TISSUE ENGINEERING: Part A

Volume 20, Numbers 3 and 4, 2014

Mary Ann Liebert, Inc.
DOI: 10.1089/ten.tea.2013.0184

Generation of Bioartificial Heart Tissue by Combining

a Three-Dimensional Gel-Based Cardiac Construct with
Decellularized Small Intestinal Submucosa
Zlata Vukadinovic-Nikolic, PhD,1,* Birgit Andree, PhD,1,* Suzanne E. Dorfman, PhD,1 Michael Pflaum, MSc,1
Tibor Horvath, BSc,1 Marco Lux, MSc,1 Letizia Venturini, PhD,2 Antonia Bar, PhD,1 George Kensah, PhD,1
Angelica Roa Lara, PhD,1 Igor Tudorache, MD,1 Serghei Cebotari, MD,1
Denise Hilfiker-Kleiner, PhD,3 Axel Haverich, MD,1 and Andres Hilfiker, PhD1

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of
ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main
drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of
this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch
by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering
an artificial tissue of 11 cm2 in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in
SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were coisolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize preexisting vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC
contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to
undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with
well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped
and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode
array measurements. A dense network of CD31 + /eNOS + cells was detected as permeating the whole construct.
Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC,
resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHEA cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as
distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact
with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM
represent a promising step toward engineering a functional perfusion system, enabling the engineering of
vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Introduction

ngineering of artificial cardiac constructs (CC) has

gained increasing interest in recent years, as it may offer
an alternative method for the repair of ischemic or damaged
heart tissue. Diverse attempts of generating cardiac grafts
have been reported. These include stacking cell sheets of
cardiomyocyte monolayers,1 casting gel-based CC,2 or
seeding cardiomyocytes on scaffolds and matrices.3 So far,
artificial CC created by the methods mentioned earlier are

restricted in size. To create a suturable CC of therapeutic

relevant dimensions, we combined a gel-based CC with decellularized porcine small intestinal submucosa (SIS).
Since its first utilization in 1987, SIS has been extensively
used as a scaffold for tissue engineering approaches and
represents a suitable biological material that is approved for
clinical application (reviewed by Andree et al.).4 After decelullarization, a detectable content of VEGF, bFGF, TGF-b,
and TNF-a is still preserved.5,6 Due to the retention of these
factors, SIS has the potential to support cellular attachment,

1
Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and
Vascular Surgery, Hannover Medical School, Hannover, Germany.
Departments of 2Haematology, Haemostaseology, Oncology and Stem Cell Transplantation and 3Cardiology and Angiology, Hannover
Medical School, Hannover, Germany.
*These authors contributed equally as first authors.

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migration, proliferation, and differentiation.7 In our own
studies, a positive impact on the cellular organization of rat
neonatal cardiomyocytes (rCMs) seeded on the submucosal
surface of SIS was observed, leading to oriented contractions
of rCM parallel to the longitudinal axis (LA) of the SIS.8 It
was assumed that the rCM align along the collagen fibers of
the SIS which are mainly oriented parallel to the SIS LA.
One of the main challenges in the field is the generation of
a vital three-dimensional (3D) construct of clinically relevant
dimensions, as nourishment of cells by diffusion only becomes insufficient after a distance of *100200 mm from the
surface.9,10 Attempts to overcome this issue include the use
of porous scaffolds,3 channeled matrices,11,12 implementation
of oxygen-carrying molecules13,14 or intrinsic vascularization
stimuli15 within the tissue, or prevascularization16,17 of the
bioartificial tissue to promote diffusion and enhance oxygen
concentration. In a recent study, in situ vascularization of gelbased CC by co-cultivation with arteriovenous loop chambers in vivo was investigated.18
The small intestinal segment can also be processed by
preserving the mesenteric arterial and venous pedicles,
providing a perfusable matrix termed biological vascularized
matrix (BioVaM).19,20 Here, we describe the construction of a
large, organized, and functional gel-based CC in combination with SIS or BioVaM. Porcine decellularized small intestinal segments served not only as a support for the cellular
organization of CM but also as a vascular bed for the in vitro
vascularization of the CC. Implementing the BioVaM as a
backbone of the bioartificial CC could lead to the generation
of a functional perfusion system, and thus a vascularized and
well-organized 3D CC.
Materials and Methods
Animal care
This work was approved by the Institutional Review
Board and the local Animal Care. It was conducted according to local government policy (#05/937) and Committee
protocols of Hannover Medical School and the Research
Advisory Committee. All animals received humane care in
compliance with the European Convention on Animal Care.
Isolation of neonatal rat heart cells
Neonatal rat heart cells were isolated as previously reported.21 In brief, hearts from 1- to 3-day old SpragueDawley
rats were minced and enzymatically digested with 0.44 mg/
mL type II collagenase (Worthington Laboratories) and
0.1 mg/mL pancreatin (Sigma-Aldrich) at 37C. rCM population in the primary isolate was enriched either by centrifugation through a discontinuous Percoll gradient using two
density solutions, 1.062 and 1.082 g/mL, made from Percoll
reagent (GE Healthcare), or by using a preplating method, as
previously described.8 In brief, isolates were cultivated for 1 h
in culture medium and humidified at 37C in an atmosphere
with 5% CO2. After the incubation period, the supernatant
enriched for rCM was collected. Determined by fluorescenceactivated cell sorting (FACS) analysis for troponin T (Supplemental Material and Methods; Supplementary Data are
available online at www.liebertpub.com/tea), the resulting
cell isolates contained 65% rCM after preplating and 77% rCM
after Percoll gradient (Supplementary Fig. S1A, B). The com-

