Non-enzymatic cryogenic isolation of therapeutic cells: Novel approach for enzyme-free isolation of pancreatic islets using In Situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.
Author: M. J. Taylor, S. Baicu
Source: Cell Transplantation
Publisher: Cognizant Communication Corporation
Abstract:Cell-based therapies, which all involve processes for procurement and re-implantation of living cells, currently rely upon expensive, inconsistent and even toxic enzyme-digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of Type I diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods we have conceptualized an alternative approach based on the hypothesis 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 situpreferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2. Retrograde ductal infusion of water to distend the acinar; 3. Freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4. Mechanically crushing and pulverizing the frozen Px into small fragments; 5. Thawing the frozen fragments, filtering and washing to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and with Syto 13/PI for fluorescence viability testing and 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 and viable (>90%) embedded islets. Islets were typically larger (range 50-500μm diameter)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. Conclusion: An enzymefree 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.
Appeared or available online: August 29, 2013