Bioe Paper

Generation of Bioartificial Heart Tissue by Combininga 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 ofischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The maindrawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus ofthis study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patchby combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineeringan artificial tissue of 11 cm2 in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) inSIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CCcontracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast toundirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology withwell-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shapedand random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrodearray 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 RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well asdistribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contactwith RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaMrepresent a promising step toward engineering a functional perfusion system, enabling the engineering ofvascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Introduction

Engineering of artificial cardiac constructs (CC) hasgained increasing interest in recent years, as it may offeran alternative method for the repair of ischemic or damagedheart tissue. Diverse attempts of generating cardiac graftshave been reported. These include stacking cell sheets ofcardiomyocyte monolayers,1 casting gel-based CC,2 orseeding 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 therapeuticrelevant dimensions, we combined a gel-based CC with de-cellularized porcine small intestinal submucosa (SIS).

Since its first utilization in 1987, SIS has been extensivelyused as a scaffold for tissue engineering approaches andrepresents a suitable biological material that is approved forclinical application (reviewed by Andree et al.).4 After de-celullarization, a detectable content of VEGF, bFGF, TGF-b,and TNF-a is still preserved.5,6 Due to the retention of thesefactors, SIS has the potential to support cellular attachment,

1Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation andVascular Surgery, Hannover Medical School, Hannover, Germany.

Departments of 2Haematology, Haemostaseology, Oncology and Stem Cell Transplantation and 3Cardiology and Angiology, HannoverMedical School, Hannover, Germany.

*These authors contributed equally as first authors.

TISSUE ENGINEERING: Part AVolume 20, Numbers 3 and 4, 2014 Mary Ann Liebert, Inc.DOI: 10.1089/ten.tea.2013.0184

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migration, proliferation, and differentiation.7 In our ownstudies, a positive impact on the cellular organization of ratneonatal cardiomyocytes (rCMs) seeded on the submucosalsurface of SIS was observed, leading to oriented contractionsof rCM parallel to the longitudinal axis (LA) of the SIS.8 Itwas assumed that the rCM align along the collagen fibers ofthe SIS which are mainly oriented parallel to the SIS LA.

One of the main challenges in the field is the generation ofa vital three-dimensional (3D) construct of clinically relevantdimensions, as nourishment of cells by diffusion only be-comes insufficient after a distance of *100200 mm from thesurface.9,10 Attempts to overcome this issue include the useof porous scaffolds,3 channeled matrices,11,12 implementationof oxygen-carrying molecules13,14 or intrinsic vascularizationstimuli15 within the tissue, or prevascularization16,17 of thebioartificial tissue to promote diffusion and enhance oxygenconcentration. In a recent study, in situ vascularization of gel-based CC by co-cultivation with arteriovenous loop cham-bers in vivo was investigated.18

The small intestinal segment can also be processed bypreserving the mesenteric arterial and venous pedicles,providing a perfusable matrix termed biological vascularizedmatrix (BioVaM).19,20 Here, we describe the construction of alarge, organized, and functional gel-based CC in combina-tion with SIS or BioVaM. Porcine decellularized small in-testinal segments served not only as a support for the cellularorganization of CM but also as a vascular bed for the in vitrovascularization of the CC. Implementing the BioVaM as abackbone of the bioartificial CC could lead to the generationof a functional perfusion system, and thus a vascularized andwell-organized 3D CC.

Materials and Methods

Animal care

This work was approved by the Institutional ReviewBoard and the local Animal Care. It was conducted accord-ing to local government policy (#05/937) and Committeeprotocols of Hannover Medical School and the ResearchAdvisory Committee. All animals received humane care incompliance with the European Convention on Animal Care.