VUKADINOVIC-NIKOLIC ET AL.
position of culture medium used for all experiments was as
follows: DMEM:M199 in ratio 1:4 supplemented with 10%
fetal calf serum (PAA), 5% horse serum (Gibco), 2 mM LGlutamin, 100 U/mL penicillin, and 100 mg/mL streptomycin.
Preparation of porcine small intestine
Porcine small intestine segments were isolated from German landrace pigs (1825 kg) and stored in undiluted Braunol
(B. Braun) at 4C. Tunica mucosa and tunica serosa of intestinal
segments were mechanically removed, and this was followed
by a chemical decellularization in 4% sodium deoxycholate
and 0.1% sodium azide solution in Millipore Water (for BioVaM: 0.5% sodium deoxycholate and 0.5% sodium dodecyl
sulfate) under continuous shaking (90 rpm) at room temperature (for BioVaM at 4C) for 2 h. SIS/BioVaM were washed
with phosphate-buffered saline (PBS) supplemented with
vancomycin (1 g/L; Ratiopharm) and 1% patricin (50 mg/mL;
Biochrom AG) solution under continuous shaking for 10 days
at 4C, changing the solution daily. Finally, SIS/BioVaM
were sterilized by 150 Gy gamma-ray irradiation. Before use,
SIS/BioVaM were cut open along the LA, fixed in a metal
frame (45 25 mm) with the submucosal side facing up
(Supplementary Fig. S2A), and covered with culture medium.
Preparation of gel-based cardiac construct
Cells collected after Percoll gradient were seeded on top of
the submucosal side of SIS/BioVaM in a density of 3.6 105
per cm2 and incubated for 12 h. 4 106 preplating-enriched
neonatal rat heart cells dissolved in 621 mL culture medium
were mixed with 292 mL 3D culture matrix collagen type I from
rat tails (Trevigen), 71 mL H2O, 370 mL gel medium (25% horse
serum, 4% fetal calf serum, 2 mM L- Glutamin, 100 U/mL
penicillin, 100 mg/mL streptomycin, 69% 2 DMEM (1.348 g
DMEM powder [Gibco, Invitrogen], and 0.037 g NaHCO3
dissolved in 30 mL water), 158 mL Matrigel (Basement
Membrane Matrix; BD), and neutralized with 63 mL 0.4 M
NaOH. The gel-based CC with a final volume of 1.575 mL was
cast either into a silicon mold to generate CC (Supplementary
Fig. S2D, E) or onto the SIS preseeded with the Percoll-enriched cell suspension leading to SIS-CC (Supplementary Fig.
S2B, C). After 1 h of incubation in a humidified 37C atmosphere with 5% CO2, solidified SIS-CC and CC were covered
with culture medium.
Macroscopic and microscopic observation
of construct contractility
Rate and direction of construct contractions were observed
using an inverted optical microscope (CKX41; Olympus). A
motorized inverted research microscope (Axio Observer Z1;
Zeiss) was used to record microscopic videos of contracting
SIS-CC and CC.
Microelectrode array analysis
Electrophysiological analyses were performed using microelectrode arrays (MEA) type 200/30iR-Ti. The MEA device allowed for the determination of the construct
contraction behavior, that is, rhythm, frequency, strength,
velocity, and transduction direction through the construct. A
piece of SIS-CC was placed on the MEA plate. Warm medium or alternatively 10 mM isoproterenol hydrochloride

