Optimizing Engineered Heart Tissue for Therapeutic
Applications as Surrogate Heart Muscle
Hiroshi Naito, MD; Ivan Melnychenko, MD; Michael Didié, MD; Karin Schneiderbanger, MD;
Pia Schubert, MD; Stephan Rosenkranz, MD; Thomas Eschenhagen, MD; Wolfram-Hubertus Zimmermann, MD
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Background—Cardiac tissue engineering aims at providing heart muscle for cardiac regeneration. Here, we hypothesized
that engineered heart tissue (EHT) can be improved by using mixed heart cell populations, culture in defined serum-free
and Matrigel-free conditions, and fusion of single-unit EHTs to multi-unit heart muscle surrogates.
Methods and Results—EHTs were constructed from native and cardiac myocyte enriched heart cell populations. The
former demonstrated a superior contractile performance and developed vascular structures. Peptide growth factorsupplemented culture medium was developed to maintain contractile EHTs in a serum-free environment. Addition of
triiodothyronine and insulin facilitated withdrawal of Matrigel from the EHT reconstitution mixture. Single-unit EHTs
could be fused to form large multi-unit EHTs with variable geometries.
Conclusions—Simulating a native heart cell environment in EHTs leads to improved function and formation of primitive
capillaries. The latter may constitute a preformed vascular bed in vitro and facilitate engraftment in vivo. Serum- and
Matrigel-free culture conditions are expected to reduce immunogenicity of EHT. Fusion of single-unit EHT allows
production of large heart muscle constructs that may eventually serve as optimized tissue grafts in cardiac regeneration
in vivo. (Circulation. 2006;114[suppl I]:I-72–I-78.)
Key Words: cardiomyocyte 䡲 myocardium 䡲 regeneration 䡲 tissue engineering 䡲 transplantation
C
ell-based therapies aim at reconstructing biologically
active tissues in vivo to restore the function of failing
organs.1,2 Experimental and first clinical trials support the
notion that hearts can at least partially be repaired by cell
implantation. An alternative approach that might overcome
some limitations of cell therapy is the implantation of in vitro
engineered myocardium.3 Tissue engineered myocardium
may not only function as an add-on to existing structures but
also could theoretically be used as true myocardial replacement or in de novo construction of myocardium in congenital
malformations (eg, Tetralogy of Fallot, single and hypoplastic ventricles). Several principally different cardiac tissue
engineering approaches have been developed. These are: (1)
seeding of cardiac myocytes on preformed polymeric scaffolds, which may function as organ blueprints;4 –7 (2) stacking
of cardiac myocyte monolayers to form cardiac muscle-like
tissue without additional matrix material;8 and (3) entrapping
of cardiac myocytes in a cardiogenic growth environment to
support self-assembly into functional myocardium.9 –11 All
these tissue engineering concepts have been tested in animal
models showing survival and growth of engrafted heart
muscle surrogates.6 – 8,12
Yet several caveats must be considered before tissue
engineering-based cardiac regeneration may be applied clinically. Optimal myocardial structure and function depends not
only on the cardiac myocyte fraction but also on nonmyocytes, which compose 70% of the total cell content of a
heart.13,14 Consequently, engineered myocardium may benefit
from a “physiological” cell composition. Culture in the
presence of xenogenic serum may lead to impregnation of
cell and matrix components or induce autoantigen presentation. Thus, identification of serum-free culture conditions will
be of paramount importance, particularly when considering
good laboratory practice and good manufacturing practice
guidelines. So far, several culture medium supplements including peptide growth factors have been identified to support cardiac myocyte growth in vitro.15 Yet a serum-free
cardiac tissue engineering approach has not been developed.
Extracellular matrix from Engelbreth-Holm-Swarm tumors
(also known as Matrigel) has been identified as an essential
component in engineered heart tissue (EHT)10 and has recently also been used by others to support cardiac muscle
development in vitro.16 However, its replacement with defined factors is equally essential as the replacement of serum.
Eventually replacement of large myocardial tissue defects as
well as reconstruction of missing myocardial structures will
require large artificial heart muscle patches.
This study aimed at providing some solutions to the
aforementioned bottlenecks. Here, we focus on improving a
tissue engineering technology, namely the generation of
From the Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Germany.