Isolation of neonatal rat heart cells

Neonatal rat heart cells were isolated as previously re-ported.21 In brief, hearts from 1- to 3-day old SpragueDawleyrats were minced and enzymatically digested with 0.44mg/mL type II collagenase (Worthington Laboratories) and0.1mg/mL pancreatin (Sigma-Aldrich) at 37C. rCM popu-lation in the primary isolate was enriched either by centrifu-gation through a discontinuous Percoll gradient using twodensity solutions, 1.062 and 1.082g/mL, made from Percollreagent (GE Healthcare), or by using a preplating method, aspreviously described.8 In brief, isolates were cultivated for 1 hin culture medium and humidified at 37C in an atmospherewith 5% CO2. After the incubation period, the supernatantenriched for rCM was collected. Determined by fluorescence-activated cell sorting (FACS) analysis for troponin T (Sup-plemental Material and Methods; Supplementary Data areavailable online at www.liebertpub.com/tea), the resultingcell isolates contained 65% rCM after preplating and 77% rCMafter Percoll gradient (Supplementary Fig. S1A, B). The com-

position of culture medium used for all experiments was asfollows: DMEM:M199 in ratio 1:4 supplemented with 10%fetal calf serum (PAA), 5% horse serum (Gibco), 2mM L-Glutamin, 100U/mL penicillin, and 100mg/mL streptomycin.

Preparation of porcine small intestine

Porcine small intestine segments were isolated from Ger-man landrace pigs (1825kg) and stored in undiluted Braunol(B. Braun) at 4C. Tunica mucosa and tunica serosa of intestinalsegments were mechanically removed, and this was followedby a chemical decellularization in 4% sodium deoxycholateand 0.1% sodium azide solution in Millipore Water (for Bio-VaM: 0.5% sodium deoxycholate and 0.5% sodium dodecylsulfate) under continuous shaking (90 rpm) at room temper-ature (for BioVaM at 4C) for 2 h. SIS/BioVaM were washedwith phosphate-buffered saline (PBS) supplemented withvancomycin (1 g/L; Ratiopharm) and 1% patricin (50mg/mL;Biochrom AG) solution under continuous shaking for 10 daysat 4C, changing the solution daily. Finally, SIS/BioVaMwere sterilized by 150Gy gamma-ray irradiation. Before use,SIS/BioVaM were cut open along the LA, fixed in a metalframe (45 25mm) 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 ofthe submucosal side of SIS/BioVaM in a density of 3.6 105

per cm2 and incubated for 12 h. 4 106 preplating-enrichedneonatal rat heart cells dissolved in 621mL culture mediumweremixedwith 292mL 3D culturematrix collagen type I fromrat tails (Trevigen), 71mL H2O, 370mL gel medium (25% horseserum, 4% fetal calf serum, 2mM L- Glutamin, 100U/mLpenicillin, 100mg/mL streptomycin, 69% 2DMEM (1.348 gDMEM powder [Gibco, Invitrogen], and 0.037 g NaHCO3dissolved in 30mL water), 158mL Matrigel (BasementMembrane Matrix; BD), and neutralized with 63mL 0.4MNaOH. The gel-based CCwith a final volume of 1.575mLwascast either into a silicon mold to generate CC (SupplementaryFig. S2D, E) or onto the SIS preseeded with the Percoll-en-riched cell suspension leading to SIS-CC (Supplementary Fig.S2B, C). After 1 h of incubation in a humidified 37C atmo-sphere with 5% CO2, solidified SIS-CC and CC were coveredwith culture medium.

Macroscopic and microscopic observationof construct contractility

Rate and direction of construct contractions were observedusing an inverted optical microscope (CKX41; Olympus). Amotorized inverted research microscope (Axio Observer Z1;Zeiss) was used to record microscopic videos of contractingSIS-CC and CC.

Microelectrode array analysis

Electrophysiological analyses were performed using mi-croelectrode arrays (MEA) type 200/30iR-Ti. The MEA de-vice allowed for the determination of the constructcontraction behavior, that is, rhythm, frequency, strength,velocity, and transduction direction through the construct. Apiece of SIS-CC was placed on the MEA plate. Warm me-dium or alternatively 10mM isoproterenol hydrochloride

800 VUKADINOVIC-NIKOLIC ET AL.

dissolved in medium was added, and construct contractionswere recorded. Using the MC_Rack (Version 4.0.0; MultiChannel System) visual mapping option, the direction ofcontractile transduction through the construct was recorded.

