Nonenzymatic 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

摘要

Cell-based therapies, which all involve processes for procurement and reimplantation 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 1 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 (P.) to release islets that are selectively cryopreserved in situ. P.ncreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CP.) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire P. solid to 90%) embedded islets. Islets were typically larger (range 50-500 μm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index?=?3.3?±?0.7. 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.