Presented at the American Heart Association Scientific Sessions, Dallas, Tex, November 13–16, 2005.
Correspondence to Wolfram-Hubertus Zimmermann, Institute of Experimental and Clinical Pharmacology and Toxicology, University Medical Center
Hamburg-Eppendorf, Martinistr. 5, 20246 Hamburg, Germany. E-mail [email protected]
© 2006 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.org
DOI: 10.1161/CIRCULATIONAHA.105.001560
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Naito et al
EHT, which has been developed by our group over the past
10 years.9,11 This study demonstrates that: (1) EHTs exhibit
an improved function when constructed from mixed rather
than cardiac myocyte enriched heart cell populations; (2)
supplementation of culture medium with growth factors
allows EHT culture under serum-free conditions; (3) Matrigel
can be replaced by insulin and triiodothyronine; and (4) EHTs
can be generated in various geometries and sizes.
Methods and Materials
Cell Isolation and Identification
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Cells were isolated by fractionated DNase/Trypsin digestion as
described earlier.10 Isolated cells were either plated on tissue culture
dishes or Petri dishes that facilitated attachment of non-myocytes or
did not allow cell attachment at all, respectively. Plating was
performed for 1 hour. Cells in suspension, being depleted off
non-myocytes (tissue culture dishes) or not depleted (Petri dishes),
were pelleted by centrifugation, reconstituted in culture medium,
counted in a Neubauer cell chamber, plated on collagen coated cover
slips (100 000 cell/cm2), and cultured in the presence of BrdU
(0.1 mmol/L) for 48 hours (DMEM, 1 g/L glucose, and supplements
as indicated). Staining for cell type-specific antigens (␣-sarcomeric
actinin: cardiac myocytes; prolyl-4␤-hydroxylase: fibroblasts;
␣-smooth muscle actin: smooth muscle cells), filamentous actin
[TRITC-phalloidin], and DNA (propidium iodide; 5 ␮g/mL) was
performed after formaldehyde fixation as described earlier.11 Finally,
400 to 1500 cells per group were analyzed to identify the respective
cell species by laser scanning microscopy (LSM; LSM 5 Pascal;
Zeiss). Endothelial cell (CD31-positive) number was assessed by
FACS (3 independent counts of 10 000 cells; FACScan, Becton
Dickinson).
EHT Construction
EHTs were constructed as described previously.11 Briefly, acetic
acid solubilized collagen type I was mixed with concentrated culture
medium (2x DMEM, 20% horse serum, 4% chick embryo extract,
200 U/mL penicillin, 200 ␮g/mL streptomycin). The pH was
neutralized by titration with 0.1 N NaOH. Matrigel was added (10%
v/v) if indicated. Finally, cells were added to the reconstitution
mixture, which was thoroughly mixed before casting in circular
molds (inner diameter, 8 mm; outer diameter, 16 mm; height, 5 mm).
Within 3 to 7 days, EHTs coalesced to form spontaneously contracting circular structures and were transferred on automated stretch
devices11 or flexible holders for continuous culture under chronic
strain.
Isometric Force Measurements
Force of contraction (twitch tension) of EHTs was measured under
isometric conditions in standard organ baths as previously described
to assess responsiveness to calcium (0.2 to 2.8 mmol/L) and
isoprenaline (0.1 to 1000 nmol/L).11
RNA and DNA Isolation
RNA and DNA were isolated with Trizol (Invitrogen) and quantified
by spectrophotometry.
Protein Isolation and Western Blotting
EHTs were washed thoroughly in ice cold phosphate-buffered saline
and homogenized in lysis buffer containing 30 mmol/L Trishydroxyl aminomethane (pH 8.8), 5 mmol/L EDTA (pH 8), 3% SDS,
10% glycerol, 30 mmol/L NaF, and 2 ␮g/mL aprotinin. Protein was
measured by a modified Lowry assay (Bio-Rad DC Protein Assay)
using rat IgG as standard. Similar quantities of total proteins were
loaded on 7% SDS-polyacrylamide gels and separated electrophoretically. Similar loading was confirmed by Ponceau staining
after transfer onto polyvinylidene fluoride membranes. Blots were
probed with monoclonal antibodies directed against sarcomeric actin
Optimizing Engineered Heart Tissue
I-73
(clone 5C5; Sigma). Primary antibodies were detected with appropriate horseradish peroxidase coupled secondary antibodies. Signals
were enhanced with the ECL-plus kit (Amersham) and recorded with
a ChemiDoc system (Syngene). Signals were quantified with GeneTools software (Syngene).