Histological analysis

Samples were embedded in TissueTek O.C.T. compoundand frozen. Horizontal sections of 7mm thickness were fixedwith acetone for 5min at - 20C, blocked, and permeabilizedwith donkey serum diluted at 1:10 in PBS containing 1% BSAand 0.25% Triton X-100 for 20min at room temperature. Forstaining against CD31, the permeabilization step was omit-ted. Primary antibodies were incubated overnight at 4C.Sections were washed thrice with PBS followed by incuba-tion with a secondary antibody for 1 h at room temperature.Sections were washed and a nuclear stain was performedwith 4, 6-diamidino-2-phenylindole, dichloride (DAPI).Stained sections were analyzed and documented using aninverted research microscope (Axio Observer A1, Filter Set43: Cy3, Filter Set 44: Cy2, Filter Set 49: DAPI; Zeiss). Thefollowing primary antibodies were used: mouse anti-CD31(Millipore), mouse anti-endothelial nitric oxide synthase(eNOS; Becton, Dickinson and Company), monoclonal anti-a-sarcomeric actinin (Sigma), and monoclonal anti-connexin43 (Sigma). Mouse anti-rat CD31 (Serotec) and rabbit anti-sarcomeric a actinin (Abcam) antibodies were used for co-staining of cardiomyocytes and endothelial cells. Negativecontrols were performed by omission of the primary anti-bodies or with the appropriate isotype antibody. Cyanine-conjugated donkey anti-mouse and anti-rabbit antibodies( Jackson Research) as well as Alexa Fluor 488 goat anti-mouse (Invitrogen) served as secondary antibodies.

Cellular orientation on SIS surface

Live/Dead cell analysis was performed using Live/Dead,Viability/Cytotoxicity Kit (Invitrogen). Cellular orientationwithin the CC or SIS-CC was assessed using either AlexaFluor 488 phalloidin staining (200 units/mL; MolecularProbes, Invitrogen) to visualize all cells or tetra-methylrhodamine methyl ester staining (TMRM + , 25 nM;Invitrogen) to visualize vital rCMs. TMRM + is kown to ac-cumulate in mitochondria. Due to their high metabolic ac-tivity, rCMs contain a high number of mitochondria and canbe, therefore, distinguished by TMRM + staining from othercell types with a low content of mitochondria.22,23 Stainedconstructs were analyzed using an inverted microscope(Axio Observer A1, Filter Set 43: Cy3, Filter Set 44: Cy2, FilterSet 49: DAPI; Zeiss). Evaluation of the cellular orientation inthe constructs stained with phalloidin-DAPI was performedwith ImageJ Version 1.34s by applying the cell-countingfunction (Plugins/Analysis/Cell counter). Four cell classeswere distinguished according to their orientation: parallel tothe SIS LA, shifted between 3060 from the LA, and shifted*90 from the LA or without orientation. The cells wereassigned to their respective cell class, while the results win-dow 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 pro-vided by PD Dr. Wulf D. Ito, Department of Cardiology,

University Hospital Hamburg-Eppendorf, Hamburg. RHE-Acells were expanded and labeled with green and red fluo-rescent proteins (GFP/RFP) by lentiviral transduction.

Preparation of lentiviral supernatantsand cell transduction

VSV.G-pseudotyped lentivirus particles were generated in293T cells by calcium phosphate cotransfection of the self-inactivating lentivirus plasmid pHR-SIN-SEW (for eGFPexpression)25 or pHR-SIN-SR (for RedEXPRESS expression)26

