Cardiac tissue engineering involves the use of synthetic and biological materials to build organs and improve health through organ transplantation. Researchers have made great progress in cardiac tissue engineering, including the manufacture of synthetic extracellular matrix scaffolding and the complete recellularization of small mammalian hearts.1 Decellularizing porcine hearts and recellularizing the cardiac extracellular matrix (cECM) with human cells represent a preferred strategy for future human cardiac transplantation because a natural scaffold allows cells to function and proliferate more effectively than a synthetic scaffold. This method would also reduce the need for antirejection medications post-transplant, normally used to suppress the immune response to the transplanted organ.2
To achieve success, it is necessary to obtain very pure cECM samples that will be able to support cell growth without any cytotoxic effects. It is crucial to develop decellularization techniques that produce intact cECM for cardiac tissue engineering and to learn more about the biology of cECM to take advantage of cECM molecules that result in a better recruitment of the cells.3
Because of the use of detergents in the automated decellularization process, it is necessary to remove harmful chemical residues that could cause cells to become apoptotic, decrease in function, or cause thrombosis in cardiac tissue samples.4 Sodium dodecyl sulfate (SDS), a strong detergent used to remove cellular material, was the primary concern for causing cytotoxicity in these experiments.
This study examined the utility of a hemolysis assay, the lysing of red blood cells,5 as a novel method to measure cytotoxicity in cECM samples. To test this method, porcine hearts were harvested and then decellularized using an automated bioreactor.6 After decellularization, samples were taken from the left ventricle (LV) and rinsed in phosphate-buffered saline (PBS). At various time points, samples were removed from the washes and tested for cytotoxicity. Human blood was purchased from a local blood bank and tested for quality and suitability. Erythrocytes from this blood were then added to the samples of washed cECM, and percent hemolysis was calculated. This was done by calculating the percentage of ruptured erythrocytes in each sample by detecting and measuring the amount of hemoglobin present via spectrophotometry.7
To further assess cytotoxicity, mouse endothelial cells (MS1) were seeded onto the cECM samples. With the assistance of cell staining, it was possible to visualize the healthy and apoptotic cells on each sample. In addition to increased cytotoxicity, another negative side effect of prolonged SDS exposure is the degradation of collagen and other proteins in the cECM. Collagen is an abundant protein in cardiac tissue and is essential for providing structure to the heart.8 The amino acid hydroxyproline was used as a marker to measure the amount of collagen contained within each sample.9 Once the amount of collagen was quantified, it was compared against a standard curve of known collagen concentrations to determine the amount of collagen preserved after decellularization.
Materials and Methods
Harvesting and Decellularization
The harvesting and initial decellularization protocol was similar to previous studies performed by Momtahan et al.6 Porcine hearts were harvested from 6 month-old swine at a local abattoir according to approved protocols for safety and animal care. To obtain cECM, the hearts were decellularized in an automated pressure and temperature-controlled apparatus designed and built by our group as described previously.6 In the SDS group, hearts were rinsed by antegrade perfusion with 1X PBS and type 1 distilled water (DW) for 1 h each, followed by three sets of 0.5% (w/v) SDS solutions for 2 h each with 1 h of nonrecycled DW between each set. In the Triton X-100 (TX-100, Fisher Scientific, Fair Lawn, NJ) group, hearts were perfused with 1× PBS, and type 1 DW for 1 h each, followed by three sets of 0.5% SDS solutions for 2 h each with 1 h of nonrecycled DW between each set. After the last 2 h wash with SDS, the hearts were perfused with 10 L of DW, recycled for 8 h overnight, followed by 2 h of 1% (v/v) TX-100 perfusion as described in Table 1.
