Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Abstract

Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

Progress in individualizing autologous islet isolation techniques for pediatric islet autotransplantation after total pancreatectomy in children for chronic pancreatitis

“…Whether our approaches apply to pediatric deceased donor islet isolations is uncertain. An increased protease dose also reduces the percentage of embedded islet tissue from young deceased donors, 16 as for our approach, but some modifications introduced for young donors in TPIAT are specific to pancreatic fibrosis and cannot be summarily applied to deceased donor pancreata.…”

“…These can be more or less aggressive depending on the digested tissue's profile at the time of addition (ie, fragile/fragmented islets or fine acinar may require a more conservative approach). This technique of enzyme bolusing has been used infrequently to avoid overdigesting the tissue, but recent reports suggest that far more enzyme than previously deemed appropriate can be used safely in human islet isolation 12,16,17 . The process of adding supplemental enzyme was further refined in 2012 by adding an enzyme recirculation option (based on ref.…”

“Old School” Islet Purification Based on the Unit Gravity Sedimentation as a Rescue Technique for Intraportal Islet Transplantation—A Case Report

“…Additionally, a modified digestion system was proposed to enhance enzymatic action over mechanical digestion by slow shaking of the Ricordi chamber 3 . Next, investigators tried optimizing the concentration of collagenase 16 or more recently blending different enzymes (e.g., increasing the dose of protease relative to that of collagenase 17 ). As the intraportal infusion of digested, but unpurified pancreatic tissue led to frequent portal vein thrombosis, the need for the elimination of excessive acinar tissue from the islet preparation became obvious.…”

Evolution of Enzyme Requirements for Human Islet Isolation

“…The range of optimal activities is likely to be dependent on the dose of a specific protease. The cost to perform these optimization/validation experiments can be reduced by adopting a human islet isolation method that recovers islets from the head, body, or tail of the same pancreas [70]. This trisected pancreas islet isolation method has recently been used to determine an optimal dose of collagenase and protease for use in isolating human islets from young donors.…”

“…Today's knowledge of the composition and dose of collagenase-protease enzyme mixtures for human islet isolation is based primarily on donor pancreata recovered from older individuals. Islet recovery from younger donors (< 35 years old) often results in suboptimal islet yields because of the high proportion of embedded and mantled islets [70]. Mantled islets, defined as those islets surrounded by acinar cells, are difficult to purify by density gradient centrifugation because of their different density when compared to non-mantled islets.…”