along with the multideleted pCMV-DR8.91 packaging plas-mid and the pMD.G envelope plasmid.27 Schematic repre-sentation of the lentiviral vectors is shown in SupplementaryFigure S3. Virus supernatant, collected 36 and 48h aftertransfection, was cleared by centrifugation and filteredthrough a 0.45 mm-pore filter. Lentivirus particles were con-centrated about 100-fold by overnight centrifugation at8000 rpm and 10C. Virus pellet was resuspended in serum-free 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 withserial dilution of concentrated virus stock and FACS analysis72 h post-transduction to assess the number of GFP- andRFP-positive cells. Concentrated lentivirus stock (5mL di-luted in 2mL medium/well) was used to infect endothelialcells in a six-well plate. After overnight incubation, the cellswere 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 topof the SIS-CC. For the generation of BioVaM-CC, the BioVaMpedicles were infused with 800 mL of 6 106/mL of GFP + orRFP+ RHE-A into venous or arterial pedicle, respectively.After 2 h of incubation, a monolayer of Percoll-enriched rCMwas seeded onto the BioVaM submucosal side and after anadditional 1 h of incubation, the gel-based construct was ad-ded. BioVaM-CC was cultured under static conditions inculture medium used for neonatal rat heart isolates. The 3Darrangement of endothelial cells within the gel CC combinedwith SIS was recorded with Confocal Laser-Scanning Micro-scopy (CLSM; Leica Microsystems). For the detection of GFP-labeled cells, excitation/emission filter was set to 488nm/500600nm. RFP fluorescence was detected at an excitation of543 nm and an emission between 555620nm. An un-seeded/unstained SIS analyzed using the same exposure and excita-tion settings served as a negative control. Observation of RHEcell distribution in SIS-CC was conducted with an uprightfluorescence microscope (BX40, Filter set GFP and RFP;Olympus). Population of the BioVaM vessel bed was docu-mented with a fluorescence stereomicroscope (MZ FL III, RFPExcitation 546/10, Emission 570LP; Leica).

Statistics

All experiments were repeated at least thrice unless oth-erwise stated. Summaries of numeric data are given asmeans and standard deviation. One-way ANOVA test wasperformed using GraphPad Prism. A probability value of0.05 or less was considered significant.

SIS-BASED BIOARTIFICIAL HEART TISSUE 801

Results

Improved cellular alignment and morphologyof rCM in SIS-CC compared with CC

In previous studies, we demonstrated that rCM seeded asa monolayer on the submucosal side of the SIS re-align alongthe collagen fibers oriented parallel to the LA of the SIS.8 In

order to determine whether the SIS morphology affects thearrangement of rCM seeded in a 3D manner, a gel-based CCwas cast onto SIS preseeded with a monolayer of neonatal ratheart cells enriched for rCMs by Percoll gradient. Seeding theSIS with a monolayer of rCM served as an adhesive betweenSIS and CC. The CC without supporting SIS (Fig. 1A) wasanalyzed in comparison to the SIS-CC combination (Fig. 1B).

FIG. 1. Compared with gel cardiacconstruct (CC) (A, C, E, G), combina-tion of gel CC with SIS preseeded witha monolayer of Percoll purified neo-natal rat heart cell isolates (B, D, F, H)leads to improved cellular alignment,morphology, and contractility after 10days of cultivation. (A) Gel-based CCin silicon mold after 10 days of culti-vation. Culture medium was removedto visualize the construct. (B) CCcasted on top of preseeded SISmounted in a metal frame after 10 daysof cultivation (SIS-CC). Enlargementshows the rim of the gel-based CC(indicated by *). (C, D) Phalloidinstaining of whole constructs against allcell types. (E, F) TMRM + staining ofwhole constructs to visualize cardio-myocytes. (G, H) Staining of cryosec-tions against a-sarcomeric actinin after10 days of cultivation. Nuclei werecounterstained with DAPI. (I) Evalua-tion of cell orientation based on phal-loidin and DAPI staining within SIS-CC over 14 days of incubation (n = 3,bars represent mean SD, PLA: paral-lel to SIS longitudinal axis, 3060%:3060% shifted to SIS longitudinalaxis, 90%: 90% shifted to the SIS lon-gitudinal axis, without: without anyorientation). ( J) Contractile activity ofCC vs. SIS-CC. One-way ANOVA;asterisk: p< 0.05; n = 10, bars representmean SD. Arrows indicate the longi-tudinal axis of the SIS. DAPI, 4, 6-diamidino-2-phenylindole, dichloride;SIS, small intestinal submucosa. Colorimages available online at www.liebertpub.com/tea