SIS-BASED BIOARTIFICIAL HEART TISSUE

dissolved in medium was added, and construct contractions
were recorded. Using the MC_Rack (Version 4.0.0; Multi
Channel System) visual mapping option, the direction of
contractile transduction through the construct was recorded.
Histological analysis
Samples were embedded in TissueTek O.C.T. compound
and frozen. Horizontal sections of 7 mm thickness were fixed
with acetone for 5 min at - 20C, blocked, and permeabilized
with donkey serum diluted at 1:10 in PBS containing 1% BSA
and 0.25% Triton X-100 for 20 min at room temperature. For
staining against CD31, the permeabilization step was omitted. Primary antibodies were incubated overnight at 4C.
Sections were washed thrice with PBS followed by incubation with a secondary antibody for 1 h at room temperature.
Sections were washed and a nuclear stain was performed
with 4, 6-diamidino-2-phenylindole, dichloride (DAPI).
Stained sections were analyzed and documented using an
inverted research microscope (Axio Observer A1, Filter Set
43: Cy3, Filter Set 44: Cy2, Filter Set 49: DAPI; Zeiss). The
following primary antibodies were used: mouse anti-CD31
(Millipore), mouse anti-endothelial nitric oxide synthase
(eNOS; Becton, Dickinson and Company), monoclonal antia-sarcomeric actinin (Sigma), and monoclonal anti-connexin
43 (Sigma). Mouse anti-rat CD31 (Serotec) and rabbit antisarcomeric a actinin (Abcam) antibodies were used for costaining of cardiomyocytes and endothelial cells. Negative
controls were performed by omission of the primary antibodies or with the appropriate isotype antibody. Cyanineconjugated donkey anti-mouse and anti-rabbit antibodies
( Jackson Research) as well as Alexa Fluor 488 goat antimouse (Invitrogen) served as secondary antibodies.
Cellular orientation on SIS surface
Live/Dead cell analysis was performed using Live/Dead,
Viability/Cytotoxicity Kit (Invitrogen). Cellular orientation
within the CC or SIS-CC was assessed using either Alexa
Fluor 488 phalloidin staining (200 units/mL; Molecular
Probes, Invitrogen) to visualize all cells or tetramethylrhodamine methyl ester staining (TMRM + , 25 nM;
Invitrogen) to visualize vital rCMs. TMRM + is kown to accumulate in mitochondria. Due to their high metabolic activity, rCMs contain a high number of mitochondria and can
be, therefore, distinguished by TMRM + staining from other
cell types with a low content of mitochondria.22,23 Stained
constructs were analyzed using an inverted microscope
(Axio Observer A1, Filter Set 43: Cy3, Filter Set 44: Cy2, Filter
Set 49: DAPI; Zeiss). Evaluation of the cellular orientation in
the constructs stained with phalloidin-DAPI was performed
with ImageJ Version 1.34s by applying the cell-counting
function (Plugins/Analysis/Cell counter). Four cell classes
were distinguished according to their orientation: parallel to
the SIS LA, shifted between 3060 from the LA, and shifted
*90 from the LA or without orientation. The cells were
assigned to their respective cell class, while the results window counted the number of cells related to each group.
Rat heart endothelial cell line cells
Rat heart endothelial cell line (RHE-A)24 was kindly provided by PD Dr. Wulf D. Ito, Department of Cardiology,

