

(1:1, v/v) per well to dissolve the formazan crystals formed

within the cells. Seventy microliters of the dissolved for-

mazen solution were transferred to a 96-well plate and the

absorbance was read on a microtiter plate spectrophotometer

at 570 nm. The readings were then correlated to the number of

live cells through a calibration curve of readings of known

cell number absorbance. Blank empty scaffolds were sub-

jected to the same processes as cell-seeded scaffolds.

Histology, immunohistochemistry,

and immunofluorescence

Samples for histology and immunohistochemistry were

fixed in a graded series of water– ethanol solutions (70%,

95%, and 100%) for 30 min each, then subjected to 100%

ethanol for 2 more hours, paraffin embedded, and serial

cross-sectioned (5 mm thick). The thin sections were stained

with hematoxylin and eosin (H&E), for detecting nucleus

and cytoplasm, respectively. Immunohistochemistry for cell

proliferation was achieved by reacting the serial sections

with Ki-67 (Sigma, St. Louise, MO). Immunofluorescence

for a specific cardiac cell marker was achieved by incubating

the samples with 1 mL of the primary antibody, a-sarcomeric

actin (Sigma, Israel), for 12 hours and than a further 4-hour

incubation with the secondary antibody, prior to sample

analysis a propidium iodide (PI; Molecular Probes, Eugene,

OR) solution containing 3.75 mg/mL of PI was added.

Fluorescence microscope analysis

for viable cell distribution

The cardiac cell constructs were placed for 3–5 min in a

well containing 10 mL of their respective culture media

and 10 mL from an acetone solution containing 5 mg/mL

fluorescein diacetate (FDA; Sigma, Israel). The cardiac con-

structs were imaged under a fluorescence microscope (FDA,

excitation—494 nm, emission—520 nm; PI, excitation—

536 nm, emission—617 nm).

Statistical analysis

As often is the case in in vitro and in vivo trials, the

individual level observations in this experiment are mea-

sured irregularly, and we are faced with a repeated mea-

sured data with fixed measurement occasions but where the

data are incomplete. This poses a challenge; most statistical

procedures that successfully deal with repeated measure-

ment questions require that the data conform to a particular,

balanced structure. Taking advantage of the particular

flexibility of multilevel modeling in dealing with such

unbalanced repeated datasets, we consider these data as a 2-

level hierarchical structure, thus providing statistical effi-

cient parameter estimation.15

Using version 1.10.0007 of MLwiN, a random effect

2-level structure of cell count measurements within ‘‘in-

dividual cell samples’’ was modeled. A natural logarithmic

transformation was applied on cell counts measurements to

reach the necessary normality assumption that missing

cases are assumed to be missing at random.16

Dependence on time was modeled as cubic, quadratic,

and linear; however, only a linear relationship was found

significant. Comparison of the different serums was based

on significance tests of differences in fitted regression coeffi-

cients. The data are graphically displayed with the fitted

data. Optimal models were chosen using the likelihood

ratio test, where the values of the statistics were compared

to a w2 distribution on 1 degree of freedom at a 95% sig-

nificance level.

RESULTS

Effect of post-MI serum supplementation

on the viability of cardiac cell constructs

Cardiac neonatal cell constructs seeded with various cell

densities (6.4�106 to 51�106 cells/cm3) were cultivated

either with post-MI adult rat sera, normal adult rat sera, or

FCS as a supplementation to the culture media. At different

days during cultivation, 2–4 scaffolds per time point, in a

given experiment, were removed from the culture plates for

analysis. The cardiac cell constructs were maintained in a

static culture for up to 16 days.