Morphological Evaluation of EHT
Formaldehyde-fixed EHTs were embedded in paraffin, sectioned,
and stained with hematoxylin and eosin for light microscopic
evaluation or processed for LSM as described previously.11 Living
EHTs were incubated for 4 hour at 37°C with DiI-LDL (3,3⬘dioctadecylindocarbocyanine iodide–low-density lipoprotein; 10 ␮g/
mL) and analyzed by LSM after thorough washout off free DiI-LDL.
Statistical Analysis
Data are presented as mean⫾standard error of the mean. Statistical
differences were determined using paired 2-tailed Student t tests or
ANOVA when appropriate. P⬍0.05 was considered statistically
significant.
Statement of Responsibility
The authors had full access to the data and take full responsibility for
their integrity. All authors have read and agree to the manuscript as
written.
Results
Assessment of Cell Composition
Cell species were identified by staining for ␣-sarcomeric
actinin (cardiac myocytes; Figure 1a), prolyl-4-hydroxylase
(fibroblasts; Figure 1b), ␣-smooth muscle actin (smooth
muscle cells; Figure 1c), and CD31 (endothelial cells; analyzed by FACS). The native cell isolate contained 47⫾1%
cardiac myocytes and 49⫾3% fibroblasts. Preplating increased the cardiac myocyte fraction to 63⫾2% and decreased the fibroblast fraction to 33⫾3% (Figure 1d, 1e).
Smooth muscle cell (3% to 4%) and endothelial cell (2% to
3%) fractions were not affected by this procedure.
Differences of EHTs With Native and Cardiac
Myocyte-Enriched Cell Fractions
Baseline force of contraction (systolic force) was markedly
higher in EHTs with native heart cell populations (n⫽17)
when compared with EHTs with cardiac myocyte enriched
heart cells (n⫽13; 0.7⫾0.06 versus 0.3⫾0.03 mN; Figure
2a). At the same time, resting tension (diastolic force) was
increased in EHTs with native heart cell populations
(0.4⫾0.04 versus 0.2⫾0.04 mN; Figure 2a). In addition,
inotropic responses to calcium (Figure 2b) and isoprenaline
(Figure 2c, 2d) were higher in the latter. DNA content was
higher in native heart cell EHTs (14⫾0.5 versus 12⫾0.7 ␮g;
P⬍0.05) despite a similar input cell number (2.5⫻106 cells/
EHT), but no microscopically distinguishable difference in
EHT cell number was observed. The only apparent morphological difference was an increased number of vascular
structures in native heart cell EHTs (Figure 3a to 3d).
Serum Replacement
Withdrawal of horse serum and chick embryo extract after
development of contractile EHTs (starting on culture day 7)
resulted in rapid ceasing of contractile activity.17 Earlier
studies had shown that EHTs benefit from addition of growth
factors.11 Thus, we performed an initial series of experiments
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July 4, 2006
Figure 1. Assessment of cell fractions in
isolated heart cells. Staining of actinin
(green; a), prolylhydroxylase (perinuclear:
green; b), and ␣-smooth muscle actin
(green; c) served to identify cardiac myocytes (CM), fibroblasts (FB), and smooth
muscle cells (SMC) in native and cardiac
myocyte-enriched cell populations.
Counterstaining was performed with propidium iodide and TRITC-phalloidin to
label nuclei and filamentous actin in red,
respectively. Cell fractions (d, e) in native
and cardiac myocyte enriched heart cell
populations. EC indicates endothelial
cells (analyzed by FACS). Bars: 50 ␮m.
