Cell-seeded scaffolds after the 24- and 48-h growth periods were snap-frozen and stored at −20°C. Scaffolds were then freeze-dried overnight using a FreeZone 4.5 freeze-drier (Labconco) to remove any residual water content before DNA extraction. The scaffolds were then digested in a solution of D-PBS/CaCl2 and MgCl2 free (Sigma), containing 2.5 U/mL papain extract (Sigma), 5 mM cysteine-HCl (Sigma), and 5 mM EDTA (Sigma), and samples were incubated overnight at 60°C. Cell extracts (n = 4) of 5 × 105 cells frozen at −20°C when scaffolds were seeded, served as the control. Samples (n = 4 scaffolds) were mixed thoroughly before assay. A Quant-IT™ PicoGreen® dsDNA Assay Kit (Life Technologies) was used and performed according to the manufacturer's protocol based on 200 μL volume for microplate reader analysis. Samples were analyzed in a Modulus II microplate multimode reader using a filter of 490 nm Ex/510–570 nm Em.
Freezone 4.5 freeze drier
Manufactured by Labconco
The FreeZone® 4.5 freeze-drier is a laboratory equipment designed to remove moisture from samples through the process of freeze-drying. It features a 4.5-liter capacity and is capable of accommodating various sample containers.
Automatically generated - may contain errors
4 protocols using freezone 4.5 freeze drier
1
Quantification of Cell Proliferation in Scaffolds
2
Decellularization and ECM Lyophilization
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Decellularization was confirmed using the Quant-IT™ Picogreen® dsDNA assay kit (Life Technologies™), performed according to manufacturer’s instructions. The ECM was lyophilized in a FreeZone® 4.5 freeze-drier (Labconco®) before milling in a PM100® planetary ball mill (Retsch®).
3
Characterizing Collagen Scaffold Porosity
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Scanning electron microscopy (SEM) characterized the porous architecture of the collagen scaffolds postmodification due to crosslinking and/or soaking, and then shaking conditions. The scaffolds tested, PBS unshaken, PBS shaken, PBE E/N unshaken, and PBS E/N shaken. Scaffolds were snap-frozen and then freeze dried using a FreeZone® 4.5 freeze-drier (Labconco®). The samples were then mounted on to metal stubs with double-sided carbon tape. Thin layers of a gold and palladium alloy were applied to each sample with an automated sputter coater (Polaron SputterCoater). The samples were then examined at ×60 low magnification at 5 kV (Hitach S-4700 SEM) as previously shown.15 (link)
4
Bovine Tissue Decellularization Protocol
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Bovine heart and aorta were harvested from a 2‐year‐old female. Samples were frozen within 4 hr of harvesting at −80°C. Heart tissue was sliced up into 5‐mm‐thick slices using a meat slicer. The aorta was opened up and flattened out and subsequently had 40‐mm‐circular samples punched out. This was repeated for the heart slices. Slices were cleaned with ethanol and placed in water for 30 min to remove excess blood. Samples were then cut up into 2 mm × 2 mm square pieces to increase surface area for decellularization. Pieces were perfused with 0.5% w/v sodium dodecyl sulfate (SDS) in deionized water for 36 hr at 170 mL/min. Samples were then perfused with 10 L of deionised water at 170 mL/min. Decellularized samples were then frozen at −80°C before being lyophilized in a FreeZone® 4.5 freeze‐drier (Labconco®). Dry ECM was then crushed up into ~0.5 mm pieces before being milled in a planetary ball mill PM 100 (Retsch). ECM powder was then collected in water, frozen at −80°C, and lyophilized leaving behind ECM powder. Powder was stored at 4°C.
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