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University Hospital Hamburg-Eppendorf, Hamburg. RHE-A
cells were expanded and labeled with green and red fluorescent proteins (GFP/RFP) by lentiviral transduction.
Preparation of lentiviral supernatants
and cell transduction
VSV.G-pseudotyped lentivirus particles were generated in
293T cells by calcium phosphate cotransfection of the selfinactivating lentivirus plasmid pHR-SIN-SEW (for eGFP
expression)25 or pHR-SIN-SR (for RedEXPRESS expression)26
along with the multideleted pCMV-DR8.91 packaging plasmid and the pMD.G envelope plasmid.27 Schematic representation of the lentiviral vectors is shown in Supplementary
Figure S3. Virus supernatant, collected 36 and 48 h after
transfection, was cleared by centrifugation and filtered
through a 0.45 mm-pore filter. Lentivirus particles were concentrated about 100-fold by overnight centrifugation at
8000 rpm and 10C. Virus pellet was resuspended in serumfree X-VIVO10 medium (LONZA) and stored in aliquots at
- 80C until use.
Virus titer, typically ranging between 1 and 5 108 IU/
mL, was determined by infection of K562 cells with 1:10 with
serial dilution of concentrated virus stock and FACS analysis
72 h post-transduction to assess the number of GFP- and
RFP-positive cells. Concentrated lentivirus stock (5 mL diluted in 2 mL medium/well) was used to infect endothelial
cells in a six-well plate. After overnight incubation, the cells
were washed with PBS and fresh medium was added.
Seeding of SIS and BioVaM with GFP +
and RFP + RHE-A
GFP + RHE-A cells (2.5 104 per cm2) were seeded on top
of the SIS-CC. For the generation of BioVaM-CC, the BioVaM
pedicles were infused with 800 mL of 6 106/mL of GFP + or
RFP + RHE-A into venous or arterial pedicle, respectively.
After 2 h of incubation, a monolayer of Percoll-enriched rCM
was seeded onto the BioVaM submucosal side and after an
additional 1 h of incubation, the gel-based construct was added. BioVaM-CC was cultured under static conditions in
culture medium used for neonatal rat heart isolates. The 3D
arrangement of endothelial cells within the gel CC combined
with SIS was recorded with Confocal Laser-Scanning Microscopy (CLSM; Leica Microsystems). For the detection of GFPlabeled cells, excitation/emission filter was set to 488 nm/
500600 nm. RFP fluorescence was detected at an excitation of
543 nm and an emission between 555620 nm. An un-seeded/
unstained SIS analyzed using the same exposure and excitation settings served as a negative control. Observation of RHE
cell distribution in SIS-CC was conducted with an upright
fluorescence microscope (BX40, Filter set GFP and RFP;
Olympus). Population of the BioVaM vessel bed was documented with a fluorescence stereomicroscope (MZ FL III, RFP
Excitation 546/10, Emission 570LP; Leica).
Statistics
All experiments were repeated at least thrice unless otherwise stated. Summaries of numeric data are given as
means and standard deviation. One-way ANOVA test was
performed using GraphPad Prism. A probability value of
0.05 or less was considered significant.

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Results
Improved cellular alignment and morphology
of rCM in SIS-CC compared with CC
In previous studies, we demonstrated that rCM seeded as
a monolayer on the submucosal side of the SIS re-align along
the collagen fibers oriented parallel to the LA of the SIS.8 In

FIG. 1. Compared with gel cardiac

construct (CC) (A, C, E, G), combination of gel CC with SIS preseeded with
a monolayer of Percoll purified neonatal rat heart cell isolates (B, D, F, H)
leads to improved cellular alignment,
morphology, and contractility after 10
days of cultivation. (A) Gel-based CC
in silicon mold after 10 days of cultivation. Culture medium was removed
to visualize the construct. (B) CC
casted on top of preseeded SIS
mounted in a metal frame after 10 days
of cultivation (SIS-CC). Enlargement
shows the rim of the gel-based CC
(indicated by *). (C, D) Phalloidin
staining of whole constructs against all
cell types. (E, F) TMRM + staining of
whole constructs to visualize cardiomyocytes. (G, H) Staining of cryosections against a-sarcomeric actinin after
10 days of cultivation. Nuclei were
counterstained with DAPI. (I) Evaluation of cell orientation based on phalloidin and DAPI staining within SISCC over 14 days of incubation (n = 3,
bars represent mean SD, PLA: parallel to SIS longitudinal axis, 3060%:
3060% shifted to SIS longitudinal
axis, 90%: 90% shifted to the SIS longitudinal axis, without: without any
orientation). ( J) Contractile activity of
CC vs. SIS-CC. One-way ANOVA;
asterisk: p < 0.05; n = 10, bars represent
mean SD. Arrows indicate the longitudinal axis of the SIS. DAPI, 4, 6diamidino-2-phenylindole, dichloride;
SIS, small intestinal submucosa. Color
images available online at www
.liebertpub.com/tea

VUKADINOVIC-NIKOLIC ET AL.
order to determine whether the SIS morphology affects the
arrangement of rCM seeded in a 3D manner, a gel-based CC
was cast onto SIS preseeded with a monolayer of neonatal rat
heart cells enriched for rCMs by Percoll gradient. Seeding the
SIS with a monolayer of rCM served as an adhesive between
SIS and CC. The CC without supporting SIS (Fig. 1A) was
analyzed in comparison to the SIS-CC combination (Fig. 1B).

