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. Author manuscript; available in PMC: 2023 Jan 4.
Published in final edited form as: Methods Mol Biol. 2022;2485:55–70. doi: 10.1007/978-1-0716-2261-2_4

Acellular Myocardial Scaffolds and Slices Fabrication, and Method for Applying Mechanical and Electrical Simulation to Tissue Construct

Bo Wang 3, Mickey Shah 2, Lakiesha N Williams 4, Amy L de Jongh Curry 5, Yi Hong 1, Ge Zhang 2,*, Jun Liao 1,*
PMCID: PMC9811994  NIHMSID: NIHMS1856532  PMID: 35618898

Summary/Abstract

Cardiac tissue engineering/regeneration using decellularized myocardium has attracted great research attention due to its potential benefit to myocardial infarction (MI) treatment. Here we described an optimal decellularization protocol to generate 3D porcine myocardial scaffolds with well-preserved cardiomyocyte lacunae, myocardial slices as a biomimetic cell culture and delivery platform, and a multi-stimulation bioreactor that is able to provide coordinated mechanical and electrical stimulations for facilitating cardiac construct development.

Keywords: Cardiac tissue engineering/regeneration, acellular myocardial scaffolds, acellular myocardial slices, decellularization, mechanical simulation, electrical simulation, bioreactor

1. Introduction:

Myocardial infarction (MI) and heart failure are major causes of mortality worldwide (1). Currently, the only successful treatment for end-stage heart failure is whole heart transplantation, which is unfortunately limited by the persistent shortage of suitable heart donors. Newer strategies, including cellular transplantation, intra myocardial gene transfer, and cardiac tissue engineering (TE), have come to the forefront as alternative therapeutic approaches (2–4).

The purpose of cardiac tissue engineering is to develop functional cardiac tissue through integrating cellular components within scaffolds that serve as a structural guide (5–8). Two major types of scaffold materials have been commonly used for cardiac tissue engineering: synthetic biodegradable material and tissue-derived acellular scaffolds (9–14). The use of synthetic biodegradable polymers still faces challenges, including inflammatory response, mismatched material properties, nonpliability, and difficulty in controlling the degradation rates (13, 15, 16). Acellular scaffolds, which are derived from native tissues or organs via decellularization, are able to preserve the extracellular matrix (ECM) compositions, overall ultrastructure, shape compatibility, ECM mechanical integrity, and bioactive molecules that benefit cell-ECM adhesion, cell-cell interaction, and de novo ECM formation (17–23).

In practice, it is important to determine the optimal decellularization protocol that can mostly remove cells, cell debris, chromosome fragments, and xenogeneic antigens in order to diminish immunogenicity while at the same time preserve the needed structural and mechanical integrity of the native tissue ECM, which is important for target tissue functionalities (23–26). For the applications of myocardial ECM scaffolds, Ott et al. decellularized whole rat heart and were able to keep the intact chamber geometry, perfusable vasculature, and competent acellular valves (27). Badylak et al. and Taylor et al. attempted scaled-up research on whole porcine heart (21, 28). Yet, many challenges still exist in whole-heart regeneration, such as preservation of myocardial ECM structure, reseeding homogeneity and thoroughness across the ventricle wall thickness, feasibility of reviving the existing vasculature network, and functional integration (21, 22, 29, 30). Thus, our group has undertaken an effort to harness the potential of decellularized porcine myocardium as a TE scaffold material and focus on tissue-level application (31–33).