During cultivation, a strong shrinkage of the CC in all threedimensions was observed when cast into the silicon mold (Fig.1A). In combination with SIS, marginal shrinkage in lengthand width was detected (Fig. 1B), while shrinkage in heightwas not prevented (data not shown). Although the dimensionsof the constructs CC and SIS-CC differ after 10 days of culti-vation, cell viability determined by Live/Dead assay was un-affected (Supplementary Fig. S4). Cell alignment in the CC wasrandomly oriented (Fig. 1C, E, G) compared with the parallelalignment along the LA of the SIS in the SIS-CC (Fig. 1D, F, H).Cell alignment analysis of the SIS-CC stained with Phalloidinresulted in approximately 11%6% cells oriented along theLA on day 2, increasing to 41% 4% on day 4, and 54%6%on day 10 (Fig. 1I). The morphology of the rCMwithin the SIS-CC exhibited an elongated, typical rod-shaped structure withproperly developed cross-striations (Fig. 1H).

rCM exhibit higher contractile activity in SIS-CCcompared with CC

CC generated on the SIS maintained their contractility overthe entire incubation time with a rate of 20878 beats/min onday 3, and 15448 beats/min on day 10 (Fig. 1J). The con-traction frequency of rCM in the CC was significantly lowerand peaked at 4329 beats/min on day 7 of cultivation. SIS-

CC contracted synchronously, suggesting the formation of afunctionally coupled syncytium throughout the entire construct(*11 cm2) in the direction of the LA of the SIS (SupplementaryVideo A). In contrast, contractions of the CC were arrhythmicand spatially not organized (Supplementary Video B).

rCM in SIS-CC show electric couplingand react to b-adrenergic stimulation

Electrophysiological analyses of the SIS-CC were per-formed at day 10 of cultivation using an MEA device. Mea-surements revealed rhythmic contractions at a rate of *200beats/min. The signal was transduced parallel to the LA ofthe SIS with a velocity of 4 cm/s (Fig. 2A). b-adrenergicstimulation with 10 mM Isoproterenol led to a reversible in-crease of the contraction frequency from *200 beats/min to*360 beats/min (Fig. 2B). In addition, cells within the con-struct stained positive for connexin 43, suggesting the pres-ence of functional gap junctions (Fig. 2C).

Endothelial cells re-assemble into a networkin the cardiac compartment

In CC, the formation of a network structure became ap-parent from day 7 onward (Fig. 3A, B). In SIS-CC, this

FIG. 2. Cells within the SIS-CC transduce electrical sig-nals and respond to chemicalstimulation. (A) Microelec-trode array analyses of thecontraction rhythm, contrac-tion frequency, and contrac-tion strength and signaltransduction direction on day10 of cultivation. Direction ofsignal transduction is shownas spatiotemporal color map-ping of excitation propaga-tion from a trigger electrode(red: early signal, blue: latesignal). (B) Contractile activ-ity of the SIS-CC before, dur-ing, and after adding 10 mMIsoproterenol at day 10 ofcultivation. (C) Staining ofhorizontal cryosection of SIS-CC after 10 days of cultiva-tion against Connexin 43 anda-sarcomeric actinin. Nucleiwere counterstained withDAPI. Arrows indicate thelongitudinal axis of the SIS.Color images available onlineat www.liebertpub.com/tea


phenomenon was delayed (Fig. 3C), but could be detectedfrom day 10 of cultivation onward (Fig. 3D). Cells formingthis network stained positive for endothelial markers CD31(Fig. 3E) and eNOS (data not shown), suggesting self-assembly of endothelial cells co-isolated in the neonatal ratheart cell preparation. In addition, CD31-positive cells werelocated adjacent to cardiomyocytes (Fig. 3F).

RHE-A cells migrate through the CC and co-localizewith 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 co-localized with the pre-existing vascular structures of thedecellularized SIS (Fig. 4B, C). Co-localization with vascularstructures extended over several millimeters in length.