SIS-BASED BIOARTIFICIAL HEART TISSUE

During cultivation, a strong shrinkage of the CC in all three
dimensions was observed when cast into the silicon mold (Fig.
1A). In combination with SIS, marginal shrinkage in length
and width was detected (Fig. 1B), while shrinkage in height
was not prevented (data not shown). Although the dimensions
of the constructs CC and SIS-CC differ after 10 days of cultivation, cell viability determined by Live/Dead assay was unaffected (Supplementary Fig. S4). Cell alignment in the CC was
randomly oriented (Fig. 1C, E, G) compared with the parallel
alignment along the LA of the SIS in the SIS-CC (Fig. 1D, F, H).
Cell alignment analysis of the SIS-CC stained with Phalloidin
resulted in approximately 11% 6% cells oriented along the
LA on day 2, increasing to 41% 4% on day 4, and 54% 6%
on day 10 (Fig. 1I). The morphology of the rCM within the SISCC exhibited an elongated, typical rod-shaped structure with
properly developed cross-striations (Fig. 1H).
rCM exhibit higher contractile activity in SIS-CC
compared with CC
CC generated on the SIS maintained their contractility over
the entire incubation time with a rate of 208 78 beats/min on
day 3, and 154 48 beats/min on day 10 (Fig. 1J). The contraction frequency of rCM in the CC was significantly lower
and peaked at 43 29 beats/min on day 7 of cultivation. SIS-

803
CC contracted synchronously, suggesting the formation of a
functionally coupled syncytium throughout the entire construct
(*11 cm2) in the direction of the LA of the SIS (Supplementary
Video A). In contrast, contractions of the CC were arrhythmic
and spatially not organized (Supplementary Video B).
rCM in SIS-CC show electric coupling
and react to b-adrenergic stimulation
Electrophysiological analyses of the SIS-CC were performed at day 10 of cultivation using an MEA device. Measurements revealed rhythmic contractions at a rate of *200
beats/min. The signal was transduced parallel to the LA of
the SIS with a velocity of 4 cm/s (Fig. 2A). b-adrenergic
stimulation with 10 mM Isoproterenol led to a reversible increase of the contraction frequency from *200 beats/min to
*360 beats/min (Fig. 2B). In addition, cells within the construct stained positive for connexin 43, suggesting the presence of functional gap junctions (Fig. 2C).
Endothelial cells re-assemble into a network
in the cardiac compartment
In CC, the formation of a network structure became apparent from day 7 onward (Fig. 3A, B). In SIS-CC, this

FIG. 2. Cells within the SISCC transduce electrical signals and respond to chemical
stimulation. (A) Microelectrode array analyses of the
contraction rhythm, contraction frequency, and contraction strength and signal
transduction direction on day
10 of cultivation. Direction of
signal transduction is shown
as spatiotemporal color mapping of excitation propagation from a trigger electrode
(red: early signal, blue: late
signal). (B) Contractile activity of the SIS-CC before, during, and after adding 10 mM
Isoproterenol at day 10 of
cultivation. (C) Staining of
horizontal cryosection of SISCC after 10 days of cultivation against Connexin 43 and
a-sarcomeric actinin. Nuclei
were counterstained with
DAPI. Arrows indicate the
longitudinal axis of the SIS.
Color images available online
at www.liebertpub.com/tea

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VUKADINOVIC-NIKOLIC ET AL.

FIG. 3. Network formation by endothelial cells within CC and SIS-CC. (AD)

Live/Dead assay (green: viable cells, red:
dead cells). (A, B) CC on day 7 (A) and
day 10 (B) of cultivation. (C, D) SIS-CC on
day 7 (C) and day 10 (D) of cultivation.
(E) Staining of CC against CD31 after 10
days of cultivation. Nuclei were counterstained with DAPI. (F) Staining of horizontal cryosections from SIS-CC after 10
days of cultivation against CD31 and asarcomeric actinin. Nuclei were counterstained with DAPI. Arrows indicate the
longitudinal axis of the SIS. Color images
available online at www.liebertpub
.com/tea

