Cryoprotectant (CPA) cytotoxicity constitutes a challenge in developing cryopreservation protocols, specifically in vitrification where high CPA concentrations are necessary to achieve the ice-free, vitreous state. Few cytotoxicity studies have investigated vitrification-relevant concentrations of CPAs, and the benefits and disadvantages of cocktail solutions and of incorporating non-permeating solutes have not been fully evaluated. In this study, we address these issues by determining the cytotoxicity kinetics for dimethylsulfoxide (Me2SO) and 1,2-propanediol (PD) on alginate-encapsulated βTC-tet mouse insulinomas for a range of concentrations and temperatures. Cytotoxicity kinetics were also determined for two cocktails, DPS (3M Me2SO + 3M PD + 0.5M sucrose) and PEG400 (1M Me2SO + 5M PD + 0.34 M poly(ethylene)glycol with M.W. of 400). PD was found to be more cytotoxic than Me2SO at higher concentrations and temperatures. This was reflected in PEG400 being more cytotoxic at room temperature than PEG400 at 4°C or DPS at either temperature. Addition of non-permeating solutes increased the cytotoxicity of cocktails. Furthermore, results indicate that CPA cytotoxicity may not be additive and that combining CPAs may increase cytotoxicity synergistically. Finally, when comparing cytotoxic effects towards encapsulated HepG2 and βTC-tet cells, and towards βTC-tet cells in capsules and in monolayers, CPAs appear more cytotoxic towards cells with higher metabolic activity. The incorporation of these results in the rational design of CPA addition/removal processes in vitrification is discussed.

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, bio-functionalized hydrogels is receiving increasing attention, as cell-matrix interactions in 3-D 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 actively being 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 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 1 or 4 days post-encapsulation, cryopreserved, and assessed up to 3 days post-warming for metabolic activity and insulin secretion, and one day post-warming for cell morphology. Besides certain transient differences of the vitrified group relative to the Fresh control, both conventional freezing and vitrification maintained metabolism, secretion 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 in oxidized RGD-modified alginate.

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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 insulindependent 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 bTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate-encapsulated bTC-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.