Heart walls are constructed of cardiac muscles that consist of cardiomyocytes, which are connected via gap junctions and structurally organized by highly vascularized ECM (34, 35). As visually demonstrated by our previous diffusion tension MRI study (36), the heart muscles have a highly organized, multilayered helical structure (Fig. 1-a, b). The compelling structural beauty of heart muscle fibers hints at the uniqueness and importance of heart muscle ECM. Indeed, the intriguing myocardial ECM network does play key roles in maintaining structural integrity, tethering cardiomyocytes, mediating contraction/relaxation of muscle fibers, and preventing excessive stretching (37–39). As shown in Figure 1-c, d, removal of the heart muscle fibers (red staining) from the myocardial collagenous network (blue staining) will leave an ECM network that possesses a three-dimensional (3D) morphology and structural anisotropy. Hence, it is understandable that determining how to preserve the 3D ultra-structure of myocardial ECM represents a real challenge in myocardium decellularization via current available decellularization means, which are often beneficial in certain aspects but disruptive at some levels or to certain components (21–23).

Figure 1:

Figure 1:

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3D multilayered helical structure of heart muscle fibers. (a) Heart muscles have well-organized multilayered helical architecture, which is mediated by 3D myocardial ECM (Diffusion Tensor MRI image by Zhang and Liao, 2010) (36); (b) porcine heart used for DT-MRI imaging. Mason’s trichrome staining of longitudinal-section (c) and cross-section (d) of the native myocardium (red: cardiomyocytes; blue: collagen). Figures reproduced with permission (31, 36).

In addition, acellular myocardial ECM closely mimic the natural microenvironment of cardiac cells, which make it an optimal cell culture substrate for cardiac applications. However, seeded cells cannot maintain high viability and homogeneous distribution in full thickness acellular myocardial scaffold because cells in the center of thick scaffold will have insufficient access to oxygen and nutrients. To overcome this challenge, our group has explored the strategy of using a thin layer of acellular myocardial slice as a platform for cell culture and delivery (40–42). Our results demonstrated that acellular myocardial slice with the thickness of ~300 μm promote cell attachment, growth, homogeneous distribution and vascular differentiation of stem cells in vitro (Fig. 6).

Figure 6:

Figure 6:

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Acellular porcine myocardial slices. (a) Fabrication of the acellular porcine myocardial slices: Left - Native myocardial tissue, middle - decellularized myocardial scaffolds, right - acellular myocardial slice at 300 μm thickness. (b) Infiltration of pig adipose-derived stem cells (ASCs) on acellular myocardial slice. Image on right: Live and dead cells on acellular myocardial slice after 1 day culture. Image at bottom: Migration distance of pig ASCs seeded on acellular myocardial slice after 5 days culture. Scale bars in (a) = 4 mm; Scale bar in (b) live and dead cell image = 100 μm.

In this chapter, we introduce an optimal decellularization protocol to generate 3D porcine acellular myocardial scaffolds in which 3D cardiomyocyte lacunae, ECM networks, vasculature templates, and mechanical anisotropy can be well preserved (31, 32). We also include the detailed experimental protocol for create acellular myocardial slices (40–42). To further improve the effectiveness and efficiency of cell differentiation and tissue remodeling of the reseeded acellular myocardial scaffolds and slices, we also describe a bioreactor conditioning protocol that is able to apply combined mechanical and electrical stimulations to tissue constructs fabricated with the acellular myocardial scaffolds and slices (33).

2. Materials

2.1. Decellularization Stock Solution

  1. 0.1 M phenylmethylsulfonylfluoride (PMSF): 0.174 g PMSF (Sigma) dissolved in 10 ml of 1-propanol (Sigma);

  2. DNase (5 mg/ml): 50 mg of DNase (Sigma) dissolved in 10 ml 1 × PBS;

  3. RNase A (5 mg/ml): 50 mg of Ribonuclease A from bovine pancreas (RNase) (Sigma) dissolved in 10 ml 1 × PBS;

  4. 100 × antibiotic-antimycotic solution (ABAM) (Life Technologies);

  5. 1 × trypsin solution (Sigma);

    All the above solutions were stored at −20°C.

  6. 1 × phosphate-buffered saline (PBS) pH 7.4 (Life Technologies) stored at 4 °C;

  7. 1% sodium dodecyl sulfate (SDS) solution: 1 g UltraPure™ sodium dodecyl sulfate (SDS) (Life Technologies) dissolved in 100 ml 1 × PBS stored at room temperature.

