RESUMEN
Reconstructive surgery remains inadequate for the treatment of volumetric muscle loss (VML). The geometry of skeletal muscle defects in VML injuries varies on a case-by-case basis. Three-dimensional (3D) printing has emerged as one strategy that enables the fabrication of scaffolds that match the geometry of the defect site. However, the time and facilities needed for imaging the defect site, processing to render computer models, and printing a suitable scaffold prevent immediate reconstructive interventions post-traumatic injuries. In addition, the proper implantation of hydrogel-based scaffolds, which have generated promising results in vitro, is a major challenge. To overcome these challenges, a paradigm is proposed in which gelatin-based hydrogels are printed directly into the defect area and cross-linked in situ. The adhesiveness of the bioink hydrogel to the skeletal muscles was assessed ex vivo. The suitability of the in situ printed bioink for the delivery of cells is successfully assessed in vitro. Acellular scaffolds are directly printed into the defect site of mice with VML injury, exhibiting proper adhesion to the surrounding tissue and promoting remnant skeletal muscle hypertrophy. The developed handheld printer capable of 3D in situ printing of adhesive scaffolds is a paradigm shift in the rapid yet precise filling of complex skeletal muscle tissue defects.
RESUMEN
This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.
