preservation method in clinical islet transplantation.42 The two-layer (perfluorochemical and UW solution) pancreas preservation method holds great promise for islet transplants.43 The perfluorochemical is water insoluble. Its high density ensures that the pancreas floats at the interface of the perfluorochemical and preservation solution. The perfluorochemical, after being saturated with oxygen, provides ample oxygen to the pancreas during preservation, thereby allowing the oxygenated pancreas to produce adenosine triphosphate necessary for maintaining tissue integrity. It has very recently been applied to preservation of human pancreata before pancreas and islet transplantation.44-46

Preparation of Islets. On arrival at the islet isolation facility, the transport container is opened and inspected for package integrity. The pancreas is then removed and briefly exposed to an antibiotic and antifungal solution. The pancreas is placed in a cooling pan and the extraneous fat and nonpancreatic tissue carefully dissected and discarded. In preparation for the distension (enzyme loading) procedure, the pancreas is divided at the neck. Two cannulas are inserted into the main pancreatic ducts at the divided surface, one directed to the head and the other to the body and tail, and secured in place (Fig. 8.1). The pancreas is weighed and then perfused under controlled conditions using the perfusion protocol developed by Lakey et al.47 The cooled perfusion solution consists of a purified enzyme blend containing collagenase48 and serine-protease inhibitor.49 The collagenase solution is loaded retrograde into the ducts to distend the pancreas under control of a roller pump with pressure monitoring.47 The distended panceas may be cut into a few pieces and then placed into the Ricordi digestion chamber. Pancreatic dissociation is accomplished when the collagenase solution is circulated through the Ricordi chamber at a temperature of 37° to 38° C (Fig. 8.2). The chamber is agitated manually or using an automated system.16 Samples are taken from the circuit at regular intervals to monitor the breakdown of the pancreas by visual inspection of tissue via the inverted microscope. When the amount of tissue liberated from the chamber increases and intact islets are observed, and it is determined that most or all of the islets are free of the surrounding acinar tissue, the recirculation reservoir and the heating circuit are bypassed. The islet isolation continues with the temperature progressively decreased to 15° to 20° C and the collagenase diluted with tissue culture medium. The digest containing the free islets is collected in containers pre-filled with tissue culture medium supplemented with 10% human serum albumin. The pancreatic digest is washed and accumulated for the purification step.

The pancreatic digest containing endocrine and exocrine tissue is purified by placing it on a continuous gradient of sodium diatrizoate ficoll50 or with iodixanol45 using a Cobe 2991 cell separator51 usually under cooling conditions (5-10° C). Fractions with adequate islet purity are combined for immediate transplantation or for pre-transplant tissue culture.52

If islet culture is performed pre-transplant, the purified islets are placed in tissue culture flasks containing tissue culture medium. The flasks are placed in an incubator in an atmosphere of 95% air and 5% CO2. The islets are cultured overnight at 37° C and for an additional 24 to 48 hours at 22° C.

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