phenomenon was delayed (Fig. 3C), but could be detected

from day 10 of cultivation onward (Fig. 3D). Cells forming
this network stained positive for endothelial markers CD31
(Fig. 3E) and eNOS (data not shown), suggesting selfassembly of endothelial cells co-isolated in the neonatal rat
heart cell preparation. In addition, CD31-positive cells were
located adjacent to cardiomyocytes (Fig. 3F).
RHE-A cells migrate through the CC and co-localize
with the pre-existing vessel bed of the SIS
GFP + RHE-A cells were seeded on top of the SIS-CC (Fig.
4A). After 10 days of cultivation, GFP + RHE-A cells colocalized with the pre-existing vascular structures of the
decellularized SIS (Fig. 4B, C). Co-localization with vascular
structures extended over several millimeters in length.
RHE-A cells migrate from the vessel bed
of the BioVaM into the CC and connect
with rat endothelial cells
To create a perfusable construct, the SIS in the SIS-CC was
replaced by the BioVaM. The BioVaM contains the arterial
and venous pedicles in addition to the SIS (Fig. 4E). GFP +
RHE-A cells and RFP + RHE-A cells were infused into the
preserved vein and artery, respectively. The CC was cast on
top of the submucosal side of the BioVaM (Fig. 4D). After 10
days of static cultivation, GFP + and RFP + RHE-A cells were
distributed throughout the whole CC (Fig. 4F, H). A dense
network composed of GFP + and RFP + RHE-A cells was

identified (Fig. 4F). Moreover, RHE-A cells re-populated the

pre-existing vessel bed of the BioVaM (Fig. 4IK).
In order to investigate the inter-cellular connectivity between GFP + /RFP + RHE-A cells infused through the BioVaM vessel pedicles and rat endothelial cells co-isolated in
the neonatal rat heart cell preparation and thereby seeded
within the gel CC, immuno-fluorescence staining against
CD31 was performed. Since RHE-A cells showed no staining
for CD3124 and primary rat endothelial cells stained positive
for CD31, differential assessment and localization of these
three cell types was feasible. Using confocal laser scanning
microscopy, GFP + /RFP + RHE-A cells and primary rat endothelial cells were detected within the BioVaM-CC. GFP +
and RFP + RHE-A cells appeared in close proximity to the
CD31 + endothelial cells, suggesting a connection between
these cells in the CC (Fig. 4G).
Discussion
In this study, we have established a hybrid artificial tissue
comprising a gel-based CC placed on the submucosal side of
SIS, thereby engineering an *11 cm2 large, functional cardiac patch with high contractility. A cell mixture isolated
from neonatal rat hearts served as a working model to gain
insights into the cellular organization and function of such
CC under in vitro conditions. Mounting the CC to the surface
of SIS seeded with a monolayer of Percoll gradient-enriched
rCM, which was favorable to achieve a functional fusion of
both components, resulted in spontaneous, simultaneous,
and unidirectional contraction of the patch (Supplementary

SIS-BASED BIOARTIFICIAL HEART TISSUE

805

FIG. 4. RHE-A cells migrate through the cardiac compartment of the SIS-CC and co-localize with the vessel bed of the SIS. Vice
versa, RHE-A cells injected into the BioVaM vessel bed contribute to the endothelial network build in the CC. (A) Schematic
illustration of the implementation of GFP + rat heart endothelial line (RHE-A) cells as a monolayer on top of the SIS-CC. (B) Live
imaging of the SIS-CC reveals GFP + RHE-A cells re-cellularizing the pre-existing vessel bed of SIS after 10 days of cultivation. (C)
Corresponding brightfield picture of B. (D) Schematic illustration of combination of CC and BioVaM. RFP + RHE-A cells were
infused into artery and GFP + RHE-A cells into venous vessel bed of the BioVaM. Complexes were analyzed after 10 days of static
incubation. Position of the optical section through the middle of the gel CC used for the CLSM analysis shown in (F) is indicated.
(E) Macroscopic view of BioVaM with arterial and venous pedicles. BioVaM was cut open longitudinally and mounted into a
metal frame with the submucosal side facing up. (F) CLSM volume projection. Arrows indicate network built by GFP + and RFP +
RHE-A cells after 10 days of cultivation (30 steps, each 1.5 mm, 20 ). (G) CLSM of BioVaM-CC complex stained against CD31 after
10 days of cultivation. Arrows indicate contact of GFP + RHE-A, RFP + RHE-A, and CD31 + rat endothelial cells. (H) Cross-section
of BioVaM-CC complex after 10 days of incubation. (I) Fluorescence image of re-cellularization of companion venous and arterial
vessel bed of BioVaM with GFP + RHE-A and RFP + RHE-A, respectively. ( J) Corresponding brightfield picture of BioVaM shown
in (K). (K) Fluorescence image of complete BioVaM re-seeded with RFP + RHE-A in arterial vessel bed after 5 days of static
cultivation. BioVaM, biological vascularized matrix; CLSM, Confocal Laser-Scanning Microscopy; GFP, green fluorescent protein;
RFP, red fluorescent protein. Color images available online at www.liebertpub.com/tea
video A). Direct comparison of the CC with SIS-CC revealed
a tremendous improvement regarding rCM cellular alignment, morphology, and the establishment of a regular contractile rate and contractile direction in the presence of SIS
(Fig. 1). Although cardiac, 3D gel-based constructs have been
used by several other groups,2,22 most of these constructs

