Heart ECM purification
Human heart tissue from several donors was procured after consent under IRB numbers 35241 and 35242 from the University of Utah and frozen at −80°C until the decellularization process began. Heart tissue was pooled and decellularized using a gentle immersion decellularization process, and handled in a biosafety cabinet throughout the purification process to maintain sterility. After thawing tissue overnight at 4°C, tissue was minced into pieces ~1 cm3 in size. Minced tissue was rinsed with sterile water (Baxter, Deerfield, USA) to remove excess blood and fluids. Next, tissue was blended in sterile water (blender model 7010S, Waring Commercial, Torrington, USA) to create a homogenous slurry. Soluble and insoluble portions of tissue were separated by centrifugation at 3,000 rcf and 40°C (Sorvall Legend XTR centrifuge, Thermo Fisher Scientific, Pittsburgh, USA). Following centrifugation, the supernatant was discarded, new sterile water was added in a 1:4 pellet to water ratio, and the pellet was dispersed and mixed thoroughly. This slurry was centrifuged again, the water was exchanged, and water washes continued until the supernatant had minimal color. Following water washes, the tissue was then washed three times in isopropyl alcohol (Sigma Aldrich, St Louis, USA), following the same procedure. Next the tissue was washed in 3 M NaCl (Sigma Aldrich); for this and the remaining washes, centrifugation was performed at 4°C. Tissue was then washed five times with 40 mg/ml sodium deoxycholate (SDC, Sigma Aldrich); with each SDC wash, the tissue was placed on an orbital shaker (model 980001, VWR, Radnor, USA) for at least 18 h before centrifugation. If after five SDC washes the SDC supernatant had color indicative of incomplete washing, additional SDC washes were performed until there was no additional color in the supernatant. Following SDC washes, tissue was washed again with 3 M NaCl, then with 100 U/ml DNase (Worthington Biochemical, Lakewood, USA) to aid DNA removal, and next with PBS (Life Technologies, Carlsbad, USA). Finally, the tissue preparation was washed ten times in sterile water. Before each new washing solution, the tissue was washed twice in sterile water to rinse out the old solution. The entire process was ~10 days in duration with the majority of time taken by the SDC wash. The resulting insoluble material, mainly composed of extracellular matrix protein components, was frozen in sterile water and lyophilized (FreeZone 2.5, Labconco, Kansas City, USA), then stored at −20°C until use.
Prior to use in scaffold fabrication or submission for mass spectrometry, lyophilized decellularized cardiac tissue was solubilized in sterile 0.1 M acetic acid (Thermo Fisher Scientific, Pittsburgh, USA) at a concentration of 5 mg/ml. After sitting in acetic acid overnight at 4°C, the decellularized tissue was homogenized (Polytron 1200 E, Kinematica AG, Littau-Lucerne, Switzerland) for 5 min. The solution was kept on ice during homogenization to prevent denaturation at high temperature.
Porous ECM scaffolds were fabricated from decellularized cardiac tissue using a sacrificial polycaprolactone (PCL) porous scaffold as a template. The PCL scaffold was generated as a template for the ECM protein and later dissolved away. To create the sacrificial porous scaffold, PCL (Sigma Aldrich) was dissolved in acetone (Sigma Aldrich) at 50°C at a concentration of 0.15 g/ml. Next, water was added dropwise to 8% of the total volume. The PCL/acetone/water solution was then mixed with the porogen NaCl, sieved to select for salt crystals between 425 and 500 µm (for stem cell and primary cardiac cell ECM scaffolds) or <250 µm (for iCell iPSC cardiomyocyte ECM scaffolds). This mixture was added to 50 ml conical tubes (BD Biosciences, Franklin Lakes, USA) thoroughly mixed and centrifuged at 500 rcf and 35°C to create a uniform saturated salt suspension. The mixture was placed at −20°C to solidify, and the salt porogen was subsequently removed by immersion in excess water. The resulting PCL foam exhibits a well-defined pore structure with larger pores formed by the salt porogen and smaller pores formed by water. These foams were cut to a thickness of 0.65–0.7 mm (stem cell and primary cardiac scaffolds) or 0.3 mm (for iPSC cardiomyocyte scaffolds) using a Centaur Deli Slicer (model 212, Lombard, USA). Sliced porous PCL scaffolds were cut with biopsy punches (Miltenyi, Bergisch Gladbach, Germany) into 3 mm diameter slices intended for further studies.