Subsequent to the final detergent perfusion with either SDS or TX-100 in both groups, the hearts were removed from the decellularization apparatus and the LVs were dissected. Samples of decellularized myocardial cECM from the LV wall were obtained, minced and mesh-standardized specimens of 2 mm × 2 mm × 2 mm were made from each heart, and placed in a beaker filled with 4 L of 1× PBS that was continuously stirred at 60 rpm with a 5 cm stir bar. The LV wall is the thickest portion of the heart and is highly vascularized; therefore, it is most probable that the majority of cytotoxicity would be found in the LV. Three liters of the 1× PBS solution were renewed every 24 h. Ten samples were removed from the solution at various time points (approximately every 5 h) with a maximum interval of 10 h between each time point, then stored at 4°C to be used for hemolysis assay, live/dead cytotoxicity assays, collagen and residual SDS content measurements, or structural analysis by scanning electron microscopy (SEM).
Human Blood Test: Fragility of the Erythrocytes
One to three months after expiration date, anonymous human blood bags were purchased from the MountainStar Health (Ogden, UT) blood bank. To assure the quality of the blood and suitability for hemolysis experiments, the fragility of the erythrocytes were measured using a method based on a study by Parpart et al.10 Dilutions of 1× PBS with osmolality of 300 mOsm/L were made, and 50 µl of blood was added to 1 ml of dilution. After 10 min, the tubes were centrifuged for 3 min at 3,000g and the absorbance of supernatant was measured at 540 nm to calculate the percentage of hemolysis using the formula in Equation 1 below:
- A = Absorbance of supernatant of erythrocyte suspension with sample solution;
- A0 = Absorbance of supernatant of erythrocyte suspension with 100% 1× PBS;
- A100 = Absorbance of supernatant of erythrocyte suspension with 0% 1× PBS.
Human Blood Test: Hemolysis Assay
Erythrocytes were separated from human blood by centrifugation at 2,000g for 10 min, and the supernatant was extracted. Erythrocytes were then diluted in 1× PBS to create an erythrocyte suspension (ES) with 2 × 109 cells/ml. A Moxi Z mini-automated cell counter kit (Ketchum, ID) was used to assure the accuracy of the erythrocyte concentration in the ES. Specimens from each of the time points for the SDS and TX-100 groups were placed in 20 ml glass vials with 5 ml of ES. The vials were then placed on an orbital shaker with 125 rpm and maintained at room temperature. Vials without any specimens were used as negative control specimens and vials with 25 mg of SDS added to the ES were used as positive control specimens. After 60 min, 1 ml of ES was removed from each vial and centrifuged for 3 min at 3,000g and absorbance of the supernatant was measured at 540 nm. Percentages of hemolysis were calculated using Equation 1 with the positive control absorption as A100 and negative control as A0.
One of the main advantages of the cECM is its ability to support mammalian cells mechanically and chemically to function and proliferate. Chemicals used during the process of obtaining acellular scaffolds through decellularization can affect the scaffold’s supportive properties drastically and cause the cells to become apoptotic or not function well.11 To assess the cytotoxicity of the cECM, mouse endothelial cells (MS1) were cultured in a 5% CO2 incubator at 37°C with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin growth medium. Specimens from various time points of both SDS and TX-100 groups were decontaminated by soaking in a sterilization solution of 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml amphotericin B (Gibco, Gaithersburg, MD) in 75% ethanol for 10 min followed by rinsing twice with sterile 1× PBS. The specimens were added to the 6 well plates containing MS1 cells, and the plates were incubated incubator for 4 h. Wells without any specimen were used as positive control specimens. The cells were then labeled using 2 µM calcein AM and 4 µM EthD-III (Biotium Viability/Cytotoxicity Assay, Hayward, CA) to assess the cytotoxicity of the cECM specimens, and a FLoid cell imaging station was used to visualize the green and red fluorescence corresponding to healthy and apoptotic cells, respectively.
Collagen is the most abundant protein in the extracellular matrix of mammalian tissues, and collagens constitute a large family of proteins that each have at least one triple-helical domain. Fibrillar collagens consist of three α-chains that have three polyproline II helices (left-handed) twisted in a triple helix (right-handed) staggered by one residue between adjacent α-chains. Interchain hydrogen bonds, electrostatic interactions involving lysine and aspartate, and the high content of proline and hydroxyproline are the factors that influence the stabilization of the triple helix.9 Because hydroxyproline is found mainly in collagen and rarely in other proteins,12 hydroxyproline content can be used as a marker to measure the amount of collagen.