required mechanical or electrical stimulation to elicit synchronous beating and orientated alignment of introduced
CM.22,2831 In contrast, spontaneous, synchronized, and
aligned contractile activity in our hybrid approach was
achieved without mechanical or electrical stimulation. This
functional self-organization in our constructs could be an

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advantage, as mechanical stress applied by other approaches
can result in CM hypertrophy,22,32,33 a compensatory response to the change in mechanical load in vivo, ultimately
leading to adverse events.34 To investigate the impact of
mechanical and electrical stimulation and to assess mechanical performance and strength in our constructs, experiments in a bioreactor system are ongoing.
Visual mapping of the construct field potential recorded
with MEA showed a tendency of signal transduction parallel
to the LA of the SIS (Fig. 2), an observation in line with
histological data (Fig. 1) and previous findings with rCM
seeded as a monolayer on SIS as well.8 Furthermore, MEA
data confirmed rhythmic contractions of the constructs with
a rate of *200 beats/min and an electrical impulse velocity
of 4 cm/s (Fig. 2). This impulse velocity was lower than a
corresponding value of 25.4 cm/s measured in intact neonatal hearts of 2 day-old SpragueDawley rats.35 In electrical
nonstimulated constructs made of neonatal rat heart cells
cultured on highly porous collagen scaffolds, Radisic et al.36
determined an electrical impulse velocity of 8.6 cm/s. Although this value is about two-fold higher compared with
our findings, envisioned transplantation of our construct
might lead to further maturation and improvement of the
impulse propagation velocity in vivo. An augmentation of the
beating frequency of our construct was observed as a response to short-term application of Isoproterenol, indicating
a functional adrenergic response of rCM in the construct (Fig.
2). Detection of connexin 43, often used as a marker of intercellular electrical coupling,37,38 revealed the presence of
gap junctions throughout the SIS-CC (Fig. 2). Collectively,
these data suggest the formation of a functional syncytium
within the cardiac compartment with electrical impulses
propagating between communicating cells via gap junctions,
thereby building a single contractile unit.
In CC as well as in SIS-CC, an organization of co-isolated
endothelial cells into an irregular network penetrating the gel
CC was detected (Fig. 3). The self-organization of endothelial
cells is a common observation within gel-based constructs
in vitro.39,40 To explore whether this network could be supplemented by externally supplied endothelial cells, GFP +
RHE-A cells were added to the SIS-CC. It became evident
that the GFP + RHE-A cells not only connected with the rat
endothelial cells within the CC, but also migrated through
the CC and re-populated the underlying SIS vessel structures
(Fig. 4). The presence of chemoattractant molecules and
growth factors such as VEGF, bFGF, TGF-beta, and TNFalpha, which are known to be maintained in SIS after decellularization,5,6,41 could explain the rapid and well-defined
vascular re-cellularization in our constructs. Notably, Li et al.
demonstrated that small-molecular-weight peptides present
in porcine SIS are directly involved in the recruitment of
endothelial cells.42
The main challenge in generating a cardiac substitute remains the appropriate supply of tissue-forming CM with
essential nutrients and oxygen, as adequate diffusion into
solid cell aggregates is limited to *100200 mm distance
from the surface.43 A solution to this issue might be achieved
by different strategies, for example, by generating a prevascularized tissue in vitro or by promoting neo-vascularization after transplantation in vivo.16,17,44,45 Lesman et al.
demonstrated that implantation of so-called tri-culture constructs generated by seeding human embryonic stem cell-

