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 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 (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.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology/Emory University, Atlanta, Georgia 30332, USA.
Hajira F
HF
Ahmad
Nicholas E
NE
Simpson
Alison N
AN
Lawson
Athanassios
A
Sambanis
D000818:Animals; D002247:Carbon Isotopes; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology; D005971:Glutamates / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008564:Membrane Potentials / Q000187:drug effects; D051379:Mice; D010179:Pancreas / Q000187:drug effects / Q000378:metabolism*; D019397:Pancreas, Artificial; D011188:Potassium / Q000494:pharmacology; D058989:Vitrification
D002247:Carbon Isotopes; D003451:Cryoprotective Agents; D005971:Glutamates; D007328:Insulin; D005947:Glucose; D011188:Potassium