We obtained allogeneic CDCs from a Sinclair minipig heart from Sinclair Bio Resources, LLC (Columbia, MO), for the placebo‐controlled pivotal study and the mechanism assessment study to create a master cell bank (MCB). The heart arrived on ice submerged in cardioplegic solution. Hearts were grossly dissected, and biopsy‐sized pieces (≈25 mg) were seeded to create explant‐derived cells (EDCs). After ≈14 days, EDCs were harvested to create an MCB. MCB vials were thawed and cultured as cardiospheres (CSps) in suspension culture. CSps were grown on Ultra Low Cell STACK vessels (Corning Life Sciences, Tewksbury, MA). allo‐CDCs were grown by seeding CSps on Nunc Triple Flasks (Thermo Fisher Scientific, Waltham, MA) and passaging when confluent. allo‐CDCs were resuspended (1.25 M/mL for a total dose of 12.5 million CDCs in 10 mL) in CryoStorCS10 (BioLife Solutions, Inc., Bothell, WA) in cryobags (PL07 PermaLife Bags; Origen Biomedical, Inc., Austin, TX), placed directly in a CryoMed controlled‐rate freezer, and then transferred to liquid nitrogen. CDCs were thawed at the day of the infusion. Upon thawing, 1 mL of heparin (100 USP units/mL) and 0.1 mL of nitroglycerin (50 μg/mL) were added as diluents for a total 10‐mL dose for administration. Cell‐dose preparation was performed by Capricor, Inc. (Beverly Hills, CA).
Nunc tripleflasks
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Nunc TripleFlasks are a line of cell culture flasks designed to provide increased surface area for cell growth. These flasks feature three interconnected chambers, allowing for a larger total growth area compared to traditional single-chamber flasks of the same footprint.
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5 protocols using nunc tripleflasks
1
Allogeneic CDC Preparation for Cardiac Studies
2
Purification of FLAG-tagged Proteins
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Cell-free extracts containing FLAG-tagged proteins were prepared as follows: extracellular parasites from twenty 500 cm2 flasks (Nunc TripleFlasks, Thermo Fisher Scientific) were lysed in 7 mL of BC100 buffer (20 mM TrisHCl pH 8, 10% glycerol; 0.2 mM EDTA, 0.1 M KCl) with the addition of a protease inhibitor cocktail (Complete Protease Inhibitor Tablets, Roche) using a Dounce homogenizer (tight plunger). The lysate was centrifuged twice at 21,000 g for 10 min at 4°C. The supernatant was then incubated with 200 µL anti-FLAG M2 affinity gel (Sigma- Aldrich) for 1 hour. Beads were then washed with 5 column volumes (CV) washing buffer (25 mM Tris pH 7.5, 150 mM KCl, 10% glycerol, 10 mM MgCl2), followed by 5 CV of washing buffer with 500 mM KCl and finally another 5 CV of low-salt wash buffer, before bound polypeptides were eluted with two CV of wash buffer containing 250 µg/mL FLAG peptide.
3
Isolation and Characterization of Extracellular Vesicles
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MVs were isolated according to the well-established protocol developed in our laboratory [45 (link)] based on the procedure introduced in the previous study [46 (link)]. HATMSC2 cells were cultured in multi-layer cell culture flasks (Nunc TripleFlasks, Thermo Scientific, Carlsbad, CA, USA) using DMEM + 10% FBS until they reached 75% confluence. Next, the cells were cultured in serum-free media in hypoxic conditions (1% O2) for 48 h to enhance the release of MVs. The conditioned media collected from the HATMSC2 cultures were mixed to obtain a homogenous starting material before the isolation of MVs. In the next step, the conditioned media were centrifuged at 300× g for 10 min at 4 °C, and at 2000× g for 10 min at 4 °C, in order to remove cellular debris and apoptotic bodies. Subsequently, the supernatants were subjected to double centrifugation at 12,000× g for 30 min at 4 °C using a Sorvall LYNX 6000 ultracentrifuge (Thermo Scientific, Carlsbad, CA, USA) with an intermediate washing step in PBS (IIET, Wroclaw, Poland). The obtained MV pellets were resuspended in 150 μL of PBS and stored at −80 °C.
4
Electron Microscopy of Immunoprecipitated Protein
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Immunoprecipitation eluents of YRS-HA-FLAG, from 17 500 cm2 flasks (Nunc TripleFlasks, Thermo Fisher Scientific) using 0.4 µl anti-FLAG resin, were concentrated to 100 µl (from an original volume of 1500 µl) by ultrafiltration with a 10 kDa MWCO centrifugal unit (as described above). Samples were then diluted by 1:5 in 25 mM Tris-HCl pH 7.5, 150 mM NaCl before application of 3.5 µl onto copper electron microscope grids coated with a plasma-treated thin carbon support films (Electron Microscopy Sciences). Staining was accomplished with 2% uranyl acetate solution using the droplet method and electron micrographs were recorded by a Gatan ORIUS 2.7 k×2.7 k CCD camera at a nominal magnification of 25 000 times using a JEOL 1200 EX II microscope operating at 100 kV. Particle images (1030) were manually selected and windowed (128 pixel) from 50 micrographs (sampling of 2.7 Å/pixel) using SIGNATURE [32] (link). Rotational averages were calculated in EMAN1 [33] (link) following pre-centring and the radial profile was plotted from the resulting total average in ImageJ [34] . Iterative reference-free averaging in EMAN was used to generate the representative average of a manually selected sub-set of images (137 out of 1030) and iterative reference-free classification produced the class averages for heterogeneity assessment.
5
Allogeneic Cardiosphere-Derived Cell Isolation
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All allogeneic CDCs were isolated and manufactured at the Cedars-Sinai Medical Center using, as the starting material, both ventricles of a male Sinclair pig donor as described [42] (link). Briefly, the heart was dissected into approximately 25 mg pieces which were seeded to obtain explant derived cells. After about 14 days, these cells were harvested to create a master cell bank (MCB). MCB vials were thawed and cultured as primary cardiospheres in suspension culture, on Ultra Low Cell STACK vessels (Corning Life Sciences). CDCs were then grown from primary cardiospheres seeded on fibronectin in Nunc Triple Flasks (Thermo Fisher Scientific, Waltham, MA) and passaged when confluent. CDCs at the 5th passage were resuspended (1.25 M CDCs/mL for a total of 12.5 million CDCs in 10 mL) in CryostorCS10 (BioLife Solutions, Inc., Bothell, WA) in cryobags (PL07 PermaLife Bags; Origen Biomedical, Inc., Austin, TX), placed directly in a CryoMed controlled-rate freezer, and then transferred to liquid nitrogen. CDCs were shipped frozen to Inserm U 955, where they were stored at -80° C.
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