Cryopreservation Effects on Intermediary Metabolism in a Pancreatic Substitute: A 13 C Nuclear Magnetic Resonance Study

Abstract

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 ¹³C 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 βTC-tet cells; the secretory function of the encapsulated cells postpreservation was also evaluated. Specifically, calcium alginate–encapsulated βTC-tet cells were frozen or vitrified with a cryoprotectant cocktail. Beads were warmed and ¹³C 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 ¹³C 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.