Decellularized and solubilized cardiac ECM was then coated onto these porous PCL scaffolds. PCL scaffolds were immersed in a turbid solution of ECM solubilized in 0.1 M acetic acid. The PCL foam and solution were placed in a vacuum chamber (Space Saver Vacuum Desiccator, Bel-Art, Wayne, USA) to remove gas pockets and facilitate full penetration of the solution into the PCL pores. Once all air bubbles were eliminated, scaffolds were air dried in a biosafety cabinet, forming a single, contiguous coating. This process was repeated to create a total of five ECM coats. Following coating, the bulk PCL matrix was removed by dissolution in 95% acetone at 40°C for 2 days. Acetone was replaced twice daily. Scaffolds were then transferred into 95% ethyl alcohol (Decon Labs, King of Prussia, USA) and then slowly exchanged into sterile water using several serial solutions of decreasing ethanol content (70, 50, and 25% ethyl alcohol). Finally, the PCL-free scaffolds were washed 10 times in sterile water. The resulting scaffolds, comprising porous, templated decellularized cardiac tissue, were frozen in 100% water, lyophilized, sterilized with ethylene oxide gas (University of Utah Hospital, Salt Lake City, USA), and stored at −20°C until use.
Primary derived cardiac cells
Human hearts were collected after research consent was obtained, under approved institutional review board of the University of Utah, IRB numbers 35241 and 35242. Left ventricle tissue from multiple donor hearts was mechanically minced into 1 mm3 pieces and placed in Dulbecco’s phosphate buffered saline DPBS(–) for 30 min. Minced tissue was placed into DPBS(–) containing 0.45 mg/ml collagenase (Worthington Biochemical Corp.) and 1 mg/ml pancreatin (Life Technologies, Carlsbad, USA). Digestion was carried out for 60 min at 37°C with gentle shaking every 5 min. Equal volume of DPBS(–) containing 10% XcytePLUS™-Xenofree Media (iBiologics, Phoenix, USA) was added to the digestion, then filtered using a 100 µm filter. Filtered cells were then washed 3× with DPBS(–) containing 10% XcytePLUS™ by centrifugation and plated into tissue culture plates (Nunc, Thermo Fisher Scientific, Pittsburgh, USA) for differential adhesion overnight at 5% CO2/37°C. After 12 h, non-adherent cells were discarded and adherent cells, likely consisting of a heterogeneous mixture of cardiomyocytes and fibroblasts, were passaged using TryplE (Life Technologies, Carlsbad, USA) and frozen till use using CryoStore CS10® (BioLife Solutions, Bothell, USA).
Induced pluripotent stem cell-derived cardiomyocytes
Pure human iCell cardiomyocytes containing monomeric red fluorescent protein (RFP) expressed under control of the endogenous Myh6 promoter were purchased from Cellular Dynamics (Madison, USA) and cultured according to manufacturer’s instructions. Briefly, immediately upon receipt, the vial of iCell cardiomyocytes was washed, cells were counted using a hemocytometer (Baxter, Deerfield, USA) and then resuspended at a concentration of 10 × 106 cells/100 µl, which in our hands yielded 100% confluence (data not shown). Cells were seeded onto scaffolds (vide infra) and placed into iCell cardiac plating medium for 48 h at 37°C and 7% supplemental CO2. After 48 h, media was replaced with iCell cardiac maintenance media that was changed every 48 h throughout the duration of the experiment.
Lyophilized and sterilized porous ECM scaffolds were added to a 50 ml conical tube containing cell suspensions of 106 cells/100 l for primary cardiac cells (on our scaffolds and Matristem®, Acell, Columbia, USA), and 107 cells/100 l for iPSC cardiomyocytes (on our scaffolds), and allowed to rehydrate for 10 min. During this time, a spatula was used to immerse the buoyant scaffolds continuously in the cell slurry. Air bubbles were removed to ensure homogeneous scaffold seeding; the vial cap was loosened and allowed to de-gas in a vacuum chamber for 10 min, a sufficient time to eliminate air bubbles without removing all dissolved oxygen in the cell slurry. Six scaffolds per well were added to a 12-well plate (Nunc). After 12 h non-adherent cells were discarded and adhered cells were cultured in iCell Cardiomyocyte media (cellular dynamics) for both iCell iPSC cardiomyocytes and primary cardiac cells. Media was changed every other day and cells were cultured in a 37 incubator with 5% supplemental CO2 for a maximum of 7 days.
