In modern day medicine, cellular therapies, regenerative medicine and tissue engineering all involve technologies for harvesting, expanding, modifying and re-implanting live viable cells and tissues. Processes for preparing the therapeutic products that incorporate living cells are critical for the stability and potency of th products but may be inherently injurious to the component cells. For example, the widely practiced technique of collagenase digestion of tissues to obtain isolated cells such as pancreatic islets from pancreata, or hepatocytes from liver is fraught with detrimental side-effects and other associated problems. This widely practiced procedure has recognized pitfalls due principally to the difficulty of controlling the digestive process to yield an optimum quantityof viable cells. Moreover, the process is harsh and even toxic, causing some inevitable loss of valuable cells. Furthermore, the process relying upon the purest forms of the enzymes are very expensive and may be subject to batch variations that have led to frustrating variability and inconsistency in attempts to optimize and standardize these processes. A totally new approach is proposed here that minimizes and potentially eliminates the need for enzymatic digestion of the tissue. Instead, the proposed process relies upon known susceptibilities of cells to freezing injury, to affect the separation of different cell types by virtue of a facilitated differential frezing and cryopreservation techniques. Feasibility for this novel approach has been demonstrated for isolating porcine pancreatic islets, which is a widely accepted model for research into the treatment of type I diabetes by islet transplantation. To obtain islets for cell-based therapies, te field of islet transplantation relies totally upon enzymatic digestion processes that destroy the extracellular matrix of the donor tissue releasing the entrapped islets for further processing and purification. In contrast, we propose to pre-treat the pancreas by differential perfusion of the endocrine and exocrine tissue in a way designed to maximize the destruction of the exocrine tissue at the same time as preserving the islets. More specifically, this new cryo-isolation approach involves an initial perfusion of the endocrine tissue (islets) with cryoprotective agents via a vascular access and after adequate equilibration of the islets only, the exocrine component (acini) is infused with a purely aqueous solution (distilled water or saline) via the ductal system The entire pancreas is then cooled under conditions that promote ice formation and destruction of the acinar tissue while preserving the endocrine portion by virtue of the cryoprotectant infiltration. The solid frozen pancreas is then amenable to indefinite storage and biobanking and subsequent processing to pulverize and fracture the gland into tiny fragments containing the cryopreserved islets. Finally, the freeze-disrupted tissue is thawed to release functional islets and destroyed acinar tissue. Having completed the initial proof-of-concept of this innovative new approach, this Phase I study proposes to develop a device prototype for cryo-isolation and evaluate its performance to establish baseline protocols. The approach combines basic research tools with recent advances in cryobiology science to systematically optimize the baseline technique, while developing a method to promote tissue fracturing by means of thermo-mechanical stresses, thereby increasing the effectiveness of differential freeze disruption and viable islet isolation. The study brings together a unique combination of expertise in cryobiology and thermo-mechanical engineering necessary to take this novel concept from feasibility to routine practice and subsequently validation in human tissue in a Phase II study.
Cell-based therapies in regenerative medicine and tissue engineering, 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 potential treatment of Type I diabetes by transplantation. This research is focused on the development of a new and novel alternative technique to enzymatic digestion by relying instead on differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ.
| Taylor, Michael J; Baicu, Simona C (2014) 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. Cell Transplant 23:1365-79 |
