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.
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.
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.
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.
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.
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.
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).
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.
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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Keywords:Copyright © 2014 by the American Society for Artificial Internal Organs
tissue engineering; heart valve replacement; cord blood cells; cardiac disease; transcatheter aortic valve implantation