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to test whether growth factors (alone or in combination) can
maintain cardiac myocytes in monolayer culture in the
absence of serum. All tested factors prevented atrophy of
cardiac myocytes to some extent, but only the combination of
all factors resulted in a cardiac myocyte phenotype essentially
undistinguishable from cardiac myocytes cultured in 10%
fetal calf serum (Figure 4). Consequently, a mixture of these
Figure 2. Contractile function of EHT with native and cardiac
myocyte enriched heart cell populations. Systolic and diastolic
force (a) of EHTs with native (n⫽17) and cardiac myocyte
enriched heart cell populations (n⫽13) at 0.4 mmol/L calcium.
Calcium concentration response curves (b). Isoprenaline concentration response curves (c, d) indicating absolute EHT forces
and the increase in twitch tension from baseline values at
0.4 mmol/L calcium. White bars/boxes indicate data from EHTs
with cardiac myocyte enriched heart cell populations. Black
bars/boxes indicate data from EHTs with native heart cell populations. *P⬍0.05 vs enriched heart cell population.
factors with additional baseline supplements maintained contractile EHTs in the absence of serum (Figure 5).
Withdrawal of Matrigel
Matrigel has been essential for the construction of EHTs with
cardiac myocyte enriched heart cell populations.10 The latter
effect is most likely explained by the high growth factor
content in Matrigel. Based on our observation that insulin
elicited strong beneficial effects on EHT development and
function,11 we tested whether its addition to the culture
medium would suffice to substitute Matrigel. Triiodothyronine (T3) supports sarcomere development and was also
tested as a Matrigel replacement. Interestingly, addition of
Matrigel was not essential for EHTs with native heart cell
populations. However, contractile force and responsiveness
Figure 3. Vascular structures in native heart cell EHTs. Hematoxylin and eosin staining (a, b) of vascular structures in EHTs.
Live labeling of endothelial cells (c, d) with DiI-LDL (red).
Naito et al
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Figure 4. Testing of growth factor effects
in monolayer cardiac myocyte cultures.
Freshly isolated cells were grown for 48
hours in the presence of the indicated
supplements.
to calcium and isoprenaline was markedly better if Matrigel
was included in the original EHT reconstitution mixture. T3
and insulin alone did not or only slightly increase force of
Matrigel-free EHTs (Figure 6a); however, simultaneous addition of both factors allowed generation of strongly contracting EHTs. Notably, addition of insulin and T3 during the first
culture day (24 hours) was sufficient to elicit its long-term
effects (Figure 6b). Essentially, T3 and insulin supplements
not only compensated the Matrigel effect but improved EHT
contractility above control (EHTs with Matrigel) levels (Figure 6c, 6d). These effects did not stem from enhanced EHT
cell number (no difference in DNA content and no apparent
structural differences; data not shown) but may be the result
of enhanced protein content in T3/insulin-treated EHTs when
compared with nontreated Matrigel-free EHTs (593⫾13 ver-
sus 479⫾15 ␮g/EHT, n⫽4 per group; P⬍0.05). Notably,
sarcomeric actin content was higher in the former EHTs
(Figure 6e) and we observed a clear positive correlation of
actin content and force of contraction (Figure 6f).
Constructing Complex EHTs
Small tissue-engineered cardiac muscle may not suffice to
function as surrogate heart tissue in vivo. Here, we used the
propensity of EHTs to fuse during in vitro culture to create
several EHT geometries, namely, star-shaped EHT patches
(fusion predominately in the center of the EHTs; Figure 7a;
supplemental video 1), tubular constructs (fusion along the
side of EHT rings; Figure 7b; supplemental video 2), EHT
networks by cutting open single EHTs in one point and
weaving them together (Figure 7c; supplemental video 3), or
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Figure 5. EHT culture in serum-free culture medium. Force of
contraction at 2.8 mmol/L calcium of EHTs cultured under standard conditions (DMEM, 10% horse serum, 2% chick embryo
extract; n⫽7) and with serum supplementation during the final 5
days of EHT culture (n⫽8). EHTs without growth factor supplements did not develop active contractions (n⫽4). Bottom panel:
Composition of the developed serum-free culture medium (in
DMEM).