RHE-A cells migrate from the vessel bedof the BioVaM into the CC and connectwith rat endothelial cells

To create a perfusable construct, the SIS in the SIS-CC wasreplaced by the BioVaM. The BioVaM contains the arterialand venous pedicles in addition to the SIS (Fig. 4E). GFP +

RHE-A cells and RFP + RHE-A cells were infused into thepreserved vein and artery, respectively. The CC was cast ontop of the submucosal side of the BioVaM (Fig. 4D). After 10days of static cultivation, GFP + and RFP + RHE-A cells weredistributed throughout the whole CC (Fig. 4F, H). A densenetwork composed of GFP + and RFP+ RHE-A cells was

identified (Fig. 4F). Moreover, RHE-A cells re-populated thepre-existing vessel bed of the BioVaM (Fig. 4IK).

In order to investigate the inter-cellular connectivity be-tween GFP +/RFP + RHE-A cells infused through the Bio-VaM vessel pedicles and rat endothelial cells co-isolated inthe neonatal rat heart cell preparation and thereby seededwithin the gel CC, immuno-fluorescence staining againstCD31 was performed. Since RHE-A cells showed no stainingfor CD3124 and primary rat endothelial cells stained positivefor CD31, differential assessment and localization of thesethree cell types was feasible. Using confocal laser scanningmicroscopy, GFP +/RFP + RHE-A cells and primary rat en-dothelial cells were detected within the BioVaM-CC. GFP +

and RFP+ RHE-A cells appeared in close proximity to theCD31 + endothelial cells, suggesting a connection betweenthese cells in the CC (Fig. 4G).

Discussion

In this study, we have established a hybrid artificial tissuecomprising a gel-based CC placed on the submucosal side ofSIS, thereby engineering an *11 cm2 large, functional car-diac patch with high contractility. A cell mixture isolatedfrom neonatal rat hearts served as a working model to gaininsights into the cellular organization and function of suchCC under in vitro conditions. Mounting the CC to the surfaceof SIS seeded with a monolayer of Percoll gradient-enrichedrCM, which was favorable to achieve a functional fusion ofboth components, resulted in spontaneous, simultaneous,and unidirectional contraction of the patch (Supplementary

FIG. 3. Network formation by endothe-lial cells within CC and SIS-CC. (AD)Live/Dead assay (green: viable cells, red:dead cells). (A, B) CC on day 7 (A) andday 10 (B) of cultivation. (C, D) SIS-CC onday 7 (C) and day 10 (D) of cultivation.(E) Staining of CC against CD31 after 10days of cultivation. Nuclei were counter-stained with DAPI. (F) Staining of hori-zontal cryosections from SIS-CC after 10days of cultivation against CD31 and a-sarcomeric actinin. Nuclei were counter-stained with DAPI. Arrows indicate thelongitudinal axis of the SIS. Color imagesavailable online at www.liebertpub.com/tea


video A). Direct comparison of the CC with SIS-CC revealeda tremendous improvement regarding rCM cellular align-ment, morphology, and the establishment of a regular con-tractile rate and contractile direction in the presence of SIS(Fig. 1). Although cardiac, 3D gel-based constructs have beenused by several other groups,2,22 most of these constructs

required mechanical or electrical stimulation to elicit syn-chronous beating and orientated alignment of introducedCM.22,2831 In contrast, spontaneous, synchronized, andaligned contractile activity in our hybrid approach wasachieved without mechanical or electrical stimulation. Thisfunctional self-organization in our constructs could be an

FIG. 4. RHE-A cells migrate through the cardiac compartment of the SIS-CC and co-localize with the vessel bed of the SIS. Viceversa, RHE-A cells injected into the BioVaM vessel bed contribute to the endothelial network build in the CC. (A) Schematicillustration of the implementation of GFP+ rat heart endothelial line (RHE-A) cells as a monolayer on top of the SIS-CC. (B) Liveimaging 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 wereinfused into artery andGFP+ RHE-A cells into venous vessel bed of the BioVaM. Complexes were analyzed after 10 days of staticincubation. 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 ametal framewith 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 1.5mm, 20 ). (G)CLSMof BioVaM-CC complex stained against CD31 after10 days of cultivation. Arrows indicate contact of GFP+ RHE-A, RFP+ RHE-A, and CD31+ rat endothelial cells. (H)Cross-sectionof BioVaM-CC complex after 10 days of incubation. (I) Fluorescence image of re-cellularization of companion venous and arterialvessel bed of BioVaMwithGFP+ RHE-A andRFP+ RHE-A, respectively. ( J)Corresponding brightfield picture of BioVaM shownin (K). (K) Fluorescence image of complete BioVaM re-seeded with RFP+ RHE-A in arterial vessel bed after 5 days of staticcultivation. BioVaM, biological vascularizedmatrix; CLSM,Confocal Laser-ScanningMicroscopy;GFP, green fluorescent protein;RFP, red fluorescent protein. Color images available online at www.liebertpub.com/tea


