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Magnetically Guided Recellularization of Decellularized Stented Porcine Pericardium-Derived Aortic Valve for TAVI

Ghodsizad, Ali*; Bordel, Viktor; Wiedensohler, Herrman; Elbanayosy, Ali*; Koerner, Michael Martin*; Gonzalez Berjon, Jose M.; Barrios, Roberto; Farag, Mina*; Zeriouh, Mohamad§; Loebe, Matthias; Noon, George Peter; Koegler, Gesine; Karck, Matthias; Ruhparwar, Arjang

doi: 10.1097/MAT.0000000000000110
Tissue Engineering\Biomaterials

Application of somatic stem cells for growth, proliferation, and differentiation in a three-dimensional pattern is an important aspect in tissue engineering. Here, we report on our bioreactor, which we applied for magnetically guided recellularization of nitinol-stented valve. Human-derived unrestricted somatic stem cells were cultured in medium in our pulsatile dynamic bioreactor for 4–6 days. Stented valves were prepared by decellularization of porcine pericardium and construction of stented tissue-engineered valves (n = 8). A magnetic field was created around the bioreactor to prevent the loss of cells. In the control group, no magnetic device was used (n = 4). Morphological characterization was assessed by immunohistochemical staining of paraffin sections and electron microscopy. The bioreactor enabled the preservation of physiologic culture conditions with aerobic cell metabolism and physiological pH values. Histological analysis showed homogeneous seeding of the pericardium with progenitor cells in the recellularized samples, whereas no cell seeding could be observed in the nonmagnetic group. Our magnetically guided multifunctional bioreactor allows for an efficient three-dimensional culturing of somatic stem cells on decellularized organ-specific matrix.

From the *Department of Surgery, Heart and Vascular Institute, Milton S. Hershey, Medical Center, College of Medicine, Hershey; Department of Cardiac Surgery, University of Heidelberg, Heidelberg, Germany; Department of Pathology, The Methodist Hospital, Joan and Sanford I. Weill Medical College, Houston, Texas; §Department of Cardiac Surgery, University of Cologne, Cologne, Germany; Department of Cardiac Surgery, The Methodist Hospital, Joan and Sanford I. Weill Medical College, Houston, Texas; and Institute for Cell Therapeutics, Heinrich-Heine University Duesseldorf, Duesseldorf, Germany.

Submitted for consideration October 2012; accepted for publication in revised form May 2014.

Disclosures: The authors have no conflicts of interest to report.

Reprint Requests: Ali Ghodsizad, MD, PhD, FETCS, Department of Surgery, Heart and Vascular Institute, Milton S. Hershey Medical Center, College of Medicine, The Pennsylvania State University, 500 University Drive, Hershey, PA 17033-0850. Email: aghodsi@gmx.org.

Since the first prosthetic treatment of heart valve disease in early 1960, valve replacement represents the most common therapy.1

Cell culture technology has made relevant progress in the field of three-dimensional cell cultures and tissue engineering.2–4 In addition to conventional, surgical valve replacement as the standard of care for several decades, transcatheter implantation techniques have entered the clinical routine application and can be seen as an efficient alternative therapy for treatment of elderly high-risk patients.5 These minimally invasive techniques will have major influence on the treatment of patients with valvular heart disease and can also be a future therapeutic solution for younger patients.

Unfortunately, the currently available prosthesis for transcatheter approach is bio-prosthetic and shows progressive calcification and degeneration after implantation. As shown by previous publications, even an accelerated degeneration can follow the structural distortions caused during the crimping procedure. So, still the transcatheter valves are primarily used in elderly high-risk patients.5,6 Tissue-engineered living heart-valve prosthesis with the ability to regenerate could solve these mentioned problems.

In this article, we describe an efficient cell seeding protocol in a pulsatile bioreactor by using magnetic cell guidance. A high-gradient magnetic field produced by a strong permanent magnet is used to retain the magnetically labeled cells at the level of the pericardium inside the bioreactor chamber.