Figure 1 shows the results of the statistical analysis for cell

viability in the cardiac cell constructs, in both post-MI- and

FCS-supplemented cultures, as assessed by metabolic ac-

tivity via MTT assay. To statistically analyze the results of

the different cell seeding densities in the various experi-

ments, the results were normalized through a calibration

curve to account for the different spectrophotometer absor-

bance readings. The results reveal a steady decline in via-

bility over time for both cultures. However, when comparing

the post-MI adult rat serum supplemented culture to the

FCS-supplemented culture, we clearly see that there is a

constant increase in the difference in culture viability, as

assessed by metabolic activity, per time point. As seen, the

lack of a viability measurement on day 0 is due to the con-

straints of the MTT assay, which needs a 24-hour incubation

period. The amount of viable cells seeded was equal and

3D CARDIAC CELL CULTURE 3469

randomly assigned to be supplemented with either FCS or

post-MI adult rat serum. It is possible to infer from the gap in

viability on day 1 (the initial gap between the 2 lines is

0.209), that the post-MI adult rat serum has a significant

effect during the first 24 hours of culture incubation. This

effect is probably achieved by increasing cell survival after

the trauma of harvesting and seeding, thus yielding a larger

cohort of viable cells. However, with time the difference

between the 2 slopes increases in a rate of 0.014 per time unit

(the data are within the 95% confidence limit). The inter-

pretation of these results is that the cardiac cells in the FCS-

supplemented culture have a lower viability, as assessed by

metabolic activity, compared to the cardiac cells in the post-

MI adult rat sera supplemented culture.

By altering the CM with the addition of either post-MI

adult rat serum or normal adult rat serum, we tried to de-

termine whether the higher cardiac cell culture viability com-

pared to FCS-supplemented media was due to the post-MI

quality of the supplementation or solely to the autospecies

characteristic. The results reveal a steady decline in viability

over time for all 3 differently supplemented cultures (Fig. 2).

Although there is no apparent difference between the slopes

of the post-MI adult rat serum and the normal adult rat serum

supplemented cultures, we can definitely perceive the grow-

ing gap between the slopes of the 2 previously mentioned

cultures and the FCS-supplemented culture. The differences,

representing an equal rate of decline in viability among the

post-MI adult rat and the normal adult rat supplemented

cultures, compared to a much faster rate of decline in viability

of the cardiac cell culture supplemented with FCS.

The fluorescence analysis after staining the cardiac cell

cultures with FDA provides a qualitative assay for cell via-

bility and may present the different organization and dis-

tribution of viable cells within the cardiac cell constructs.

As can be seen in Fig. 3A, a 7-day cardiac cell construct

supplemented with FCS is without any large cell clusters

and sparsely laden with round immature cells, compared

with the normal rat serum and post-MI supplemented cul-

tures, which exhibit large, tissue-like clusters (Fig. 3B, C).

When examined under higher magnification (Fig. 3D), cells

with a typical elongated muscle like morphology are seen

within what resembles muscle-like tissue. These results go

hand in hand with the previously presented data that cardiac

cells cultivated with normal rat and post-MI supplemented

media maintain viability and undergo maturation, to a better

extent, compared to FCS-supplemented culture. Support for

the specific lineage of these cell cultures can be seen in the

following immunofluorescence images.

Morphology of cardiac cell constructs cultivated

with different medium supplementation

Neonatal cardiac cells seeded within the weakly adhesive

alginate scaffolds are organized within a day or 2 (de-

pending on seeding density) in cell clusters, which grew in

size with culture time. Cardiac cell constructs, cultivated

with post-MI adult rat serum supplemented media, ex-

hibited a different morphology compared to cardiac cells cul-

tivated with FCS-supplemented media. In the post-MI adult

rat serum supplemented cardiac cell construct, formation of

large cell clusters could be seen as early as the day 3 in

culture (Fig. 4A), compared with FCS-supplemented car-

diac cell constructs, which did not present any substantial

cell clusters at this time. Similar differences between the 2

cultures were observed on days 6 and 13 of the cell cultures

(Fig. 4B, C). In comparison to previous work in static

cultivation, which exhibited cell cluster size range between

50 and 100 mm in diameter,4 the cell clusters in post-MI

adult rat serum supplemented media ranged from 150 mm to

as large as 300 mm in diameter at day 6 and kept their size

FIG. 1. Statistical analysis of the results of the metabolic ac-

tivity of cardiac cell cultures as a function of time and different

serum supplementation. The MTT uptake assay was performed

daily on 2–4 scaffolds. Each data point on the curve is an average

of 5–10 separate experiments (95% significance level, p< 0.05).

The x-axis in Fig. 1 represents the log viability of the cardiac cells

(refer to Methods), and the y-axis the time frame.

