Water and Cryoprotectant Permeability Characteristics of Isolated Human and Canine Pancreatic Islets

Abstract

Cryopreservation allows accumulation of the necessary islet transplantable mass as well as adequate time for tissue typing and infectious disease screening. Cryopreservation protocols may be optimized by modeling the osmotically induced volume excursions that occur during the addition and removal of cryoprotective agents (CPAs). To that end, three transport parameters were measured at 22°C in canine and human islets isolated by collagenase digestion and euroficoll purification: (i) the apparent hydraulic conductivity (L p ), (ii) the permeability coefficient of the CPA (P s ), and (iii) the associated reflection coefficient (σ). The parameters were determined by volumetric analysis of islets upon abrupt exposure to 1, 2, and 3 M dimethyl sulfoxide (DMSO), ethylene glycol (EG), glycerol (GLY), and propylene glycol (PG). The parameters were calculated using the Kedem-Katchalsky theory to describe islet volume excursion kinetics (assuming islets to be single equivalent osmotic units with the same volume and surface area of the actual islet) and a three-parameter curve fit was performed using the Marquardt-Levenberg method. It was determined that the permeability characteristics of pancreatic islets are species specific, and based upon the measured parameters, the highest P s values for canine islets were observed following exposure to 2 M EG, and the highest P s values for human islets were observed following exposure to 2 M PG. The permeability parameters were analyzed adjusting for islet radius using ANCOVA procedures to acquire least square means. For canine islets exposed to 2 M EG these values were determined to be 0.936 μm/min/atm, 2.47 μm/s, and 0.90 (for L p , P s , and ϕ, respectively) and for human islets exposed to 2 M PG the values were determined to be 1.56 μm/min/atm, 3.48 μm/s, and 0.85 (for L p , P s , and σ, respectively). These parameters were used in a model to calculate osmotically induced islet volumetric response upon addition/dilution of the optimum CPAs, taking into consideration critical volume excursion limits at which irreversible damage occurs.