Protocols for the preparation and characterization of decellularized tissue and organ scaffolds for tissue engineering

Extracellular matrix (ECM) scaffolds are extensively used in tissue engineering studies and numerous clinical applications for tissue and organ reconstructions. Due to the global severe shortage of human tissues and organs, xenogeneic biomaterials are a common source for human tissue engineering and regenerative medicine applications. Traditional methods for decellularization often disrupt the 3D architecture and damage the structural integrity of the ECM scaffold. To efficiently obtain natural ECM scaffolds from animal tissues and organs with intact architecture, we have developed a platform decellularization process using supercritical CO and tested its potential application in tissue engineering. A combination of human mesenchymal stem cells with a decellularized dermal matrix scaffold allowed complete regeneration of skin structure in a porcine full-thickness wound model. Porcine tissues and organs were sliced to the appropriate thickness, placed in muslin bags and subjected to decellularization by supercritical carbon dioxide extraction technology. The decellularized tissue and organ ECM scaffolds were freeze-dried and cut to the appropriate size, thickness and according to different size specification in Petri dishes for the use in the experiments and clinical tissue engineering applications. The decellularized tissue and organ ECM scaffolds were also freeze-milled to a 45–200 μm powder for potential 3D bioprinting application. The decellularized tissue and organ ECM scaffolds were characterized by hematoxylin and eosin and DAPI staining. Scanning electron microscopy was performed to examine the collagen fibers and cellular debris.