Cryo-isolation: A novel new method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue

M.J. Taylor, S.C. Baicu
Cell and Tissue Systems, N. Charleston, USA

Objective : A critical component of treating Type I diabetes by transplantation is the availability of sufficient high quality islets. Currently, islets can only be obtained by reliance upon expensive, inconsistent and toxic enzyme-digestion processes. As an alternative we hypothesize that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ.

Methods : Pancreases were procured from juvenile pigs using approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1. Infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries. 2. Retrograde ductal infusion of water (or saline) to fully distend the gland. 3. The entire Px was frozen solid to -160°C and stored in liquid nitrogen. 4. The frozen pancreas was mechanically crushed and pulverized into small fragments. 5. The frozen fragments were 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 samples were taken for static glucose-stimulated insulin release assessment.

Results: As predicted the cryo-isolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets (figure). The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells was variable. Islets were typically larger than their counterparts isolated from juvenile pigs using conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3±0.7 (n=3).

Conclusion: 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.