| In the first part of this thesis, we studied the effects of cryopreservation on the intermediary metabolism of encapsulated betaTC-tet cells by 13C nuclear magnetic resonance (NMR) and isotopomer analysis, as well as the effects of cryopreservation on insulin secretory function. In order to use the 13C NMR and isotopomer analysis method, we first developed the appropriate parameters and procedures for 13C labeling, extraction, and isotopomer analysis from Fresh and Cryopreserved encapsulated betaTC-tet cells. After the appropriate parameters and procedures were determined, relative metabolic fluxes in the tricarboxylic acid (TCA) cycle were measured from Fresh, Frozen and DPS-vitrified encapsulated cells. It was found that intermediary metabolism was maintained, as measured by 13C NMR and isotopomer analysis, in cryopreserved encapsulated betaTC-tet cells. Although insulin secretory function was maintained in the Conventionally Frozen group, it was impaired in the DPS-vitrified group. Small-scale glucose-stimulated and K+-induced depolarization experiments also indicated impaired secretion from the DPS vitrified group, but not from the Frozen encapsulated cells. Similar stimulation indices between glucose-stimulated and depolarization-induced secretion tests post-warming, similar intracellular insulin content to Fresh controls, and impaired depolarizationinduced secretion compared to Fresh controls, indicate a possible defect in late-stage insulin secretion for the DPS-vitrified group.;In the second part of this thesis, we investigated the effect of cell-matrix interactions on cellular response to cryopreservation by assessing metabolic activity and insulin secretion in Cryopreserved Stable C2C12 cells encapsulated in RGD and RGEalginate hydrogels up to one day post-warming. In addition, to address the longer-term cellular response to cryopreservation of cells encapsulated in an adhesive hydrogel environment, Stable C2C12 cells were encapsulated in RGD-alginate hydrogels, cultured for 1 or 4 days, cryopreserved, and assayed up to 3 days post-warming for insulin secretion and metabolic activity. As cell spreading has been associated with myoblast differentiation and fusion into myotubes in RGD-alginate, cell circularity in the beads was also measured one day post-warming. Results indicate that the presence of cellmatrix interactions did not affect cellular response in terms of metabolic activity or insulin secretion up to one day post-warming. Additionally, irrespective of culture time pre-preservation, metabolic activity, insulin secretion rate, and cell morphology were maintained in Frozen, RGD-alginate encapsulated Stable C2C12 cells. Similarly, although there were differences immediately post-warming, overall, metabolic activity, insulin secretion, and cell morphology were maintained in DPS-vitrified, RGD-alginate encapsulated cells.;In conclusion, the work in this thesis presents a systematic comparison of the two cryopreservation methods, conventional freezing and vitrification, on the in vitro cellular aspects of insulin-secreting cells encapsulated in an adhesive vs. non-adhesive alginate environment. Results on cryopreservation effects on intermediary metabolism in encapsulated betaTC-tet cells offer insight into the effects of cryopreservation on cellular bioenergetics, indicating that relative carbon flow through the TCA cycle pathways examined is unaffected by cryopreservation. Additionally, DPS-vitrification led to impaired insulin secretion from encapsulated betaTC-tet cells, possibly due to a defect in late-stage insulin secretion. The results from Stable C2C12 cells encapsulated in RGD vs. RGE-alginate indicate that up to one day post-warming, cell-matrix interactions do not affect cellular response for these cells after vitrification or freezing. Although there are transient differences from the Fresh control in terms of metabolic activity and insulin for DPS-vitrified RGD-encapsulated Stable C2C12 cells, metabolic activity and insulin secretion are maintained at all time points assayed for Frozen constructs. Overall, due to results comparable to Fresh controls and simplicity of procedure, conventional freezing is appropriate for cryopreservation of betaTC-tet cells encapsulated in unmodified alginate or Stable C2C12 cells encapsulated in partially oxidized, RGD-modified alginate. (Abstract shortened by UMI.). |