To prepare cECM samples for quantification of hydroxyproline, decellularized specimens from various time points from both the SDS and the TX-100 groups were collected and then lyophilized for 2 days. Samples were tested for the presence of hydroxyproline using BioVision’s Hydroxyproline Assay kit (BioVision, San Francisco, CA) based on the manufacturer’s protocol. First, 5 mg of the lyophilized samples were homogenized in 500 ml DW. Then the samples were digested in 500 ml of 12 N HCl at 60°C for 1 day. Chloramine T and 4-(Dimethylamino)benzaldehyde reagents from the assay were added to each sample in a 96 well plate and incubated for 30 min at 60°C, and then the absorbance of the samples was measured at 560 nm. A standard curve was prepared with a known hydroxyproline solution to determine the amount of collagen in each sample.
Structural Analysis with Scanning Electron Microscopy
The decellularization process makes the cECM porous by removing cellular components from the tissue; however, overprocessing or soaking the cECM in PBS for too long, to remove detergent residues, can alter the porosity. Scanning electron microscopy was used to analyze the porosity and protein conformation of the cECM samples at different time points from both the SDS and the TX-100 groups. Biological sample preparation for SEM can also alter the structure of the sample.13,14 To avoid significant alteration, samples were minimally processed by soaking the specimens in 50% ethanol (v/v) followed by flash freezing the specimens by placing them in 1 L of 100% ethanol, cooled down to about −150°C using liquid nitrogen. Frozen specimens were immediately placed in a lyophilizer and dried for 5 days. Specimens were then attached to SEM stubs using carbon tape, coated with 3 nm of platinum, and placed in a Helios NanoLab 600 Focused Ion Beam/SEM (FEI, Hillsboro, OR) for image acquisition.
Sodium Dodecyl Sulfate Measurement Using a Colorimetric Method
As previously reported by Jurado et al.15 and Rusconi et al.,16 amounts of SDS in hydrophilic solutions can be quantified using colorimetric methods. In this study, specimens from both SDS and TX-100 groups, collected at different time points, were placed in 1 ml DW on a shaker for 24 h at room temperature. Standards solutions of 0 to 10 mg/L SDS in DW were created for calibration. A 50 mM buffer solution of sodium tetraborate (Na2B4O7, Sigma-Aldrich, St. Louis, MO) was prepared, and the pH was adjusted to 10.5 by using a dilute solution of sodium hydroxide. To color the SDS, 0.1 g of methylene blue reagent was dissolved in 100 ml of borate buffer solution (10 mM) with adjusted pH 5–6. The methylene blue solution was kept in a topaz-colored flask. In a glass test tube, 10 µl of phenolphthalein was added to 5 ml of diluted SDS sample or standard solutions. The solution were alkalinized with addition of 200 µl of sodium tetraborate at pH 10.5 to the color change of phenolphthalein. Next, 100 µl of stabilized methylene blue solution and 4 ml chloroform (CHCl3; Sigma-Aldrich) were added to the SDS solutions. Before each measurement, the glass test tube was stirred vigorously for 30 s and allowed to rest for 10 min. The chloroform phase was used for colorimetric measurements with UV-vis quartz cuvettes at 650 nm against pure chloroform.
Reported data are average ± sample standard deviation, and the number of samples for quantification experiments was n = 5 unless mentioned otherwise. For the statistical analysis of data, SAS JMP 11 (Cary, NC) software was used, and α = 0.05 was considered as the threshold of p values for determining the statistical significance of the data. Student’s standard t-test was used for analyzing the data in two groups of SDS and TX-100. In the cytotoxicity assay, ImageJ v1.49 (NIH, Bethesda, MD), which is available in the public domain, was used to calculate the percentages of live and dead cells in random acquired images.
Whole Heart Decellularization
A total of 10 hearts were processed for this study. Five hearts were processed for the SDS group and 5 for the TX-100 group based on the protocol shown in Table 1. Figure 1 shows a processed heart from the TX-100 group after 2 h of TX-100 perfusion. After processing, all hearts were dissected open, and the LVs were minced to obtain at least 200 specimens as shown in Figure 1.