EHT “ropes” by twirling several EHTs around each other
(Figure 7d; supplemental video 4). Fusion was established
within 24 to 48 hours, yielding in-unison contracting novel
heart muscle constructs. EHTs could be cultured for at least 1
month under the described in vitro conditions. Single and
complex EHTs developed thin cardiac myocyte networks (10
to 20 ␮m) and thick myocardial structures (⬇150 ␮m), which
were localized preferentially in the center and close to the
surface, respectively (Figure 8).
Discussion
The present study addressed several issues pertaining to the
improvement of so far developed tissue engineering concepts
and eventually its clinical application. Namely, use of native,
ie, not enriched, cell populations improved the contractile
performance of EHT. Serum-free and Matrigel-free culture
conditions could be established to reduce the fraction of
immunogenic EHT components. Finally, new means to construct more complex EHT structures were exploited demonstrating the possibility to generate large contractile cardiac
surrogate tissue in vitro.
The use of mixed cell populations in cardiac tissue engineering appears straightforward if one wishes to construct a
true heart muscle equivalent. Yet, non-myocytes tend to
overgrow cardiac myocytes in monolayer cultures. This is
apparently not the case in EHTs, which can be cultured in the
Figure 6. Supplementation of Matrigel. Force of contraction of
EHTs (a) at 2.8 mmol/L calcium. Medium supplements (I: 10
␮g/mL insulin and/or T: 0.1 nmol/L T3) were added during the
initial 7 days of EHT culture. Force of contraction of EHTs (b) at
2.8 mmol/L calcium. Supplements were added for 24 hours
starting at the day of EHT construction (ITd0), for 7 days (ITd7),
or for the total duration of EHT culture (ITd12). Calcium and isoprenaline concentration response curves (c, d) of control (Ctr),
Matrigel-free (-M), and ITd7-supplemented EHTs. Actin content
(e) of EHTs (bottom panel: representative blot of respective
samples). Correlation of EHT actin content (f) and maximal
twitch tension at 2.8 mmol/L. *P⬍0.05 vs -M
absence of otherwise commonly used cytostatic agents. Interestingly, large vascular structures formed in EHTs containing native heart cell populations, which may facilitate engraftment in vivo. The finding that non-myocytes facilitate
EHT development also imposes a potential caveat for future
application of stem cells in cardiac tissue engineering. Essentially, our data suggest that stem cells would have to be
differentiated into various cardiac cell types to establish and
maintain a cardiogenic environment.
The development of a serum-free chemically defined tissue
engineering concept will be essential to correspond to good
laboratory practice and good manufacturing practice guidelines. The choice of growth factors in our serum-free culture
mix was based on previous experience11 and findings by
others.15 Further optimization of the described medium is
warranted. This relates especially to the identification of the
“right” concentration of a single factor at the “right” time and
eventually the “right” combination of factors. The importance
of timing and combination of growth supporting factors is
exemplified by the finding that Matrigel can be replaced by
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Optimizing Engineered Heart Tissue
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Figure 7. Generation of different EHT
geometries. EHTs fuse after sustained
contact to form in-unison contracting
complex cardiac muscle constructs.
Star-shaped EHTs (a) were generated by
stacking 5 EHTs on a custom-made
holder. Single-unit EHTs fused in the
center. 5 EHTs (b) were grown on horizontal glass pipettes. Adjacent EHTs
fused to form a tubular construct. 6
EHTs (c) were cut open and layered to
form a contracting network. 3 EHTs (d)
were twirled together to form a longitudinal “rope” structure. Bars: 10 mm.
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adding insulin and T3 for only 24 hours at the beginning of
EHT culture. In fact, the necessity to only transiently stimulate cells within EHTs with T3 and insulin could indicate a
crucial phase for EHT development. We did indeed observe a
marked cell loss during the first three culture days in standard
EHT cultures (unpublished observation). The cause of cell
loss appeared to be mostly apoptotic cell death; however, an
involvement of necrosis and autophagia cannot be ruled out.
Consequently, substances that protect cells, and especially
cardiac myocytes, from apoptotic cell death may impose a
beneficial effect on EHT development. Several groups have
described anti-apoptotic and hypertrophic effects of insulin.18
In contrast, T3 may not directly protect from cell death but
induce expression of ␣-myosin heavy chain molecules and
thereby play a role in re-establishing sarcomere structures in
freshly isolated heart cells.19 Eventually, large-scale screening will be necessary to define an optimal cardiogenic
cocktail to facilitate the generation of structurally and functionally optimal EHTs. A “physiological” cell population in
EHTs may also contribute to the allocation of essential
factors. This could explain why contractile EHTs could be
generated in the current study also in the absence of Matrigel,
albeit with a low calcium and isoprenaline responsiveness.