VUKADINOVIC-NIKOLIC ET AL.
derived CM, human umbilical vein endothelial cells, and
mouse embryonic fibroblasts on biodegradable porous scaffolds onto rat hearts resulted in the functional integration of
the donor- and host-derived vasculature.16 Although these
strategies are promising, induction of angiogenesis in the
artificial tissue alone is probably insufficient to create constructs of clinically relevant dimensions in vitro if not perfused. Synthetic or biological scaffolds with an inherent
vascular bed can be used to provide a perfusable system that
is already functional in vitro. Attempts to create cardiac tissues containing endothelial cells in combination with perfusable, micro-channeled matrices have been reported.12
Decellularization of vascularized tissues results in the generation of perfusable, biological matrices. For example, decellularized whole hearts or parts thereof exhibited potential
for re-cellularization with different cell types.4650 These matrices could be favorable for cardiac tissue engineering, as
they provide the appropriate surface for cardiomyocyte attachment and survival. However, in our own studies, compatibility of cardiomyocytes and SIS has been demonstrated.8
Furthermore, SIS and products thereof are approved by the
U.S. Food and Drug Administration and are already applied
in the clinic. It has been demonstrated that a native, nondecellularized BioVaM can be used as an autologous transplant for myocardial reconstruction.51
Here, we have used the BioVaM derived from the porcine
small intestine as a perfusable, biological matrix.19,20 By
substituting SIS for BioVaM as a support for the CC, we have
generated a perfusable cardiac patch exhibiting the same
properties as the SIS-CC. By infusing GFP + /RFP + RHE-A
cells through the BioVaM vessel pedicles, we achieved recellularization of the BioVaM vessel bed and observed migration and network formation of the infused cells
throughout the gel CC placed on top of the submucosal side
of the BioVaM (Fig. 4). The emerging network was composed
from RHE-A cells as well as from rat endothelial cells coisolated with the rCMs, strongly suggesting a cellular integration of both cell types. However, further improvement of
the prevascularization of the cardiac compartment and of the
functional connection to endothelial cells infused through the
BioVaM pedicles with endothelial cells in the CC is necessary
to obtain a completely perfusable construct. Subsequently,
the height of the construct could be successively increased by
casting several layers of a gel-based CC on top of each other.
In between each step, the construct should be cultivated
under perfusion to enhance vascularization before adding
the next layer. By this stepwise procedure, a construct with
clinically relevant dimensions could be engineered. For
therapeutic applications, our approach has to be translated to
the human system, ideally utilizing autologous, patient derived cells.
Discovery of human embryonic stem cells and induced
pluripotent stem cells (hiPSC)52 and the derivation of cardiomyocytes thereof offer new perspectives for the generation
of transplantable human cardiac tissue in vitro.53 Successful
generation of constructs based on cell sheet technology54,55 as
well as gel-based CC56 by using hiPSC-derived CM have
been reported. Repopulation of the BioVaM vessel bed with
human cord blood-derived endothelial cells has been previously demonstrated by our group.19 The ideal solution
would be the re-endothelialization by hiPSC-derived endothelial cells.

SIS-BASED BIOARTIFICIAL HEART TISSUE

Translation of our approach to a humanized system by
using cardiomyocytes, endothelial and mural cells, ideally
derived from human pluripotent stem cells, would be a
definite goal. Till now, this approach has been hampered by
the limited amount of cells available after hiPSC differentiation and their degree of maturation. When the problem of
mass production and maturation of hiPSC-derived cells is
solved, the constructs generated from human cells should be
tested in vivo by transplantation into immuno-suppressed
pigs, a large animal model with a cardiovascular system
similar to those of humans.
In summary, we successfully engineered a cardiac patch
by combining a gel-based construct with a decellularized,
biological matrix. Application of the BioVaM represents a
first step toward generating an in vitro prevascularized and
perfusable construct with the potential to create clinically
relevant dimensions.

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10.

11.

12.

13.

Acknowledgments
Rat heart endothelial cell line (RHE-A) was kindly provided
by PD Dr. Wulf D. Ito, Department of Cardiology, University
Hospital Hamburg-Eppendorf, Hamburg. This work was
supported by Deutscher Akademischer Austausch Dienst
(DAAD) foundation, the CORTISS foundation, REBIRTH
Cluster of Excellence, and Deutsche Forschungsgemeinschaft
(Project HA 13 06/9-1).

14.

15.

Disclosure Statement
No competing financial interests exist.

16.

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Address correspondence to:

Andres Hilfiker, PhD
Leibniz Research Laboratories for Biotechnology
and Artificial Organs (LEBAO)
Department of Cardiothoracic, Transplantation
and Vascular Surgery
Hannover Medical School
Carl Neuberg Str. 1
D-30625 Hannover
Germany
E-mail: hilfiker.andres@mh-hannover.de
Received: March 22, 2013
Accepted: September 25, 2013
Online Publication Date: November 18, 2013