The decellularization process was performed on whole porcine hearts through retrograde aortic perfusion. Similar methods have been used by Robertson et al.17 on rat hearts and Remlinger et al.18 on porcine hearts. We have previously shown that our decellularization process results in more than 98% DNA removal from various sections of the hearts, and the produced cECM has very limited immunogenicity.6
To assure the quality of blood used in the hemolysis experiments, erythrocyte fragility was determined by suspending the blood in different osmotic solutions and the percentage of hemolysis was recorded using light absorption at 540 nm. As shown in Figure 2, solutions of 40%, 60%, and 80% 1× PBS (120, 180, and 240 mOsm/L) produced hemolysis percentages of 97.57% ± 4.23, 58.74% ± 8.82, and 6.94% ± 5.96, respectively. Median osmotic fragility was observed to take place with 62% ± 4 (187 ± 11 mOsm/L) concentration of PBS. A representative image of the supernatant color obtained after centrifugation with the various 1× PBS solutions with respect to the percentage of hemolysis produced is also shown in Figure 2. To assure the quality of the results from this assay, the data were compared with an established fragility standard curve for verification.10
Various chemicals used in the process of decellularization can have different effects on the tissue ultrastructure, mechanical behavior, and the biochemical composition, which may affect the subsequent host response to the material. Chemicals are specifically chosen for use in the process because of their inherent potential to damage cell membranes. Thus, if they remain in the cECM after the decellularization process, it is likely that the cECM will be cytotoxic.11,19 In the cytotoxicity assessment of the cECM in this study, specimens (n = 6) collected at different time points from both the SDS and the TX-100 groups were placed with ES for 1 h. It was observed that periods shorter than 60 min led to unreliable results caused by incomplete reactions between the residual surfactants and the erythrocytes. This observation was also reported by Krzyzaniak and Yalkowsky.20
As shown in Figure 3, A and B, both SDS and TX-100 samples with no wash time produced greater than 90% hemolysis (90.16 ± 25.66 and 91.42 ± 13.84, respectively). No statistically significant difference between the SDS and the TX-100 groups (p > 0.05) was observed until the 5 h time point, at which point the SDS group produced 72.38% ± 10.37 hemolysis whereas the TX-100 group produced 25.61% ± 16.12 hemolysis. The statistical difference between the SDS and the TX-100 groups continued to be significant until the 40 h time point at which time the specimens in the SDS group produced 8.19% ± 4.31 hemolysis. It was observed that the minimum amount of required wash to reach no statistical significant difference with the negative control specimen was 10 h for the TX-100 and 40 h for the SDS group. As shown in Figure 3C, a linear decrease in the hemolysis percentages was observed in both groups during the first 10 h for TX-100 and 40 h for SDS.
Cytotoxicity of the cECM was measured by counting the number of dead versus live cells using the Biotium Viability/Cytotoxicity Assay. The polyanionic dye calcein provided in the assay is absorbed by live cells and produces a uniform green fluorescence. The EthD-III enters cells with damaged membranes and after binding to mammalian cell nucleic acids, the fluorescence intensity increases by 40-fold and produces a red fluorescence in dead cells. Sodium dodecyl sulfate and TX-100 chemicals used for decellularization in this study are inherently able to damage the cell membranes and cause toxicity, thus the Viability/Cytotoxicity Assay can be used to determine the level of cytotoxicity.
Although the FBS used in growth medium is known to have neutralizing inhibitors and digestive capacities,21 it was observed that specimens (n = 4) from both SDS and TX-100 groups at 0 h produced 97.35% ± 1.52 and 94.75% ± 3.42 dead cells, respectively, as shown in Figure 4, A and B. A statistically significant difference between SDS and TX-100 groups was observed at the 10 h time point with the SDS group producing 51.94% ± 10.46 dead cells, whereas the TX-100 group produced 7.86% ± 2.53 dead cells. The percentage of dead cells was observed to be higher for the SDS group until the 40 h time point, when no statistically significant difference was observed between the SDS and the TX-100 groups (p > 0.05). For the TX-100 group after 10 h, no statistically significant difference was observed compared with the negative control specimen (p > 0.05), whereas the time point at which there was no statistically significant difference was 40 h for the SDS group. Representative images of the cells in contact with the specimens from both groups, labeled with Viability/Cytotoxicity Assay are shown in Figure 4, C to J.