FIG. 2. Statistical analysis of the results of the metabolic ac-

tivity of cardiac cell cultures as a function of time and different

serum supplementation. The MTT uptake assay was performed

daily on 2– 4 scaffolds. Each data point on the curve is an average

of 5–10 separate experiments (95% significance level, p< 0.05).

The x-axis in Fig. 1 represents the log viability of the cardiac cells

(refer to Methods), and the y-axis the time frame.

3470 SCHWARZKOPF ET AL.

FIG. 3. Seven-day-old cardiac cell constructs supplemented with FCS (A), normal adult rat serum (B) and post-MI adult rat serum

(C, D) as seen under the fluorescence microscope after being stained by FDA, which stain viable cells in green. Color images available

online at www.liebertpub.com /ten.

FIG. 4. Morphology of large cardiac cell clusters from post-MI adult rat serum supplemented culture. (A) Day 3 in culture. (B) Day 6

in culture. (C) Day 13 in culture (original magnification,�40). The scaffolds were paraffin embedded, cross-sectioned, and stained with

H&E. Color images available online at www.liebertpub.com /ten.

3D CARDIAC CELL CULTURE 3471

of at least 150 mm at day 13 of cell culture (Fig. 4A–C).

There are a few possible explanations for this phenomenon;

the first is the added qualities of autospecies serum–

supplemented medium compared to the standard FCS

supplementation. The second may be that the post-MI adult

rat serum helps to maintain the number and viability of the

cardiac cells longer than the FCS supplementation. The

third explanation is the possible minute amount of cardiac

cell proliferation induced by the post-MI adult rat serum,

which is demonstrated with the positive Ki-67 im-

munoassaying; this amount of proliferation together with

the increased viability helped to keep the cardiac cell con-

struct in a constant size for such a long period of time.

In contrast with previous work in static culture,4 in which

the cardiac cells aggregated into small spherical shapes, in

the work presented here, cells cultivated with post-MI adult

rat serum supplementedmediapresented inavarious morpho-

logic architectures, which included large elongated and

spherical clusters (Fig. 5A–D).

Immunofluorescence and immunohistochemistry

of cardiac constructs cultivated with different

medium supplementation

Immunofluorescence staining of the cardiac cell constructs

from days 6 and 14, supplemented with FCS, normal rat

serum, or post-MI adult rat serum, for a typical cardiac

contracting protein, namely, a-sarcomeric actin, is presented

in Fig. 6. a-Sarcomeric actin is specific for I bands of cardiac

and skeletal muscle cells but is not found in other cells17;

therefore, the distinction between myocyte and nonmyocyte

cell is simple. The samples display an interesting variation

among the different cultures; in the FCS-supplemented cul-

tures, small isolated round myocytes are seen with no visual

intracellular striping or any mature tissue-like features (Fig.

6A, B). On the other hand, the normal adult rat and post-MI

serum supplemented cultures exhibit aligned, elongated

cells that contain centrally elongated nuclei and visual

intracellular striping typical of mature muscle cells (Fig. 6C–

F). The cardiac cells are not in isolated groups like the

FCS-supplemented cultures, but rather in organized, inter-

connected-looking cell clusters resembling mature muscle

tissue. Viewed at a lower magnification, we can see the tis-

sue-like constructs of the positively stained myocytes re-

sembling mature muscle tissue (Fig. 6G, H).

Ki-67 is a nuclear antigen expressed in all phases of the

cell cycle except G0.18 Ki-67 is apparent mainly in the late

S phase, increases further in G2, persists during prophase and

metaphase, and decreases in anaphase and telophase. Ki-67

is preferable to thymidine, bromodeoxyuridine, and pro-

liferating-cell nuclear antigen for labeling, because it is not

involved in DNA repair.19 Expression of Ki-67 is a re-

quirement for cells to traverse the cell cycle and undergo

cell division.20,21 Ki-67 was measured in cardiac cells from

both post-MI adult rat serum and FCS-supplemented cul-

tures; although no positive staining was observed in the FCS-

supplemented culture, an abundance of positive staining

nuclei were observed in the post-MI adult rat serum sup-

plemented culture (Fig. 7).