advantage, as mechanical stress applied by other approachescan result in CM hypertrophy,22,32,33 a compensatory re-sponse to the change in mechanical load in vivo, ultimatelyleading to adverse events.34 To investigate the impact ofmechanical and electrical stimulation and to assess me-chanical performance and strength in our constructs, exper-iments in a bioreactor system are ongoing.

Visual mapping of the construct field potential recordedwith MEA showed a tendency of signal transduction parallelto the LA of the SIS (Fig. 2), an observation in line withhistological data (Fig. 1) and previous findings with rCMseeded as a monolayer on SIS as well.8 Furthermore, MEAdata confirmed rhythmic contractions of the constructs witha rate of *200 beats/min and an electrical impulse velocityof 4 cm/s (Fig. 2). This impulse velocity was lower than acorresponding value of 25.4 cm/s measured in intact neo-natal hearts of 2 day-old SpragueDawley rats.35 In electricalnonstimulated constructs made of neonatal rat heart cellscultured on highly porous collagen scaffolds, Radisic et al.36

determined an electrical impulse velocity of 8.6 cm/s. Al-though this value is about two-fold higher compared withour findings, envisioned transplantation of our constructmight lead to further maturation and improvement of theimpulse propagation velocity in vivo. An augmentation of thebeating frequency of our construct was observed as a re-sponse to short-term application of Isoproterenol, indicatinga functional adrenergic response of rCM in the construct (Fig.2). Detection of connexin 43, often used as a marker of in-tercellular electrical coupling,37,38 revealed the presence ofgap junctions throughout the SIS-CC (Fig. 2). Collectively,these data suggest the formation of a functional syncytiumwithin the cardiac compartment with electrical impulsespropagating between communicating cells via gap junctions,thereby building a single contractile unit.

In CC as well as in SIS-CC, an organization of co-isolatedendothelial cells into an irregular network penetrating the gelCC was detected (Fig. 3). The self-organization of endothelialcells is a common observation within gel-based constructsin vitro.39,40 To explore whether this network could be sup-plemented by externally supplied endothelial cells, GFP +

RHE-A cells were added to the SIS-CC. It became evidentthat the GFP + RHE-A cells not only connected with the ratendothelial cells within the CC, but also migrated throughthe CC and re-populated the underlying SIS vessel structures(Fig. 4). The presence of chemoattractant molecules andgrowth factors such as VEGF, bFGF, TGF-beta, and TNF-alpha, which are known to be maintained in SIS after de-cellularization,5,6,41 could explain the rapid and well-definedvascular re-cellularization in our constructs. Notably, Li et al.demonstrated that small-molecular-weight peptides presentin porcine SIS are directly involved in the recruitment ofendothelial cells.42

The main challenge in generating a cardiac substitute re-mains the appropriate supply of tissue-forming CM withessential nutrients and oxygen, as adequate diffusion intosolid cell aggregates is limited to *100200 mm distancefrom the surface.43 A solution to this issue might be achievedby different strategies, for example, by generating a pre-vascularized tissue in vitro or by promoting neo-vasculari-zation after transplantation in vivo.16,17,44,45 Lesman et al.demonstrated that implantation of so-called tri-culture con-structs generated by seeding human embryonic stem cell-

derived CM, human umbilical vein endothelial cells, andmouse embryonic fibroblasts on biodegradable porous scaf-folds onto rat hearts resulted in the functional integration ofthe donor- and host-derived vasculature.16 Although thesestrategies are promising, induction of angiogenesis in theartificial tissue alone is probably insufficient to create con-structs of clinically relevant dimensions in vitro if not per-fused. Synthetic or biological scaffolds with an inherentvascular bed can be used to provide a perfusable system thatis already functional in vitro. Attempts to create cardiac tis-sues containing endothelial cells in combination with perfu-sable, micro-channeled matrices have been reported.12