Stabilization of the triple helix in collagen is primarily caused by interchain hydrogen bonds, electrostatic interactions involving lysine and aspartate, and the high content of proline and hydroxyproline.9 Soaking the cECM in PBS for too long, to remove detergent residues, can potentially deteriorate the factors involved in the stabilization of the collagen and solubilize this protein. To investigate the changes in the amounts of collagen, hydroxyproline content in specimens (n = 3) at different time points were measured per milligram of dried tissue in both SDS and TX-100 groups. Although the averages of hydroxyproline content in both the SDS and the TX-100 groups declined over 110 and 75 h, respectively, the statistical difference was not significant (p > 0.05). As shown in Figure 5, the contents of hyroxyproline in the SDS group started at (0 h) 24.16 ± 6.93 µg/mg of lyophilized tissue and reduced to 18.88 ± 8.33 µg/mg of lyophilized tissue after 110 h. In the TX-100 group, the contents were observed to start at 23.84 ± 7.39 µg/mg of lyophilized tissue and after 75 h decreased to 20.43 ± 2.19 µg/mg of lyophilized tissue.
Scanning Electron Microscopy Structural Analysis
As previously reported,22–24 surface structural analysis can be performed using SEM techniques to assess the alignment of collagen fibers. In this study, samples at various time points were flash frozen, lyophilized for 5 days, and coated with 3 nm of platinum. Representative SEM images acquired from samples at different time points are shown in Figure 6. In both the SDS and the TX-100 groups, samples with longer wash times demonstrated more dissociation and disruption of fiber alignment.
Sodium Dodecyl Sulfate Colorimetric Assay
The colorimetric assay with UV-vis detector was used to measure the amount of SDS in different samples. To obtain the linear range of SDS solutions, the correlation between absorbance and concentration was identified. A calibration curve was prepared for different SDS amounts in the range of 0–20 mg/L. The adjusted regression of 0.996 showed a linear correlation between the absorbance and different SDS concentrations and consequently the precision of the applied procedure. Soluble SDS content in the specimens was then calculated using the calibration curve as shown in Figure 7. As shown, the concentration of SDS in the specimens started at 894.32 ± 55.45 and 753.81 ± 48.57 µg/ml in the SDS and TX-100 groups, respectively. In the TX-100 group, the concentration decreased to 6.99 ± 0.57 µg/ml after 21 h, whereas at a similar time point in the SDS group, the concentration was observed to be 98.06 ± 6.08 µg/ml. After 40 h, SDS contents in the SDS and TX-100 groups did not demonstrate a statistically significant difference, and the concentration was 5.09 ± 0.57 µg/ml in the TX-100 group.
Natural extracellular matrices have been shown to be promising scaffolds with tissue-specific potential to support cell growth and differentiation.6,25,26 As stated by Taylor et al.27 as one of the pioneers in developing whole organ decellularization, the improvement of the decellularization process to produce ECM from deceased organs is one of the most notable achievements in the field of tissue engineering within the past 7 years. Acellular ECM can be obtained by decellularization of native tissue either by perfusing detergent solutions through the vasculature or diffusion of this solution through pieces of tissue.28 Decellularization removes any cellular materials from the matrix that might cause immune reaction after transplantation.
Sodium dodecyl sulfate is the preferred agent for removing cell debris.17,22 However, as a strong ionic detergent, SDS can denature proteins29,30 and damage phospholipid membranes,31 leading to cell toxicity.32 Accordingly, one should take sufficient care to remove residual SDS after the decellularization process. This critical issue increases the need for a standard method to evaluate cECM cytotoxicity after decellularization. There were several previous chemical methods for quantification of residual SDS,15,16,33 but the previous methods measured the amount of SDS and not the activity. There was still a need to determine a threshold for SDS concentration leading to cell toxicity.