2.2. Porcine Myocardium

  1. Fresh porcine hearts were obtained from juvenile pigs (~6 months old) from a local slaughter house.

  2. The porcine hearts were transported to the laboratory in 1 × PBS on ice.

  3. A myocardium square (20 × 20 × ~ 3 mm) was dissected from the middle region of the anterior left ventricular wall of the porcine heart (Fig. 1-b). (see Note 1)

  4. All the heart samples were kept in 1 × PBS solution at −80°C for preservation. (see Note 2)

2.3. Cell culture medium, Differentiation Medium, and Complete Medium

  1. Mesenchymal stem cell (MSC) medium: Low-glucose Dulbecco’s modified Eagle’s medium (L-DMEM) with 10% fetal bovine serum (FBS), 1% mesenchymal stem cell growth supplement (Sciencell), and 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen).

  2. Differentiation medium: L-DMEM, 10% FBS, 3 μmol/L 5-azacytidine (MP Biomedicals), and 100 U/ml penicillin and 100μg/ml streptomycin. (see Note 3)

  3. Complete medium: L-DMEM, 10% FBS, 1% cardiac myocyte growth supplement (Sciencell), 100 U/ml penicillin and 100 μg/ml streptomycin.

  4. All the above media were stored at 4 °C.

3. Methods

3.1. Myocardium Decellularization Procedure,

  1. Four pieces of native myocardium samples are thawed at room temperature and washed four times with 100 ml distilled water for 10 min in a 100 ml Simax glass media storage bottle on an orbital shaker (Belly Dancer, Stovall).

  2. A frame-pin supporting system is prepared to better maintain tissue macrogeometry during decellularization (Fig. 3). Briefly, four corners of the myocardium sample are perforated with four 27G × 31/2 ″ BD Quincke spinal needles and then mounted onto customized rectangular plastic frames. (see Note 6)

  3. After being mounted onto the frame-pin system, the myocardium samples are immersed inside 100 ml decellularization working solution in a 100 ml Simax glass media storage bottle sealed with cap.

  4. Preparing decellularization working solution: For 50 ml working solution, add 41.8 ml 1 × PBS, 2 ml DNase, 200 μl RNase, 5 ml 1% SDS, 500 μl 0.1M PMSF, 500 μl ABAM, and 5 μl trypsin solution to make a final solution of 0.1% (SDS), 0.01% trypsin, 1 mM PMSF, 0.2 mg/ml DNase, and 20 μg/ml RNase A.

  5. The myocardium patches are then decellularized with agitated decellularization working solution on an orbital shaker (Belly Dancer, Stovall) at 30 revolutions per minute at room temperature.

  6. A 10-minute ultrasonic treatment (50 HZ, Branson) is applied each day, and the decellularization solution is changed every day to avoid contamination and tissue deterioration.

  7. The completeness of myocardium decellularization can be determined when the myocardium patches became a bright white color, a typical color of collagenous materials; the whole procedure lasts approximately 2.5 weeks. (see Note 7, Note 8)

  8. After decellularization, all the myocardial scaffolds are removed from the frame-pin system and washed four times with 100 ml distilled water for 10 minutes and then washed four times with 100 ml PBS for 10 min, each time in a 100 ml Simax glass media storage bottle on an orbital shaker.

Figure 3:

Figure 3:

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The frame-pin supporting system. (a) Sample morphology after 3-day decellularization; (b) sample morphology after 2.5-week decellularization. Figures reproduced with permission (32).

3.2. Preparation and Handling Tips for Acellular Myocardial Slices

  1. The obtained acellular myocardial scaffold is trimmed into a square shape with the size of 2 cm (L) × 2 cm (W) × 1cm (H).

  2. The square sample is washed with 0.01% Triton X-100 for 1 hour and then rinsed with 1X phosphate buffered saline (PBS) for three days.