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Methods

Bioreactor

The bioreactor (engineo GmbH, Kelkheim, Germany) consists of a transparent glass chamber (KGW-Isotherm GmbH, Karlsruhe, Germany) where the decellularized scaffolds can be placed in (total length: 250 mm/length of the insert: 120 mm/thickness of the wall: 1 mm). The reservoir of the medium and the chamber were connected by several silicone tubes. All components of the bioreactor were sterilized by plasma sterilization.

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Medium and Gas Perfusion

A closed pulsatile circulatory system was used to provide a pulsatile perfusion with flow rates up to 1.5 L/min and a pulsatile rate of 20 beats/min. Gas (95% ambient air, 5% CO2) exchange occurs by medium surface aeration inside the medium reservoir. Active transport of fresh gas into the reservoir is provided through a continuous active gas circulation. Low-glucose Dulbecco’s Modified Eagle’s medium (DMEM) medium/10−7 M dexamethasone and 30% good manufacturing practice-grade fetal calf serum (FCS) (GIBCO, Invitrogen, Karlsruhe, Germany) were used for culturing experiments in the bioreactor. To counteract contamination with bacteria, streptomycin/penicillin (100 μg/ml and 100 IU/ml) and gentamicin (40 μg/ml, GIBCO, Invitrogen) were added to the medium. Seven hundred milliliters of medium was used for each seeding experiment.

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Magnetic Device

A circular magnetic device (Miltenyi Biotec, Bergisch Gladbach, Germany) consisting of six magnets (each 1.1 Tesla) is placed around the bioreactor chamber. The stented tissue-engineered valves (STVs) were placed in the chamber at the level of the magnetic device (Figures 1 and 2).

Figure 1

Figure 1

Figure 2

Figure 2

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Immunophenotyping of USSCs

Flow cytometric assessment was accomplished on a BD FACSCanto flow cytometer for CD34+ (clone AC136), CD133+ CD133/2 (clone 293C3), and CD45+ (clone 30F11) (Miltenyi Biotec). All samples were incubated for 30 minutes and washed with phosphate-buffered saline (PBS) for each staining step; for fixation, paraformaldehyde (2% end concentration) was used.

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Cell Treatment

Mesenchymal cord blood cells were collected from human umbilical cord vein, called “unrestricted somatic stem cells” (USSCs). After collection, processing, and initial characterization of cord blood units, those that were suitable for banking were used after the donor mothers’ consent was collected. With the application of Sepax procedure (SEPAX Cell Processing Device, Sepax Genesis BPS, Hackensack, NJ), the isolation of highly purified mononuclear cell component with high granulocyte and red blood cell depletion by means of a density gradient medium is achieved, and cell numbers of 1 × 109 USSCs can be obtained within four to five passages.7,8

For the subsequent generation of USSC colonies, 30% GMP-grade FCS (GIBCO, Invitrogen), low-glucose DMEM medium/10−7M dexamethasone (GIBCO, Invitrogen) was used for the initiation of the cultures. Expansion of USSCs was performed in 30% GMP-grade FCS (GIBCO, Invitrogen), low-glucose DMEM medium in a closed system applying cell stacks (Costar Corning, Sigma-Aldrich, St. Louis, MO). Serum was identified as the most critical factor for generation and expansion, and FCS needs to be carefully preselected to allow for extensive amplification of multipotent USSCs.3,4 Up to 40 × 106 human cord blood-derived stem cells (USSCs) were used.

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Cell Labeling

Commercially available antibody–bead products CD34+ (clone AC136), CD133+ CD133/2 (clone 293C3), and CD45+ (clone 30F11) (Miltenyi Biotec) were used for magnetic labeling of human cord blood stem cells. The antitransferrin antibody was used as the primary antibody (Miltenyi Biotec) and anti-mouse antibody as the second antibody (Invitrogen). Each antibody/bead solution of 0.7 ml was used for labeling of 1 × 106 cells. The cell antibody suspension was incubated for 30 minutes at room temperature on an orbital shaker. After several washing steps, USSCs were added to the culture medium.