This was formerly not possible when enriched heart cell
populations were used.10
The potential to fuse single EHTs to more complex and
essentially larger contractile heart muscle units is intriguing
and may offer a solution to the common problem of size
limitation in cardiac tissue engineering. Here, large constructs
consist of single small units that remain largely diffusible for
oxygen and metabolites. Core necrosis was not observed in
single-unit or in multi-unit EHTs, and cell debris stemming
from apoptosis during early stages of EHT development may
in fact be scavenged by macrophages within EHT.11 More
detailed studies on cell survival and eventually cell seeding
efficiency in EHTs are presently underway. Single-unit EHTs
developed maximal forces of 1.5 mN in the present study.
Exemplary force measurements in multi-unit EHTs demonstrated maximal forces of 1.5 to 3 mN. However, the latter
measurements were hampered by the nonuniform geometry
of the tissue constructs. This may in fact lead to an underestimation of force development. Ultimately, the utility of
structurally and functionally optimized EHTs in cardiac
regeneration must be tested in vivo.
Conclusion and Future Aspects
The present study suggests that tissue-engineered cardiac
muscle should be composed of a “physiological” heart cell
population and can be cultured in the absence of serum as
well as Matrigel. Fusion of single EHTs may eventually allow
enlargement of EHT to a clinical scale. However, several
issues remain to be addressed before tissue engineering may
be translated from a purely experimental stage to a clinically
applicable treatment. These include the identification of
clinically applicable cells, replacement of all non-human
components, and an unambiguous proof of the cardiac tissue
engineering concept in large animals.
Figure 8. Development of muscle strands in single and complex
EHTs. Networks (a) of single cardiac myocytes in a single-unit
EHT. Thick (b) muscle bundle (diameter: ⬇150 ␮m) inside a
star-shaped EHT. Staining of filamentous actin in green
(phalloidin-Alexa 488).
Sources of Funding
This study was supported by the German Research Foundation
(Deutsche Forschungsgemeinschaft; DFG Es 88/8-2 to T.E., DFG
GRK 750 A1 to W.H.Z., and DFG RO 1306/2-2 to S.R. and W.H.Z.),
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July 4, 2006
the German Ministry for Education and Research (BMBF 01GN
0124 and BMBF 01GN 0520 to T.E. and W.H.Z.), the Deutsche
Stiftung für Herzforschung (F29/03 to W.H.Z), the Novartis Foundation (W.H.Z.), the European Union (EUGeneHeart to T.E.), and
the Foundation Leducq (T.E. and W.H.Z.).
Disclosures
10.
11.
None.
References
Downloaded from http://circ.ahajournals.org/ by guest on June 18, 2017
1. Dimmeler S, Zeiher AM, Schneider MD. Unchain my heart: the scientific
foundations of cardiac repair. J Clin Invest. 2005;115:572–583.
2. Murry CE, Field LJ, Menasche P. Cell-based cardiac repair: reflections at
the 10-year point. Circulation. 2005;112:3174 –3183.
3. Eschenhagen T, Zimmermann WH. Engineering myocardial tissue. Circ
Res. 2005;97:1220 –1231.
4. Carrier RL, Papadaki M, Rupnick M, Schoen FJ, Bursac N, Langer R,
Freed LE, Vunjak-Novakovic G. Cardiac tissue engineering: cell seeding,
cultivation parameters, and tissue construct characterization. Biotechnol
Bioeng. 1999;64:580 –589.
5. Kofidis T, Akhyari P, Boublik J, Theodorou P, Martin U, Ruhparwar A,
Fischer S, Eschenhagen T, Kubis HP, Kraft T, Leyh R, Haverich A. In
vitro engineering of heart muscle: artificial myocardial tissue. J Thorac
Cardiovasc Surg. 2002;124:63– 69.