  3. The rinsed sample is embedded in Tissue Tek OCT (Fisher Scientific, PA, USA) and snap-frozen on in liquid nitrogen.

  4. The embedded sample is cryosectioned into acellular myocardial slices of 300um thickness (Fig. 6).

  5. The acellular myocardial slices are sterilized prior to cell seeding using absolute ethanol for 45 minutes followed by three washes with sterilized DI water for 15 minutes each.

3.3. Preparation of the Multi-stimulation Bioreactor

  1. The bioreactor used in this study consists of one tissue culture chamber in which all the structural elements are machined from polysulfone that provides excellent thermal and chemical stability.

  2. Inside the tissue culture chamber, a maximum of four pieces of tissue construct (20 mm × 20 mm × ~3 mm) can be mounted between a fixed clamp and a movable clamp (Fig. 2-c).

  3. Ti-corn blue sutures (# 0) are used for connecting the sample with two clamps.

  4. The cover of the tissue culture chamber is fabricated with ¼ inch-thick, clear polycarbonate (Small Parts). A hole (2 cm in diameter) is cut on the cover and sealed with PIFE membrane with a 0.2 μm pore size (Millipore) to enable air exchange (Fig. 2-c).

  5. Linear movement is applied by the movable clamp that is driven by an Xslide assembly and a stepper motor (Velmex) (Fig. 2-c). (see Note 4, Note 5)

  6. Electrodes are made from Teflon-coated silver wire (75 μm diameter, A-M Systems). The end part (2 cm) of the Teflon insulation is stripped off, and the naked wires are inserted into the two opposite edges of the tissue construct.

  7. The frequency and amplitude of the cyclic stretch are controlled by a customized LabView program (version 2010, National Instruments). To simulate similar experiences of the myocardial tissue, electrical pulses are applied at the initial stage of each unloading cycle (Fig. 2-b). The frequency and amplitude of the electrical pulses are controlled by the LabView program, which is capable of delivering multiple protocols of mechanical stretching and various waveforms of electrical stimulation.

Figure 2:

Figure 2:

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Design of a Multistimulation Bioreactor. (a) Schematic illustration of the acellular myocardial scaffold subjected to cell injection, mechanical, and electrical stimulations; (b) wave forms of the applied mechanical stretch and electrical pulses; (c) the multi-stimulation bioreactor placed in the incubator. Figures reproduced with permission (33).

3.4. Bioreactor Setup and Sterilization for Acellular Tissue Construct

  1. After sample washing, the naked end of the positive and negative electrodes are inserted into the two opposite edges of the acellular myocardial scaffolds (20 mm × 20 mm × ~3 mm), and the other ends of the Teflon-coated silver wires are dangled for later bioreactor connection.

  2. The acellular myocardial scaffolds (with electrodes mounted) are transferred into the culture chamber of the bioreactor and mounted between a fixed clamp and a movable clamp using the surgical Ti-corn blue sutures (# 0). Two to four pieces of the acellular scaffold samples can be placed in the bioreactor at one time.

  3. The other ends of the Teflon-coated silver wires are then connected with the electrical-control module of the bioreactor.

  4. The acellular myocardial scaffolds are then sterilized in 70% ethanol in the tissue culture chamber for two hours. (see Note 9)

  5. After ethanol sterilization, all the samples are rinsed thoroughly four times with sterilized PBS while still sitting in the culture chamber. (see Note 9)

  6. For sterilizing the other parts of the bioreactor that could not be immersed in ethanol, the whole tissue culture chamber with the mounted acellular scaffold samples is further treated with UV light for 20 minutes. (see Note 9)

3.5. MSC culture, Reseeding, Differentiation, and Bioreactor Conditioning Protocol

  1. Well characterized Lewis rat mesenchymal stem cells (MSCs, fourth passage) are obtained from the Stem/Progenitor Cell Standardization Core (SPCS) at the Texas A&M Health Science Center (NIH/NCRR grant). (see Note 10)

  2. After receiving the cells, MSCs are re-suspended in mesenchymal stem cell medium and seeded onto 175-mm flasks at a density of 2 × 103 cells/cm2. The medium is changed twice a week.