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Tissue Decellularization

Porcine pericardium was harvested under sterile conditions. After the removal of the adherent tissue, the tissue was incubated in PBS solution (Mg2+, Ca2+ 100 mg/L, pH 7.4—Sigma-Aldrich, Seelze, Germany) for 24 hours with 200 U/ml penicillin (GIBCO, Invitrogen) and 200 μg/ml streptomycin (GIBCO, Invitrogen). The tissue flaps were treated with 0.25% sodium deoxycholate (GIBCO, Invitrogen) in PBS for 48 hours at 37°C followed by an intense washing with 0.5% sodium deoxycholate and sodium dodecylsulfate (Karl Roth GmbH; Karlsruhe) in PBS to remove residual cell debris. The DNA concentration in native and recellularized tissue was measured by spectrometric analysis.

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Experimental Procedure

The decellularized pericardium was reconstructed and sutured into nitinol stents to produce STVs. The STVs were placed in the designed bioreactor chamber. Afterward cells were added to the circulating volume in the bioreactor. The pericardial matrix was perfused under a pulsatile flow (up to 1.5 L/min, pulsatile rate of up to 20 beats/min) in the bioreactor for the next 4–7 days. Physiologic conditions including a temperature of 37°C, pH value of 7.4 (±0.05), an oxygen tension (pO2—150 ± 20 mm Hg) and a constant level of pCO2 with a permanent high-flow perfusion. The tissue probes were divided into two groups: group I (n = 4) was recellularized with the use of our magnetic device, “magnetically recellularized STV” (MRSTV), and group II (n = 4) was recellularized without the use of magnetic enhancement, “Conventionally Recellularized STV (CRSTV).” The USSCs used for both experiments were labeled with magnetic beads.

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Histology, Immunohistochemistry, and Electron Microscopy

Immunohistology of the scaffolds was performed. The specimen was fixed with 4% formalin, embedded in paraffin, and sectioned at 8 μm. The sections were stained with Elastin VVG and Masson’s Trichrome (Mastertechs, American MasterTech, Lodi, CA) according to the manufacturer’s instruction. To confirm the phenotype of the cells after recellularization, samples were also analyzed with anti-CD31 and anti-CD 34 antibody stains. Samples for electron microscopy (EM) were fixed in 2.5% glutaraldehyde (Sigma-Aldrich) for 24 hours, dehydrated with graded ethanol series, and dried in CO2. The dried samples were mounted on aluminum and coated with gold.

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Results

Unrestricted somatic stem cells express high levels of CD13, CD29, CD44, CD71, CD73, CD105, CD146, CD166, and HLA-ABC. All lines were negative for CD31, CD34, CD45, CD56, CD106, AC133 (CD133/1), CD271, and HLA-DR. Fluorescence-activated cell sorting analysis did not reveal any significant differences between the different USSC lines; the immunophenotype is independent of the passages used.7,8 After detergent treatment of the pericardial tissue, samples showed <5% of residual DNA compared with native tissue samples. Stented valves were constructed after decellularization of porcine pericardium by embedding the matrix in nitinol stents (n = 8) (Engineo GmbH, Hechingen GmbH). Fifteen seeding experiments were started, and eight could be finished successfully. All stented pericardial flaps showed a smooth surface, translucent leaflets without vegetations or calcifications, and were comparable with native tissue. Histology and immunohistochemistry of the decellularized flaps revealed an efficiently preserved 3-dimensional network of the scaffold including elastic fibers and glycosaminoglycans (Figure 3, A and B), comparable with native tissue. After recellularization, the luminal surface of MRSTV was covered with a confluent cell monolayer (Masson’s trichrome, Figure 4A). A high concentration of magnetic beads can be seen. Reseeded cells did only show a weak CD31 and CD34 expression (Figure 4B), so they did not demonstrate clear evidence for endothelial differentiation. In contrast, in CRSTV flaps, no cells could be found on the surface (Figure 4C). EM images showed the histological characteristics of the spindle-like cells (Figure 5, A and B). Macroscopic analysis revealed a homogeneous seeding of the valve with labeled USSCs, whereas no seeding of cells could be observed in the nonmagnetic group. The arrows highlight the area, which was not embedded in the magnetic device (Figure 6).

