Effect of DMSO concentration, cell density and needle gauge on the viability of cryopreserved cells in three dimensional hyaluronan hydrogel

For cells seeded in scaffolds, transplanted cell survival rate plays an important role for cell transplantation efficiency, and is essential for successful cell transplantation. Fibroblast viability in HyStem-C was examined by a double staining Live/Dead Viability/Cytotoxicity assay, and cell images were analyzed using MetaMorph software for calculating live cell percentage for fresh and cryopreserved cells at different incubation time points, delivery methods, differing DMSO and cell concentrations. The results of this research demonstrated that in HyStem-C, the viability of cryopreserved cells (85%) was significantly lower than fresh collected cells (96.7%). In addition, the physical force from a 27 gauge needle significantly decreased frozen cell survival rates to 83-85% compared to pipette delivered cells. Higher DMSO concentration (1.0%) and higher cell density (2 × 10⁷ per milliliter) also significantly decreased cell survival to 73%. Cryopreserved cell viability in three dimensional scaffolding can be maintained over 80% with cell density of 1 × 10⁷ per milliliter, total DMSO concentration of 0.5%, and passed through a 27-gauge needle. These results demonstrate the viability of cells seeded in hyaluronan hydrogel with commonly used storage and delivery methods can bring rather satisfactory cell transplantation efficiency.