Gross imaging and video
A Leica M165FC dissecting microscope equipped with a DFC425 camera and Leica Application Suite software v3.8.0 was used to take bright-field and fluorescent images of the empty ECM scaffolds and Matristem® (Acell, Columbia, USA) and seeded ECM scaffolds on explanted mouse heart, tissue, respectively. Videos of cell-seeded, beating ECM scaffolds were taken with an Olympus IX51microscope equipped with fluorescence and DP72 CCD camera and CellSens software v1.6.
Samples of decellularized tissue solubilized in 0.1 M acetic acid were submitted to the University of Utah Health Sciences Center Mass Spectrometry and Proteomics Core (Salt Lake City, USA) for protein identification.
Digest of proteins in solution
Proteins from heart scaffolds were digested with TPCK-modified trypsin (Promega). Trypsin (in 50 mM ammonium bicarbonate) was added to the solution (adjusted to pH 7.9) to obtain a ratio of ~1–25 (enzyme to protein). Digest reactions were allowed to continue for overnight (at 37°C) for standard protein ID analyses.
Peptides were analyzed using a nano-LC–MS/MS system comprised of a nano-LC pump (Eksigent) and a LTQ-FT mass spectrometer (ThermoElectron Corporation, San Jose, CA, USA). The LTQ-FT is a hybrid mass spectrometer with a linear ion trap used typically for MS/MS fragmentation (i.e. peptide sequence) and a Fourier transform ion-cyclotron resonance (FT-ICR) mass spectrometer used primarily for primary MS accurate mass measurement of peptide molecular ions. The LTQ-FT is equipped with a nanospray ion source (ThermoElectron Corp.). Approximately 5–20 fmoles of tryptic digest samples were dissolved in 5% acetonitrile with 0.1% formic acid and injected onto a C18 nanobore LC column for nano-LC–MS/MS and identification of peptides. The nanobore column was homemade [C18 (Atlantis, Waters Corp); 3 µm particle; column: 75 µm ID × 100 mm length] Atlantis dC18, 3 μm × 75 μm × 100 mm (Waters Corp.). A linear gradient LC profile was used to separate and elute peptides, consisting of 5–70% solvent B in 78 min with a flow rate of 350 nl/min (solvent B: 80% acetonitrile with 0.1% formic acid; solvent A: 5% acetonitrile with 0.1% formic acid). The LTQ-FT mass spectrometer was operated in the data-dependent acquisition mode controlled by Xcalibur 1.4 software, in which the “top 10” most intense peaks observed in an FT primary scan (i.e. MS survey spectrum) are determined by the computer on-the-fly and each peak is subsequently trapped for MS/MS analysis and peptide fragmentation (sequencing by collision-induced dissociation) in the LTQ linear ion trap portion of the instrument. Spectra in the FT-ICR were acquired from m/z 400 to 1,700 at 50,000 resolving power with about 3 ppm mass accuracy. The LTQ linear ion trap was operated with the following parameters: precursor activation time 30 ms and activation Q at 0.25; collision energy was set at 35%; dynamic exclusion width was set at low mass of 0.1 Da and high mass at 2.1 Da with one repeat count and duration of 10 s.
Mascot database searches
LTQ FT MS raw data files were processed to peak lists with BioworksBrowser 3.2 software (ThermoElectron Corp., San Jose, CA, USA). Processing parameters used to generate peak lists were as follows: precursor mass 351–5,500 Da; grouping was enabled allowing 5 intermediate MS/MS scans; precursor mass tolerance 5 ppm, minimum ion count in MS/MS was set to 15, and minimum group count was set to 1. Resulting DTA files from each data acquisition file were merged and the data file was searched for identified proteins against the NCBI human taxonomy sub-database, using MASCOT search engine (Matrix Science Ltd.; version 2.2.1; in-house licensed). Searches were done with tryptic specificity, allowing two missed cleavages and a mass error tolerance of 5 ppm in MS spectra (i.e. FT-ICR data) and 0.5 Da for MS/MS ions (i.e. LTQ linear ion trap). Variable modification included in the searches was oxidation of methionine, histidine and tryptophan residues. Identified peptides were generally accepted only when the MASCOT ion score value exceeded 20. Peptides identified in the MASCOT search results were all further validated by manual confirmation of molecular ions from the FTMS spectra and assigned fragment ions from the corresponding MS/MS spectra.
Surface coatings with decellularized tissue
Glass chamber slides (Chamber slide system 154534, Nunc) were coated with decellularized cardiac tissue solubilized in 0.1 M acetic acid, diluted to 1.5 mg/ml.