Figure 3

Figure 3

Figure 4

Figure 4

Figure 5

Figure 5

Figure 6

Figure 6

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Discussion

Biological valves, which are currently used in treatment of valvular disease, have certain limitations and undergo calcification and degeneration after implantation. To prevent these limitations, tissue-engineered valves can be considered an optimal solution with the potential to overcome thrombogenicity and calcification, without losing the excellent hemodynamic properties.9,10

This study demonstrates that in vitro recellularization of detergent-decellularized valves with human cord blood stem cells is feasible in a short period of time using magnetic guidance after a recellularization period of 4 to 7 days. Previous publications have already described the application of magnetic devices in the field of tissue engineering, including optimized reseeding of 3-D structures and vascular tissue-engineered grafts.11,12 The goal of tissue engineering is to develop artificial tissue, which can be used as a template for growth, repair, and remodeling. Decellularized porcine pericardium can be used as the appropriate scaffold for recellularization with stem cells. Important characteristics are the high biocompatibility, the fact that major structural components are sustained, and the high strength of the tissue-engineered organ-specific acellular matrix.13 Our technology for recellularization of decellularized pericardium can be used in different fields of research, such as cardiac tissue engineering. When using high-perfusion flows for maintenance of sufficient cell culture medium supply through physiologic tissue surface, wash-off of seeded cells is a major problem. To overcome cell wash-off phenomena, cells were labeled magnetically to obtain a high cell density. So, cell adhesion was established using a strong magnetic field in close proximity to the pericardial scaffold. An equal cell distribution can minimize the nonphysiologic surface from which fast degeneration and thromboembolic complications can emerge. The importance of bioreactor in the process of recellularization has been proven in earlier studies.14 Bioreactors generate an environment necessary for the creation of new tissues by providing biochemical and physical regulatory signals and by encouraging cell proliferation and differentiation.14,15

Mechanical stimulation of tissues in vivo has been shown to enhance the functionality and clinical applicability of tissue-engineered constructs.15 In our experiments, pulsatile perfusion was maintained in the bioreactor. Our method can be used to attain high cell concentrations on a decellularized scaffold in a limited time. It minimizes the risk of contamination and high passages of the used stem cells. We have developed an efficient method to optimize the cell seeding process in creation of recellularized biocompatible pericardial scaffolds. This approval can be extended to any cell seeding procedure in bioreactors by increasing the likelihood of cell presence close to the targeted matrix. Several aspects need to be further clarified in future experiments. The optimized protocol, which enables cell-based tissue engineering of matrices using a magnetic device, needs to be further developed. The length of the recellularization period, the number of used cells, and the flow in the bioreactor are part of the parameters, which need to be further optimized. Further histological analysis is needed to show improved recellularization of the surface and deeper layer of the decellularized matrix. Also further clear evidence for differentiation of the applied stem cells can be examined after extended cell culture treatment.

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Acknowledgments

We thank the JOTEC GmbH (Hechingen, Germany) for construction of the porcine pericardium-based stented valve after the decellularization of pericardium by our team. We thank the Miltenyi Biotec GmbH, Engineo GmbH, and KGW-Isotherm GmbH for their technical support.

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References

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Keywords:

tissue engineering; heart valve replacement; cord blood cells; cardiac disease; transcatheter aortic valve implantation

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