6. Leor J, Aboulafia-Etzion S, Dar A, Shapiro L, Barbash IM, Battler A,
Granot Y, Cohen S. Bioengineered cardiac grafts: A new approach to
repair the infarcted myocardium? Circulation. 2000;102:III56 –II61.
7. Li RK, Jia ZQ, Weisel RD, Mickle DA, Choi A, Yau TM. Survival and
function of bioengineered cardiac grafts. Circulation. 1999;100:II63–II9.
8. Shimizu T, Yamato M, Isoi Y, Akutsu T, Setomaru T, Abe K, Kikuchi A,
Umezu M, Okano T. Fabrication of pulsatile cardiac tissue grafts using a
novel 3-dimensional cell sheet manipulation technique and temperatureresponsive cell culture surfaces. Circ Res. 2002;90:e40.
9. Eschenhagen T, Fink C, Remmers U, Scholz H, Wattchow J, Weil J,
Zimmermann W, Dohmen HH, Schafer H, Bishopric N, Wakatsuki T,
12.
13.
14.
15.
16.
17.
18.
19.
Elson EL. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. FASEB J.
1997;11:683– 694.
Zimmermann WH, Fink C, Kralisch D, Remmers U, Weil J, Eschenhagen
T. Three-dimensional engineered heart tissue from neonatal rat cardiac
myocytes. Biotechnol Bioeng. 2000;68:106 –114.
Zimmermann WH, Schneiderbanger K, Schubert P, Didie M, Munzel F,
Heubach JF, Kostin S, Neuhuber WL, Eschenhagen T. Tissue engineering
of a differentiated cardiac muscle construct. Circ Res. 2002;90:223–230.
Zimmermann WH, Didie M, Wasmeier GH, Nixdorff U, Hess A, Melnychenko I, Boy O, Neuhuber WL, Weyand M, Eschenhagen T. Cardiac
grafting of engineered heart tissue in syngenic rats. Circulation. 2002;
106:I151–I157.
Brutsaert DL. Cardiac endothelial-myocardial signaling: its role in
cardiac growth, contractile performance, and rhythmicity. Physiol Rev.
2003;83:59 –115.
Nag AC, Zak R. Dissociation of adult mammalian heart into single cell
suspension: an ultrastructural study. J Anat. 1979;129:541–559.
Mohamed SN, Holmes R, Hartzell CR. A serum-free, chemically-defined
medium for function and growth of primary neonatal rat heart cell
cultures. In Vitro. 1983;19:471– 478.
Radisic M, Park H, Shing H, Consi T, Schoen FJ, Langer R, Freed LE,
Vunjak-Novakovic G. Functional assembly of engineered myocardium by
electrical stimulation of cardiac myocytes cultured on scaffolds. Proc
Natl Acad Sci U S A. 2004;101:18129 –18134.
Eschenhagen T, Didie M, Munzel F, Schubert P, Schneiderbanger K,
Zimmermann WH. 3D engineered heart tissue for replacement therapy.
Basic Res Cardiol. 2002;97(Suppl 1):I146 –I152.
Latronico MV, Costinean S, Lavitrano ML, Peschle C, Condorelli G.
Regulation of cell size and contractile function by AKT in cardiomyocytes. Ann N Y Acad Sci. 2004;1015:250 –260.
Gustafson TA, Bahl JJ, Markham BE, Roeske WR, Morkin E.
Hormonal regulation of myosin heavy chain and alpha-actin gene
expression in cultured fetal rat heart myocytes. J Biol Chem. 1987;
262:13316 –13322.
Optimizing Engineered Heart Tissue for Therapeutic Applications as Surrogate Heart
Muscle
Hiroshi Naito, Ivan Melnychenko, Michael Didié, Karin Schneiderbanger, Pia Schubert,
Stephan Rosenkranz, Thomas Eschenhagen and Wolfram-Hubertus Zimmermann
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Circulation. 2006;114:I-72-I-78
doi: 10.1161/CIRCULATIONAHA.105.001560
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2006 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
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http://circ.ahajournals.org/content/114/1_suppl/I-72
Data Supplement (unedited) at:
http://circ.ahajournals.org/content/suppl/2007/10/18/114.1_suppl.I-72.DC2
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