Human and canine pancreatic islet cryopreservation: A theoretical approach
Abstract
The transplantation of pancreatic islets of Langerhans in patients with insulin dependent diabetes mellitus (IDDM) may provide a cure for this disease. To realize these goals, long term graft storage is a necessary procedure to facilitate tissue matching, infectious disease screening, organ sharing, immunomanipulation of islets and multipledonor transplantation. Current islet cryopreservation results in post-thaw recoveries of 60 to 90% of the original volume, however decreased function of the tissue is observed and the procedure is not widely used in clinical islet transplantation centers. A major portion of this thesis was directed at evaluating key biophysical characteristics of human and canine islets, with the end goal of using them to make predictions regarding cryopreservation outcomes. The osmotic properties of human and canine islets (which dictate volumetric responses during cryopreservation) were characterized, and osmotically inactive fractions (Vbp) were determined to be different for each species. Permeability coefficients (Lp, Ps and σ) of human and canine islets were measured for four different cryoprotectants (dimethyl sulfoxide, ethylene glycol, propylene glycol and glycerol), and it was determined that different cryoprotective agents (CPA's) were optimal for each species: ethylene glycol (EG) for canine and propylene glycol (PG) for human islets. To address a current issue in studies of this nature, two different methods of determining these permeability characteristics quantitatively were compared (2 vs.3 parameter Kedem-Katchalsky curve fitting). The two parameter estimate were in closer agreement with values published for other cell types and were used for subsequent modeling. The physical-chemical properties of the ternary solution water/NaCl/ethylene glycol were determined, and these data were used in conjunction with low temperature permeability data to predict an optimum canine islet cryopreservation protocol, which is presented in a flow diagram and includes CPA addition, equilibrium cooling rate and plunging temperature, as well as thawing and CPA dilution procedures. Finally, the effects of microencapsulation on human and canine islet volumetric responses were determined to facilitate understanding of the islet-microcapsule system during cryopreservation.
Degree
Ph.D.
Advisors
Scott-Moncrieff, Purdue University.
Subject Area
Biophysics|Cellular biology|Surgery
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