Cryopreservation effects on recombinant myoblasts encapsulated in adhesive alginate hydrogels.

Cell encapsulation in hydrogels is widely used in tissue engineering applications, including encapsulation of islets or other insulin-secreting cells in pancreatic substitutes. Use of adhesive, biofunctionalized hydrogels is receiving increasing attention as cell-matrix interactions in three-dimensional (3-D) environments can be important for various cell processes. With pancreatic substitutes, studies have indicated benefits of 3-D adhesion on the viability and/or function of insulin-secreting cells. As long-term storage of microencapsulated cells is critical for their clinical translation, cryopreservation of cells in hydrogels is being actively investigated. Previous studies have examined the cryopreservation response of cells encapsulated in non-adhesive hydrogels using conventional freezing and/or vitrification (ice-free cryopreservation); however, none have systematically compared the two cryopreservation methods with cells encapsulated within an adhesive 3-D environment. The latter would be significant, as evidence suggests adhesion influences the cellular response to cryopreservation. Thus, the objective of this study was to determine the response to conventional freezing and vitrification of insulin-secreting cells encapsulated in an adhesive biomimetic hydrogel. Recombinant insulin-secreting C2C12 myoblasts were encapsulated in oxidized RGD-alginate and cultured for 1 or 4days post-encapsulation, cryopreserved, and assessed up to 3days post-warming for metabolic activity and insulin secretion, and 1day post-warming for cell morphology. Besides certain transient differences in the vitrified group relative to the fresh control, both conventional freezing and vitrification maintained the metabolism, secretory activity, and morphology of the recombinant C2C12 cells. Thus, due to a simpler procedure and slightly superior results, conventional freezing is recommended over vitrification for the cryopreservation of C2C12 cells encapsulated in oxidized, RGD-modified alginate.

Cryopreservation effects on intermediary metabolism in a pancreatic substitute: a (13)C nuclear magnetic resonance study.

Cryopreservation is important for clinical translation of tissue-engineered constructs. With respect to a pancreatic substitute, encapsulated islets or beta cells have been widely studied for the treatment of insulin-dependent diabetes mellitus. Besides cell viability loss, cryopreservation may affect the function of the remaining viable cells in a pancreatic substitute by altering fundamental processes in glucose-stimulated insulin secretion, such as pathways associated with intermediary metabolism, potentially leading to insulin-secretion defects. In this study, we used (13)C nuclear magnetic resonance (NMR) spectroscopy and isotopomer analysis to determine the effects of conventional freezing and ice-free cryopreservation (vitrification) on carbon flow through tricarboxylic acid (TCA) cycle-associated pathways in encapsulated murine insulinoma ßTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate-encapsulated ßTC-tet cells were frozen or vitrified with a cryoprotectant cocktail. Beads were warmed and (13)C labeling and extraction were performed. Insulin secretion rates were determined during basal and labeling periods and during small-scale glucose stimulation and K(+)-induced depolarization. Relative metabolic fluxes were determined from (13)C NMR spectra using a modified single pyruvate pool model with the tcaCALC modeling program. Treatments were compared with nonpreserved controls. Results showed that relative carbon flow through TCA-cycle-associated pathways was not affected by conventional freezing or vitrification. However, vitrification, but not freezing, led to impaired insulin secretion on a per viable cell basis. The reduced secretion from the Vitrified group occurred irrespective of scale and was present whether secretion was stimulated by glucose or K(+)-induced depolarization, indicating that it might be due to a defect in late-stage secretion events.