"Off-the-Shelf" Nerve Matrix Preservation.

Background: Decellularized human nerves overcome the limitations of the current treatments for large peripheral nerve injuries. However, the use of decellularized nerves requires an "off-the-shelf" availability for useful and actual clinical application. In this study, we addressed the preservation of the native and decellularized human nerve matrix in an integrative approach for tissue scaffold production. Materials and Methods: For native nerve matrix preservation analysis, we used histological examination and immunofluorescence to examine the structure, biomechanical assays to evaluate the tensile strength and Young's modulus, and analyzed the extracellular matrix (ECM) composition using enzyme-linked immunosorbent assay (ELISA) and biochemical assays for laminin, collagen and sulfated glycosaminoglycans (sGAG). After decellularization, nuclear remnants and DNA content were evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining and the picogreen quantification assay, as well as immunofluorescence or ELISA for cell rests (S100 protein and myelin staining) evaluation. Decellularized cryopreserved scaffolds were assayed for biomechanics, ECM composition, and structural maintenance. Cytotoxicity assays were performed to evaluate the biocompatibility of the nerve matrix extracts after cryopreservation. Results: We compared different strategies for native nerve storage and found that preservation up to 7 days at 4°C in Roswell Park Memorial Institute medium maintained biomechanical properties, such as Young's modulus and tensile strength, along with the structure and ECM composition, regarding laminin, collagen, and sGAG. After a successful decellularization process, that eliminated cell remnants, nerve scaffolds were frozen in an "in house" formulated cryoprotectant, using an automatic controlled rate freezer. Nerve structure, ECM composition, and biomechanical properties were maintained before and after the freezing process in comparison with native nerves. The extracts of the nerve scaffolds after thawing were not cytotoxic and the freezing process sustained good viability in 3T3 cells (graphical abstract). Conclusion: Since our approach facilitates transport, storage, and provide a ready-to-use alternative, it could be used in a clinical application for the treatment of long-gap peripheral nerve injuries in regenerative medicine.

Cryopreservation of hematopoietic progenitor cells from apheresis at high cell concentrations does not impair the hematopoietic recovery after transplantation.

BACKGROUND: The high number of nuclear cells (NCs) from hematopoietic progenitor cells-apheresis (HPC-A) requires cryopreservation in large volumes or at high NC concentrations. The effect of NC concentration during cryopreservation has yet to be examined. STUDY DESIGN AND METHODS: In the experimental arm (n = 610, Protocol B), the first HPC-A sample from the patient was cryopreserved in two cryobags and subsequent collections in one cryobag, resulting in high NC concentrations (>100 x 10(6) NCs/mL) in most cases. The effect of NC concentrations at freezing in NC recovery after thawing and engraftment kinetics was analyzed and compared with a group of HPC-A cryopreserved at standard NC concentrations (n = 455, Protocol A). RESULTS: The mean (SD) NC concentration at freezing was 78 (28) x 10(6) per mL (median, 82 x 10(6)/mL; range, 12 x 10(6)-156 x 10(6)/mL) and 183 (108) x 10(6) per mL (median, 156 x 10(6)/mL; range, 16 x 10(6)-678 x 10(6)/mL), for HPC-A cryopreserved according to Protocols A and B, respectively. The NC viabilities of the test vials and HPC-A components after thawing were 88 percent versus 85 percent and 85 percent versus 82 percent, and the cloning efficiency was 49 percent versus 33 percent for Protocols A and B, respectively (p < 0.001). Significant differences were not observed in the recovery of NCs. Days to neutrophil and platelet engraftment were not different between patients transplanted in the standard- (n = 143) or high-cell-concentration group (n = 238). CONCLUSION: The cryopreservation of HPC-A at higher than standard NC concentrations has no adverse impact on hematopoietic reconstitution after transplantation.