Comparative Study Of Islets Harvested From Non-heart-beating And Brain Death Donor For Islet Transplantation

Objective: With well developed transplantation technology,donor shortage has become the main obstacle to the development of organtransplantation. In the USA, about18people die everyday while waiting fororgan transplantation. Its ratio of organ supply to demand is approximate1:3compared with1:30in China. For completely insulin independence, onerecipient would transplant islet preparations from multiple donors which, tosome extent, enlarges the gap between pancreas supply and demand. Althoughliving-donor islet transplantation has been reported, the donor and beneficiarymay suffer from serious physiological and psychological burden respectivelywhich make it far from general acceptance. It is reported that because of warmischemia injury pancreases from non-heart-beating donors (NHBDs) are notsuitable for pancreatic transplantation, but can be an invaluable resource of islettransplantation. Our research studies the practicability of islet harvested fromwhich for transplantation, while brain death donors (BDDs) pancreas serves asa measuring stick. Methods: After organ perfusion with UW solution, liver,pancreas and other organs are procured from nine BDDs and five NHBDs withstandard multiple organ procurement technique. Then, organs are preserved inUW solution statically and transfer to operating theatre. After removal of spleenand duodenum, pancreas is sent to laboratory of GMP grade, where itundergoes pruning to separate from excessive fat tissue and fascia andperfusion with Liberase MTF C/T in the help of a syringe. When perfusion issatisfying, cut the pancreas into small tissue blocks of3×3×1cubic centimetersand put into a digestion chamber for digestion. Temperature is controlledbetween36-38℃. Sample every2minutes and observe under phase-contrastmicroscope after DTZ staining. Terminate digestion using stop buffercontaining10%human albumin when more than50%islets (the ratio of released islets to total islet number) have released. Centrifuge and discard thesupernatant several times for getting rid of residual enzyme. Islets were thenpurified with the use of continuous gradients of Biocoll in a COBE2991CellProcessor. Differences of islet equivalent (IEQ), IEQ/g pancreas weight andpurity between BDDs and NHBDs are studied. Calculate islet survival rateunder phase-contrast microscope after FDA/PI staining. Function of isolatedislet in vitro is evaluated by glucose stimulation test. In order to assess whetherdifferent batches of Liberase MTF C/T have their influence on the outcomesand reproducibility of digestion results, digestion time and islet yield of thethree batches are compared. Results: There are no marked difference in donorage, body weight, weight of pancreas and cold ischemia timestatistically(P>0.05). Islet equivalent (IEQ) and IEQ/g pancreas weight ofNHBDs pancreas pre-and post purification are814964.89±34949.52,448144.22±63230.98,10928.80±1021.73/g and6047.3707±1128.35/grespectively. Those of BDDs are817326.80±21791.49,498056.00±27319.09,11278.69±880.78/g and6866.9481±542.07/g respectively. No statisticaldifferences are observed (P>0.05). Islet purity of NHBDs and BDDs pancreaspre-and post purification are5.0±0.7%VS.5.2±0.8%, P>0.05;64.4±5.9%VS.66.4±4.7%, P>0.05.91±2%islet cells survive post-purification inNHBDs group comparing with92±3%in BDDs group, P>0.05. There are onlyfive pancreas available in the later group, which can not match the standard thateach different batch should contains at least2pancreas, so statistical analysis isdeprived. In NHBDs group, N (batch1)=4, N (batch2)=2, N (batch3)=3,there is no diffreence in islet yield. Conclusion: Donor organ shortage is avexed issue facing the whole world. This study demonstrates that islet yield,purity and survival rate of NHBDs are equal in that of BDDs when warmischemia time is restricted to less than19.1±1.8min. Islets harvested fromNHBDs are suitable for clinical islet transplantation. Since no statisticaldifferences are observed in digestion time and islet yield pre-and post- purification, variation in different batches of Liberase MTF C/T is small.