097 Non-enzymatic cryo-isolation of viable Islets from juvenile porcine pancreas after storage for >6 months at −135 °C

Abstract

Cryo-isolation of therapeutic cells such as pancreatic islets has been proposed as a novel new alternative to conventional enzymatic digestion of the pancreas (Taylor, Baicu., Transpl Proc. 43 (2011) 3181–3183. And Cell Transpl. 2013 (In Press)). Apart from avoiding the expense, inconsistencies and toxicity associated with the collagenase-digestion process, cryo-isolation embodies several inherent advantages including the convenience of biobanking for storage and shipping. This study investigates the feasibility of obtaining viable islets from juvenile porcine pancreas after storage at −135 °C for >6 months using the cryo-isolation technique. Pancreases were procured from juvenile pigs using approved procedures and processed using the cryo-isolation protocol. This involved: (1) Infiltrating islets in situ preferentially with 2M dimethyl sulfoxide as the cryoprotectant (CPA) via antegrade perfusion of the major arteries; (2) retrograde ductal infusion of water, and then (3) the entire pancreas was frozen solid to −160 °C and stored in the vapor phase of liquid nitrogen. After storage at −135 °C for 1 day to 8 months, pancreases were further processed as follows: The frozen pancreas was mechanically crushed and pulverized into small fragments which were then thawed, filtered and washed with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and with Syto13/Propidium iodide (PI) for fluorescence viability testing and assessed for function using a conventional glucose-stimulated insulin secretion (GSIS) assay. The cryo-isolate from pancreata stored for >6 months contained pancreatic fragments comprising discrete dithizone-positive islets embedded in a shell of dead amorphous material. The cryo-isolate product was similar to that obtained from frozen pancreases stored for 1 day in that there was an abundance of largely intact and viable (>90%) islets. Moreover, the cryo-isolated islets were typically larger (range 50–500 μm) than their counterparts isolated from juvenile pigs using conventional collagenase digestion techniques and demonstrated GSIS indices (>3), which was not significantly different from controls (fresh islets from collagenase digestion). An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a new and novel method that avoids the problems associated with conventional collagenase digestion methods and permits long-term biobanking of pancreases as a ready source of islets. Source of funding: This work was funded in part by a grant from NIH/NIDDK#1R43DK096773. Conflict of interest: The authors are, or have been, employed by Cell and Tissue Systems. mtaylor@celltissuesystems.com