524

Successful clinical islet isolations from donor pancreas under fifty years of age

Gregory Szot

University of California Transplantation Surgery, San Francisco, CA, United States

Pancreatic islet transplantation offers a specific, minimally-invasive approach to restore normoglycemia and insulin independence in patients with type 1 diabetes, but its clinical applicability is limited by the complexity of the islet isolation process and the lack of consensus regarding donor characteristics that can predict successful islet isolation outcomes. One characteristic previously thought to predict worse isolation outcomes is younger age of the donor (<50), but with advances in the enzymatic blends used to digest the tissue, this may no longer be true. Here we describe our experience with islet isolation from deceased donors less than 50 yrs. old.

Pancreata (n=10) were processed using a modified Ricordi method. Briefly, pancreata were perfused with a GMP grade Collagenase NB1 and Neutral Protease NB mixture (Serva) via the pancreatic duct followed by continuous chamber digestion. Islets were washed and then purified using a continuous density gradient. Eight of the ten processed pancreas were suitable for transplant with an average islet yield of 648,124 (±190,901) Islet Equivalents (IEQ). The Serva collagenase NB1 and neutral protease NB functioned within a tight range of donor ages: 33-48 years with a mean of 38 (±5) years and average BMI of 38 (±6). An average trimmed pancreas weight of 113 (±12) grams yielded 5,764 (±1,745) purified IEQ per gram of trimmed pancreas with a 90% post culture recovery. Five different lots of Serva enzyme were tested. Several factors or modifications were identified as contributing to these successful isolations. These included: 1) a brief average cold ischemia time of 8:10 (±2.0) hours; 2) Moderate average concentrations of Collagenase (1,976U) and Neutral Protease NB (238U) in the perfusate; and 3) addition of CaCl2 (11mM) per average pancreas weight of 113 (±12) grams. In addition, the digestion time was relatively brief, averaging 13 (±1) minutes at 37°C, and the digestate was diluted into cold RPMI containing 5.0% HSA, insulin, and heparin while the chamber temperature was maintained at 30°C. The resultant digest was then centrifuged and pooled into flasks containing 0.625% HSA and 2% Pentastarch solution at 4°C. Islets were then purified on a continuous density iodixanol (Optiprep) gradient after being washed in a solution containing 0.2% Pentastarch.

Using this approach, we have been able to demonstrate islet function (detectable c-peptide) in all 8 transplants 1 month after transplant, and have achieved insulin independence for at least 3 months in all but 2 patients. Current durations of independence in our patients are >12mos x 2, >10mos x 1, >3mos x 1; with 2 patients (<3 months psot-transplant) requiring half the amount of, pre-transplant, insulin and are listed for a second transplant. In summary, we provide evidence that with relatively minor modifications in the isolation protocol, high quality, clinically usable islets can be isolated from younger donors.