DISCUSSION

When approaching the challenge of ex vivo reconstruct-

ing of cardiac tissue from isolated cells seeded in polymeric

FIG. 5. Variations in cardiac cell cluster morphology in cultures supplemented with post-MI adult rat serum. (A–D) Sixth day cardiac

cell clusters ranging in largest diameter from 200 to 300 mm (original magnification,�40). The scaffolds were paraffin embedded, cross-

sectioned, and stained with H&E. Color images available online at www.liebertpub.com /ten.

3472 SCHWARZKOPF ET AL.

scaffold, 1 of the issues to be addressed is the culture media.

The aim is to supply the cardiac cell cultures with the op-

timal amount and selection of growth factors and cytokines

that will enable cardiac muscle tissue regeneration. In the

present study, cardiac cells constructs were cultured with

media supplemented with post-MI adult rat serum to try to

answer the needs of the reconstructing tissue in a superior

way than the standard protocol media, which is supple-

mented with FCS.

Anversa and Kajstura8 showed that the adult heart has a

subpopulation of myocytes that are not terminally differ-

entiated.7 These myocytes reentered the cell cycle and

underwent nuclear mitotic division early after the heart

sustained an infarction. In animal models as well, it was

shown, that conditions resembling MI are characterized by

DNA replication and myocyte division.22 These responses

peak 7–14 days after MI.

Our results show that cultivating neonatal rat cardiac cells

with serum from post-MI and normal adult rats achieved a

more viable cellular construct compared to cultivation with

FCS, as revealed by the significant difference in metabolic

activity by MTT assay during culture. The difference be-

tween the 2 cultures can be attributed not only to a decrease

in cell mortality and to an increase in viability, but also to a

greater percentage of cardiac cells surviving the cell har-

vesting and seeding trauma, all due to the effects of the

novel culture media. This presents us with a larger number

of cells cultured successfully.

A smaller portion of the difference in cell number may be

attributed to possible cell proliferation, as seen with the

positive immunohistochemical Ki-67 staining in cardiac cell

constructs supplemented with post-MI adult rat serum,

compared with a negative Ki-67 stain in cardiac cell con-

structs supplemented with FCS. Although the function of Ki-

67 is not clear, it seems to promote cell proliferation by

interfering with the binding of p53 to DNA.20,21 In addition,

it is not involved in DNA repair.19 Therefore, Ki-67 was

selected as a proliferative marker in this study

FIG. 6. Confocal microscope images of a-sarcomeric actin-

labeled cardiac cell constructs cultured for 6 (A, C, E, G) and 14

(B, D, F, H) days in FCS (A, B), normal adult rat serum (C, D),

and post-MI adult rat serum (E–H) supplemented media. Distinct

cardiac cell and tissue characteristics (intracellular striping,

elongated cell and tissue morphology, and centrally elongated

nuclei) can be seen in the normal and post-MI adult rat serum

supplemented cultures compared with the absence of such char-

acteristics in the FCS-supplemented culture. Color images avail-

able online at www.liebertpub.com /ten.

FIG. 7. Ki-67 labeling of cardiac cell construct cultivated with

post-MI adult rat serum supplemented medium. Multiple positive

stained nuclei are visible in the image. Color images available

online at www.liebertpub.com /ten.

3D CARDIAC CELL CULTURE 3473

From a morphologic point of view, as presented in pre-

vious studies of static culture conditions, cardiac cell clus-

ters within the alginate scaffold did not exceed 50–100 mm

in diameter.4 Our work, as well, shows that cardiac cell

clusters from cultures supplemented with FCS did not show

any large clusters. Compared to the large clusters presented

in the cardiac cell cultures supplemented with post-MI adult

rat serum, the cell clusters ranged by average from 150 to

300 mm in diameter and preserved such size up to 13 days

in culture. Furthermore, the large cardiac cell clusters pre-

sented in an array of 3D forms. Cardiac cell constructs

supplemented with post-MI adult rat serum and normal adult

rat serum exhibited, as observed in immunofluorescence-

stained images, myocyte cell characteristics such as intra-

cellular striping, elongated cell morphology, and centrally

elongated nuclei, compared to the FCS-supplemented

constructs, which exhibited round cells with none of the

mentioned characteristics. Moreover, the constructs sup-

plemented with post-MI and normal adult rat serum ex-

hibited a distinct muscle tissue–like appearance.