  3. The fifth to eighth passages of MSCs are re-suspended after HyQtase (Thermo Scientific) treatment and two washes with Tyrode’s balanced salt solution (Sigma).

  4. Next, the MSCs are used for scaffold recellularization. The density of MSCs used for scaffold recellularization was 106 cells/ml in mesenchymal stem cell medium.

  5. After the completion of the sterilization protocols for the bioreactor and tissue constructs, each scaffold sample is injected with totally 106 MSCs (1 ml MSC solution used; cell density of MSC solution: 106 cells/ml).

  6. An 1-ml syringe with 26G permanent needle (BD) is used for cell injection, and 1-ml MSCs are injected evenly at nine injection points (~0.1 ml/point) located as a 3 × 3 array within the middle region of the square sample (Fig. 2-a). (see Note 11)

  7. The movable clamp is adjusted to make sure no stretch applied on the reseeded scaffold sample, and this clamp-to-clamp distance is set as the reference distance to calculate the applied strain. (see Note 12)

  8. The tissue culture chamber is filled with differentiation medium until the medium fully immersed all the reseeded scaffold samples.

  9. The tissue culture chamber is covered and the top edges sealed with sterilized parafilm.

  10. The bioreactor chamber is carefully moved into the incubator with incubation condition set at 37°C in a humid atmosphere with 5% CO2. (see Note 13)

  11. After wires are connected to the bioreactor control modules, the LabView program initiates application of mechanical stretch (20% strain) and electrical pulse (5 volt).

  12. Both the triangular strain waveform and square wave electrical pulse are set at a frequency of 1 Hz (Fig. 2-b), which simulates the physiological frequency experienced by the heart muscles. (see Note 14)

  13. Note that the differentiation medium is added in the bioreactor chamber to facilitate the cardiomyocyte differentiation during the first 24 hours of tissue culture.

  14. After the 24-hour differentiation medium treatment, the medium is changed to the complete medium for the remaining bioreactor conditioning protocol, and the medium is changed every three days.

4. Notes

  1. For the square myocardium patch dissection, one edge was aligned along the muscle fiber preferred direction (PD), and the other edge aligned along the cross-fiber preferred direction (XD); note that the PD direction was determined based on overall muscle fiber texture and heart anatomy (Fig. 1-b).

  2. Snap freezing can disrupt cellular membranes by forming intracellular ice crystals and cause cell lysis (43, 44); therefore, we deep-froze the myocardium samples before decellularization treatment in order to increase the efficiency of decellularization.

  3. 5-azacytidine is a member of the cytosine analogue, which had been reported to induce uncontrolled myogenic specification by random (45–47). It was reported that treating mesenchymal stem cells with 5-azacytidine could generate a cardiomyocyte differentiation rate at ~30% (46, 47).

  4. For cyclic mechanical stretching, a stepper motor was chosen because its motion could be precisely controlled and easily programmed.

  5. To monitor the real-time tension level in the tissue construct, load cells can be applied in the bioreactor design to oversee the mechanical forces experienced by the construct during the tissue remodeling process.

  6. Frame-pin supporting system was applied for preventing scaffold contraction; this design well preserved the 3D cardiomyocyte lacunae during decellularization procedures (Fig. 5-b).

  7. The efficiency of cardiomyocyte removal could be verified by histology (Fig. 4-b,c and 5-a) and quantitative DNA analysis by ~2.5 weeks (31, 32). Xenogeneic antigens, porcine a-Gal, were found being completely removed from the acellular myocardial scaffolds (32). We also showed that the vasculature templates (acellular blood vessel structure) were preserved in the acellular myocardial scaffolds (31, 32).

  8. The decellularization protocol described here generated acellular myocardial scaffolds with thorough decellularization and ECM preservation; however, it took a relatively long treatment time (~2.5 weeks). To achieve effective decellularization within a shorter time period, the concentration of SDS can be increased to 0.5%.