Decellularization of vascularized tissues results in the gen-eration of perfusable, biological matrices. For example, de-cellularized whole hearts or parts thereof exhibited potentialfor re-cellularization with different cell types.4650 These ma-trices could be favorable for cardiac tissue engineering, asthey provide the appropriate surface for cardiomyocyte at-tachment and survival. However, in our own studies, com-patibility of cardiomyocytes and SIS has been demonstrated.8

Furthermore, SIS and products thereof are approved by theU.S. Food and Drug Administration and are already appliedin the clinic. It has been demonstrated that a native, non-decellularized BioVaM can be used as an autologous trans-plant for myocardial reconstruction.51

Here, we have used the BioVaM derived from the porcinesmall intestine as a perfusable, biological matrix.19,20 Bysubstituting SIS for BioVaM as a support for the CC, we havegenerated a perfusable cardiac patch exhibiting the sameproperties as the SIS-CC. By infusing GFP +/RFP + RHE-Acells through the BioVaM vessel pedicles, we achieved re-cellularization of the BioVaM vessel bed and observed mi-gration and network formation of the infused cellsthroughout the gel CC placed on top of the submucosal sideof the BioVaM (Fig. 4). The emerging network was composedfrom RHE-A cells as well as from rat endothelial cells co-isolated with the rCMs, strongly suggesting a cellular inte-gration of both cell types. However, further improvement ofthe prevascularization of the cardiac compartment and of thefunctional connection to endothelial cells infused through theBioVaM pedicles with endothelial cells in the CC is necessaryto obtain a completely perfusable construct. Subsequently,the height of the construct could be successively increased bycasting several layers of a gel-based CC on top of each other.In between each step, the construct should be cultivatedunder perfusion to enhance vascularization before addingthe next layer. By this stepwise procedure, a construct withclinically relevant dimensions could be engineered. Fortherapeutic applications, our approach has to be translated tothe human system, ideally utilizing autologous, patient de-rived cells.

Discovery of human embryonic stem cells and inducedpluripotent stem cells (hiPSC)52 and the derivation of cardi-omyocytes thereof offer new perspectives for the generationof transplantable human cardiac tissue in vitro.53 Successfulgeneration of constructs based on cell sheet technology54,55 aswell as gel-based CC56 by using hiPSC-derived CM havebeen reported. Repopulation of the BioVaM vessel bed withhuman cord blood-derived endothelial cells has been previ-ously demonstrated by our group.19 The ideal solutionwould be the re-endothelialization by hiPSC-derived endo-thelial cells.


Translation of our approach to a humanized system byusing cardiomyocytes, endothelial and mural cells, ideallyderived from human pluripotent stem cells, would be adefinite goal. Till now, this approach has been hampered bythe limited amount of cells available after hiPSC differenti-ation and their degree of maturation. When the problem ofmass production and maturation of hiPSC-derived cells issolved, the constructs generated from human cells should betested in vivo by transplantation into immuno-suppressedpigs, a large animal model with a cardiovascular systemsimilar to those of humans.

In summary, we successfully engineered a cardiac patchby combining a gel-based construct with a decellularized,biological matrix. Application of the BioVaM represents afirst step toward generating an in vitro prevascularized andperfusable construct with the potential to create clinicallyrelevant dimensions.

Acknowledgments

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

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Andres Hilfiker, PhD

Leibniz Research Laboratories for Biotechnologyand Artificial Organs (LEBAO)

Department of Cardiothoracic, Transplantationand Vascular Surgery

Hannover Medical SchoolCarl Neuberg Str. 1D-30625 Hannover

Germany

E-mail: [email protected]

Received: March 22, 2013Accepted: September 25, 2013

Online Publication Date: November 18, 2013


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