Immunofluorescence staining for a specific cardiac mar-

ker, a-sarcomeric actin, further supports the finding that the

cells which are presented in the images are of mature

cardiac muscle cells.

Altogether, these data indicate that cardiac cell cultures

cultivated with post-MI and normal adult rat serum achieve

a better outcome represented by a more viable and mature

cardiac cell tissue. These results that compare conventional,

FCS-supplemented media with autospecies- and growth

factor-induced cultivation, post-MI and normal adult rat

serum supplemented media, suggest that a physiologic ap-

proach need be taken while planning culture media com-

ponents in cardiac tissue engineering.

We can only assume, at this point, that the species spe-

cific growth factors, such as insulin-like growth factor-1,

angiotensin-II, and transforming growth factor-b at basal

level, normal adult rat serum or induced by the MI, and

supplemented to the cardiac cell culture via the post-MI

adult rat serum, provided the cause for the significant dis-

parity observed between the differently supplemented car-

diac cell cultures.

Recent studies have shown a negative effect of FCS

supplementation on viability and extracellular matrix pro-

duction by fibroblast and chondrocyte cell cultures.23,24

Furthermore, a novel study found that FCS inhibited the

in vitro growth of 1-cell mouse embryos and that cultivation

with FCS-supplemented media increased the percentage of

embryos that died before they reached the blastocyst stage.25

We confirm herein a significant advantage for autospecies

serum supplementation compared to standard protocol of

FCS supplementation for neonatal rat cardiac cell cultures.

We did not reveal a statistically significant advantage in

cardiac cell culture metabolic activity to the added quality of

post-MI adult rat serum compared with normal adult rat

serum. These findings may be elucidated by the overwhelming

effect of autospecies serum supplementation compared to a

much more moderate effect of the post-MI quality of the

supplemented serum, which may have been shadowed by the

former, much larger effect. Another explanation for the lack

of a significant advantage of the post-MI quality of the sup-

plemented serum may be the fact that the normal adult rat

serum was isolated from living adult rats in captivity. Such a

feature may have a strong influence on the level of stress

under which the rat is, and thus may influence the level of

acute phase reactants and growth factors in the rat’s serum,

unlike a ‘‘true’’ normal adult rat’s serum.

Although FCS posses a variety of naturally occurring

growth factors, there is substantial evidence that a lack of a

good cross-reactive response among species can hinder the

growth factors effect on the growing cardiac cell culture.26,27

Lack of a good cross-reactivity of growth factors among

different species has repeatedly been validated; for ex-

ample, humanized mouse antibodies directed to human

vascular endothelial growth factor and vascular endothe-

lial growth factor receptor were poorly cross-reactive.26 In

addition, a minimal T-cell immune response was elicited in

rodents when exposed to specific human peptides.27 Such a

lack of cross-reactivity can assist in substantiating the ad-

vantages of autospecies serum supplementation compared

to standard protocol FCS supplementation.

In conclusion, cell cultures—and cardiac cell cultures in

particular—would benefit greatly when cultured with au-

tospecies serum supplementation on standard protocol FCS

supplementation. The primary problem facing scientists in

applying such actions in small mammalian cell cultures is

the lack of an affluent serum source. However, with today’s

explosion in embryonic stem cell research, autospecies serum

supplementation can be a much-needed factor for achieving

a more viable and differentiated engineered tissue. In such

a setting, the added quality of post-MI serum, and the

specific factors within may unravel their true potential in

accelerating the progress down the much-wanted path from

embryonic stem cell to a differentiated cardiac cell, and

open the way to combining physiologic growth factor in-

cubation in cardiac cell cultures.

ACKNOWLEDGMENTS

The authors thank Ms. Parvin Zerin for her help in his-

tology work. The research was supported by a grant from

the Israel Science Foundation: No. 793/04.

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Address reprint requests to:

Smadar Cohen, Ph.D.

Department of Biotechnology Engineering

Bldg. 39, Room 222

Ben-Gurion University of the Negev

Beer Sheva

Israel 84105

E-mail: scohen@bgumail.bgu.ac.il

3D CARDIAC CELL CULTURE 3475