Measuring Cryopreservation Effects on Cellular Metabolism in a Pancreatic Substitute Using C NMR and Isotopomer Analysis

Introduction Cryopreservation is essential for bringing tissue engineered constructs from the benchtop to the clinic. Two main methods of cryopreservation that may be used are conventional freezing and vitrification (ice-free cryopreservation). For a pancreatic substitute containing beta cells, metabolism is key to construct function (glucose-stimulated insulin secretion). However, little is known about the effects of cryopreservation on intermediary metabolism. We hypothesize that cryopreservation will significantly affect intermediary metabolism in a pancreatic substitute containing encapsulated murine insulinomas. To investigate this, we have used C nuclear magnetic resonance (NMR) and isotopomer analysis. By exposing encapsulated cells to C-glucose and analyzing steady-state, cell-derived Cglutamate isotopomer patterns with metabolic models in the tcaCALC software [1], this method allows for a quantitative measure of metabolism postcryopreservation. Materials and Methods βTC-tet cells were encapsulated at a density of 7x10 cells/ml in 2% LVM alginate (FMC Biopolymer, Philadelphia, PA). Studies were first performed with fresh beads to determine isotopomeric steady-state. Beads were conventionally frozen using 10% dimethylsulfoxide (DMSO) in fully supplemented DMEM and vitrified using the DPS cryoprotectant cocktail (3 M 1,2 propanediol, 3 M DMSO, and 0.5 M sucrose). For C labeling and extraction, beads were exposed to basal medium without glucose for 1 hour prior to labeling medium containing C-glucose (Cambridge Isotopes, Andover, MA) and subsequent perchloric acid extraction [2]. After C NMR spectroscopy on extracts, glutamate C2, C3, and C4 multiplet peak areas were determined using line-fitting. Isotopomer analyis was performed using metabolic models in tcaCALC. The tcaSIM software was used to simulate spectra based on tcaCALCderived results. Statistics were performed using a one-way ANOVA in the General Linear Model in Minitab. Results Isotopomeric steady-state for encapsulated cells was reached by six hours (data not shown). Analysis with tcaCALC indicated that the modified single pyruvate pool model with a second non-pyruvate carboxylase anaplerotic entrance [2,3] to the TCA cycle fit the experimental data better than the standard model of glucose metabolism (p<0.05) (data not shown). Simulated resonances also indicate that the modified model is a good representation of the experimental data (Figure 1). Using this model, initial results from tcaCALC (Table 1) show that cells in both vitrified and frozen beads may have lower pyruvate carboxylase activity and flux through glycolysis compared to those in fresh beads. Also, cells in frozen beads may have a lower flux through a second non-pyruvate carboxylase anaplerotic entrance. However, further experiments with the frozen group need to be performed to determine statistical significance. Current studies are focusing on analyzing insulin secretory response in beads post-cryopreservation and correlating it with metabolic fluxes.