Chamber slides (Chamber slide system 154534, Nunc) were coated by incubation with cardiac ECM protein solubilized in 0.1 M acetic acid (diluted to 1.5 mg/ml) overnight at 4°C and subsequently washed three times with PBS. Coated chamber slides were air-dried and sterilized by UV exposure in biosafety cabinet for 30 min.
Mouse heart explant and in situ scaffold placement
Care of animals was in accordance with institutional guidelines. Necropsies were performed on 6 C57/BL6 mice routinely sacrificed by veterinary staff for other purposes. Hearts were aseptically removed and the pericardium was dissected away. ECM scaffolds seeded with viable cardiomyocytes and cultured for 6 days were overlaid onto the left ventricle portion of the freshly isolated hearts. Once placed, scaffolds remained untouched on this cardiac tissue surface for 3 min. After this time, forceps were used to attempt to displace the scaffold from the surface by sliding the scaffolds along heart wall.
For viability analysis, cell-seeded ECM scaffolds were stained with Calcein AM and PI (Invitrogen, Carlsbad, USA) according to manufacturer’s instructions and imaged on their external surfaces and through their center zones. To image cell viability within scaffold centers, the scaffolds were cut transversely and placed cut-side down onto a glass slide and imaged (a second slide was held perpendicular to the first glass slide to stabilize the cut scaffold). Confocal images from 3 scaffolds and 3 images per scaffold were collected in the red and green channels. Using ImageJ (imagej.nih.gov), the number of green (Calcein AM) cells were compared to the number of red (PI) cells in each frame to obtain percent cell viability on day 7. The average and standard deviation (SD) from 9 images are displayed below.
To determine cell distribution, cells were fixed in 4% paraformaldehyde (Affymetrix, Cleveland, USA) and labeled with and 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen) and rhodamine-phalloidin (Life Technologies, Carlsbad, USA) according to manufacturer’s instructions. Rabbit anti-cardiac troponin T (Abcam, Cambridge, USA 1:200), rabbit anti-connexin 43 (Abcam 1:200) and rabbit anti-N-cadherin (Abcam 1:200) were used with secondary antibody Donkey anti-rabbit-488 (Invitrogen, 1:500).
Cells within scaffolds were imaged on a Nikon AR1 confocal microscope. For all images, a z-series comprising 7 sections, 20 μm thick were stacked into a single image (spanning 140 μm total). A Prairie multi-photon confocal microscope was used to image the second harmonic signal of collagen from both the explanted native mouse heart and ECM scaffold placed onto the mouse heart.
Scanning electron microscopy
Scaffolds were fixed in 4% paraformaldehyde and 2% glutaraldehyde and post-fixed in 2% osmium tetroxide, dehydrated through a series of ethanol washes, and dried with hexamethyldisilazane. The scaffolds were then sputter-coated with gold (30 s at 40 microamps) using a Pelco SC-7 autosputter coater and imaged with a SEM (FEI Quanta 600 FEG scanning electron microscope) under high vacuum.
Optical action potential
After 6 days of culture, ECM scaffolds (3 mm in diameter, 0.3 mm thick) with a porogen size of 250 µm seeded with iCell cardiomyocytes were used for optical action potential and microelectrode array recordings. iCell media was replaced with a voltage-dependent dye solution (Infrared dye USD DI-4ANBDQBS diluted 1:1,000 in iCell media) for 7 min. After 7 min, media was replaced with dye-free iCell media. All media used was pre-warmed to 37°C. Scaffolds were transferred to an imaging chamber filled with iCell media on a Nikon microscope. Only 70 µl of media was added to keep scaffold moist, but prevent it from floating. A 50 W heat lamp was used 4 inches away from the imaging chamber to keep the cells at 37°C (a thermometer was placed in the imaging chamber to ensure a constant temperature was maintained). The heat lamp was turned off only during direct image capture.
To reduce movement artifacts, cardiomyocyte seeded scaffolds were treated with 10 µM blebbistatin (reconstituted in DMSO at 16.7 mM) diluted in HEPES to uncouple cardiomyocyte motion. Scaffolds were allowed to sit for 15 min for the uncoupler to take effect prior to imaging.
Microelectrode array recordings
Also on day 6, another set of cell-seeded ECM scaffolds was analyzed using a custom-built microelectrode array apparatus (kindly donated by A. Moreno, University of Utah). iCell cardiomyocyte seeded scaffolds were recorded in 37°C iCell media. To confirm the electrical acuity of the cell-seeded ECM scaffolds, 1 µM isoproterenol (Hospira, Lake Forest, USA) was added and the scaffolds were subsequently recorded.