A Thin Layer of Decellularized Porcine Myocardium for Cell Delivery

Decellularized porcine myocardium has shown many benefits as a cell delivery scaffold for cardiac therapy. However, using full thickness decellularized myocardium as cardiac patch may lead to poor viability and inhomogeneous distribution of delivered cells, due to perfusion limitations. In this study, we explored the feasibility of decellularized porcine myocardial slice (dPMS) to construct a vascularized cardiac patch for cell delivery. Decellularized porcine myocardium was sliced into thin layers (thickness~300 µm). Adipose-derived Stem cells (ASCs) obtained from rat and pig were seeded on dPMS. The viability, infiltration, and differentiation of seeded ASCs were examined. The mechanical properties of dPMSs of various thickness and native myocardium were tested. We noticed dPMS supported attachment and growth of rat and pig ASCs. Both rat and pig ASCs showed high viability, similar patterns of proliferation and infiltration within dPMS. Rat ASCs showed expression of early-endothelial markers followed by mature-endothelial marker without any additional inducers on dPMS. Using rat myocardial infarction model, we delivered ASCs using dPMS patched to the infarcted myocardium. After 1 week, a higher number of transplanted cells were present in the infarcted area when cells were delivered using dPMS versus direct injection. Compared with MI group, increased vascular formation was also observed.


Supplementary Methods: Hematoxylin and Eosin Staining
Harvested rat heart tissues, decellularized cardiac tissues, and native cardiac tissues were fixed with 4% paraformaldehyde (w/v) for 3 days at room temperature and then washed with 1X PBS three times for one hour each. Tissues were then dehydrated with graded series of 70%, 95%, and 100% ethanol, followed by two changes of xylenes and three changes of paraffin wax for one hour each using tissue processor (Leica ASP300S; Leica, USA). The processed tissues were embedded in paraffin wax in embedding module and sectioned into 10μm thick slices using a microtome. The tissue slices were deparaffinized with xylene, rehydrated with gradation of ethanol, and stained using Hematoxylin and Eosin kit (H&E; American MasterTech Scientific, USA) according to the manufacturer's recommendations.

Flow Cytometry Analysis of ASCs
The isolated ASCs were stained with stem cell markers and analyzed using flow cytometry.
ASCs from the second passage were used for the analysis. For each cell marker, approximately 100,000 ASCs were incubated with either fluorescein isothiocyanate (FITC), alexa fluor 647 (A647) or phycoerythrin (PE) conjugated antibodies for 30 minutes at room temperature protecting from light. The following antibodies were used: FITC conjugated rat antibody for CD90 and CD31, PE conjugated rat antibody for CD29 and CD34, FITC conjugated pig antibody for CD90, CD31 and CD14 (Abcam, USA), and A647 conjugated pig antibody for CD29 (BD Biosciences, USA). Cells were also stained with fluorescent live/dead dye (Life technologies, USA) to obtain the live cell population and only those cells were included in the analysis. The stained cells were subjected to flow cytometer analysis using a LSR Fortessa flow cytometer (BD Biosciences, USA). Data was analyzed using FlowJo software (v9.8.3).
Unstained cells were used with every run for each antibody as a negative control.

Quantiative Real Time Polymerase Chain Reaction (qRT-PCR) Analysis
qRT-PCR was performed to examine the effects of the dPMS on gene expression of rat and pig ASCs. The dPMS reseeded with ASCs was digested using collagenase type II (4 mg/mL, Sigma-Aldrich, USA) at 37 C for 15 minutes, followed by centrifugation at 600g for 5 minutes to extract cells. Then total RNA was isolated from the cells using an Absolutely RNA Microprep Kit (Agilent Technologies, USA). The total amount and purity of isolated RNA was measured using a Synergy H1 Hybrid Reader nanodrop (Bio-Tek Instruments, USA) and 1% agarose gel electrophoresis. Approximately 40 ng/µl total RNA was converted into cDNA using Verso cDNA kit (Thermo Scientific, USA) per the manufacturer's instructions. The real-time PCR reactions were performed using the SYBR GreenER qPCR SuperMix (Invitrogen, USA) and Applied Biosystems 7500 real-time PCR detection system. Relative gene expression was analyzed with the comparative Ct method. 1 The results were normalized to β-actin and GAPDH and expressed as a fold change for cells isolated from recellularized dPMS compared with cells cultured on TCP (ΔΔCt). qPCR was performed on 4 independent samples and each sample were tested in 3 pseudo-replicates. The primers used in this study are listed in Supplementary Table   S1.  hours. Finally, the tissue sections were stained using VECTASHIELD mounting medium with DAPI for nuclei staining. Fluorescence images were captured with an inverted AxioVision A1 microscope (Carl Zeiss). Our results showed that the rat ASCs did not express GATA4 and cardiac troponin I markers on day 1, day 3, or day 5. Figure S2. Expression of cardiac related markers (GATA4, cardiac troponin and SMA) at day 1, day 3 and day 5 for rat ASCs on 300 µm dPMS. Scale bar 50 µm.

GAGs Content Measurement:
GAGs content in the native myocardium and decellularized porcine myocardium was quantified using the Blyscan sulfated glycosaminoglycan assay following the manufacturer's instruction (Biocolor, Carrickfergus, UK). The total GAGs content was measured in µg/mg wet tissue using four independent samples. We found that decellularized porcine myocardium had significantly reduced GAGs content (0.22 ± 0.03 µg/mg wet tissue) as compared to native myocardium (2.04 ± 0.51 µg/mg wet tissue). respectively. We found that some of the infiltrated cells were macrophages (Fig. S4 A). Among them, M2 macrophages also were present (Fig. S4 B). M2 macrophages have been reported to promote positive tissue remodeling.  Table 2) 2 . Average vessels were counted for each region and represented in per mm 2 area. We found the higher number of vessels per mm 2 area in the MI group treated by dPMS with ASCs than the control group (MI only) at week 1 (Fig. S5)