  9. Effective sterilization is an essential step for tissue culture, especially for a bioreactor that has many complicated components and was constructed for repeated use. For our application, a combined method that used both 70% ethanol sterilization and UV light sterilization was adopted. Yet, the sterilization protocol can be further optimized to reduce sterilizing treatments to a minimal level (33). Moreover, for thinner samples, the duration for the 70% ethanol treatment should be largely reduced, and a thorough rinse must be performed to remove residual ethanol.

  10. For better cardiomyocyte differentiation, other cell sources that can be adopted include cardiac stem cells, embryonic stem cells (ESCs), or induced pluripotent stem (iPS) cells (48–52).

  11. To obtain the tissue engineered cardiac construct with a high cell density, we employed a needle injection method for cell implanting with a total cell amount of 106 cells/scaffold. However, due to the porous structure of the acellular myocardial scaffold, a small amount of leakage happened during the process of the cell injection. For this kind of situation, we injected the leaked medium back into the same region of the acellular myocardial scaffold.

  12. The applied tissue construct strain was estimated by normalizing the displacement of the movable clamp to the reference distance of two sample mounting clamps. The use of the clamp-to-clamp displacement in strain estimation was not an ideal method to accurately measure the tissue construct strain. To achieve more accurate measurement of the tissue construct strain, a camera can be used for real-time tracking of markers pasted on the tissue construct.

  13. Temperature is another important parameter in bioreactor conditioning. Heat exchange has to be carefully designed to maintain the incubator/bioreactor at a constant temperature (37°C). In our application, we placed both the culture chamber and the stepper motor inside the incubator. We noticed that the heat generated by the stepper motor after long hours of operation greatly affected the motor performance. This problem was solved by designing a water refrigeration system in which cold copper coils were wrapped around the step motor, and cooling water was circulated by a rotation pump outside of the incubator. The water refrigeration system dissipated the heat generated by the step motor effectively, and the temperature was maintained in a reasonable range without causing any motor malfunction.

  14. Previous studies have shown that cyclic mechanical stimulation can assist in the cell alignment, stimulate ECM formation (53, 54), and improve the cardiomyocytes development and function (55, 56). Electrical stimulation is believed to be able to induce transient calcium levels and facilitate cell proliferation and promote the formation and localization of electric gap junctions (57, 58). The benefit of the combined mechanical and electrical stimulations was evidenced by good cell viability, repopulation, differentiation, and positive tissue remodeling within a short period of time (2 – 4 days) (33).

Figure 5:

Figure 5:

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Details of the aligned 3D cardiomyocyte lacunae. (a) Edge-to-edge view of the acellular myocardial scaffold revealed by H&E staining; thorough decellularization and preservation of cardiomyocyte lacunae (porous structures) were verified. (b) 3D topography of the acellular myocardial scaffold revealed by SEM; enlarged view showed more details of the aligned 3D cardiomyocyte lacunae; note that arrows highlight the interconnecting openings inside the cardiomyocyte lacunae. Figures reproduced with permission (31, 32).

Figure 4:

Figure 4:

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Acellular myocardial scaffolds show well-preserved cardiomyocyte lacunae. Mason’s trichrome staining of (a) the native myocardium and (b) the acellular myocardial scaffold (red: cardiomyocytes; blue: collagen). (c) H&E staining of the longitudinal and transversal views of the acellular myocardial scaffolds showed structural anisotropy (red: collagen); arrow indicates vasculature channel was preserved after decellularization. Figures reproduced with permission (31, 32).

Acknowledgments

We greatly appreciate the support from NIH National Heart, Lung, and Blood Institute grant 1R15HL097321 to JL, 1R15HL122949 to GZ, and 1R15HL140503 to YH. The authors also would like to acknowledge the support from AHA (13GRNT17150041), NIH (1R01EB022018-01), and MAFES Strategic Research Initiative (CRESS MIS-361020).

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