In this study, we developed a novel, convenient, and rapid method to examine cytotoxicity of acellular cECM. Disruption of red blood cells caused by SDS contact is a relevant indicator that we used to measure cytotoxicity of cECM. This hemolysis assay is a practical method that directly measures cell death caused by SDS presence that can also be applied to any other tissue or scaffold. Data shown in Figures 3 and 4 suggest that the hemolysis assay results are generally consistent with the cell viability assay results on acellular tissue (e.g., samples with <20% hemolysis also show <20% dead cells), which is a critical factor in the preparation of cECM.
Hemoglobin is the main component inside erythrocytes responsible for transport of oxygen from the lungs to tissues. Four oxygen molecules can bind to the hemoglobin producing an oxygenated hemoglobin, alternating the complex refractive indexes.34 It has been shown that oxygenation of hemoglobin causes a slight decrease of approximately 0.25% in the light absorbance at 540 nm,35 which can be avoided by deactivating the hemoglobin using hydrogen peroxide.36 In the experiments performed in this study, negative control specimens of erythrocyte solutions were used to normalize the absorbance. All the samples and negative control specimens had the same amount of air exposure to assure equal hemoglobin oxygenation rates.
Our data also support the use of Triton X-100 to extract retained SDS trapped in the structure of cECM as suggested by Robertson et al.17 and Song et al.37 Sodium dodecyl sulfate is a relatively small ionic molecule that can diffuse through the collagenous fibers and get trapped there because of its ionic nature.38 As shown in Figure 6, relying just on PBS washes to remove SDS results in dissociated collagen fibers that not only decreases the mechanical strength of cardiac tissue but also might alter the seeded cell proliferation and gene expression patterns. Triton X-100 is a nonionic detergent that can bind to SDS and facilitate its removal from the cECM, as previously reported.39 Considering the data from the cytotoxicity assessment (Figure 4) and soluble SDS measurements (Figure 7), a threshold of 7 ± 2 µg of soluble SDS in 1 ml of DW can be considered as the limit for cytotoxicity.
The collagen structural analysis was included in this work to demonstrate the importance of complete removal of SDS from the cECM. In SEM images shown in Figure 6, it was demonstrated that prolonged exposure of cECM to SDS causes deformation in the collagen fibers and damages the triple helices. However, quantification of collagen (Figure 5) showed no significant decrease in the total amount of collagen in cECM samples.
To summarize, the hemolysis assay is a rapid colorimetric method that can be used to examine the cytotoxicity of a tissue or scaffold caused by residual detergents. Future studies should be performed to see whether this assay can be used for other cytotoxic agents, such as peracetic acid or ethanol, which are common sterilizing chemicals in biomedical applications. Additional optimization of the hemolysis assay (e.g., ratio of tissue to red cells, contact time) would also be useful to improve the utility and versatility of the assay.
Although advances such as achieving a whole decellularized heart scaffold that is biocompatible with erythrocytes and endothelial cells have been made in the field of tissue engineering, creating a fully functional bioartificial heart using autologous cells must yet be demonstrated.
In this study, the novel use of a hemolysis assay for determining the cytotoxicity of decellularized heart tissues because of residual chemicals used in the process was demonstrated. The removal of SDS was accelerated by introducing an additional 2 h of TX-100 wash in the decellularization process. The cytotoxicity of the cECM with 2 h of TX-100 after 10 h of PBS wash was equivalent to 40 h of PBS wash when not including TX-100 in the process. The results from the hemolysis assay were shown to be consistent with the established cell viability live/dead assay on endothelial cells in contact with cECM specimens. In addition, the effects of prolonged exposure to SDS and TX-100 on collagen and the structure of the cECM were shown to be minimal.
Holly Howarth, Brenden Herrod, and Makena Ford provided laboratory assistance. The authors gratefully acknowledge BYU for providing funding for this project.
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