Improved Islet Yields From Macaca Nemestrina and Marginal Human Pancreata After Two‐Layer Method Preservation and Endogenous Trypsin Inhibition

Abstract

We tested whether two-layer method (TLM) pancreas preservation and trypsin inhibition (Pefabloc) during processing allows longer preservation while retaining or improving viable islet recovery. Non-marginal primate (Macaca nemestrina) and marginal human (ischemic or preservation-injured) pancreata were processed with a research-oriented pan technique (Seattle method). Organs were processed upon arrival (∫ Pefabloc), or after TLM or University of Wisconsin solution (UW) preservation (π Pefabloc). Islet yield, viability, and function were assessed. Pefabloc increased M. nemestrina islet yields from 9696 ∫ 1749 IE/g to 15822∫ 1332 IE/g (p ∞ 0.01). Twolayer method preservation (∞ 6 h) further increased yields, to 23 769 ∫ 2773 IE/g (vs. π Pefabloc; p ∞0.01). Similarly, Pefabloc increased marginal human islet yields from 2473 ∫ 472 IE/g to 4723∫ 1006 IE/g (p ∞ 0.04). This increase was maintained after lengthy TLM preservation (Ͼ 30 h; 4801 ∫ 1066 IE/g). We also tested the applicability of TLM preservation (23.5 ∫ 3.2 h) to the processing of marginal human pancreata by the Edmonton/Immune Tolerance Network clinical protocol. Islet yield and function approached published results of pancreata processed 4.8 ∫ 0.8 h after organ recovery (p Ω 0.06). Pefabloc, and TLM vs. UW preservation, prolonged the tolerable interval between organ recovery and islet isolation. Islet yield, viability, and functionality improved from both marginal and nonmarginal pancreata.

Flexible Management of Enzymatic Digestion Improves Human Islet Isolation Outcome from Sub‐Optimal Donor Pancreata

“…However, despite continuous improvements, it is not yet possible to retrieve the entire pool of islets that are contained in a pancreas (6)(7)(8).…”

Serine protease inhibitors suppress pancreatic endogenous proteases and modulate bacterial neutral proteases

“…[19][20][21][22][23][24] These studies have shown an improvement of islet yield due to impairment of serine proteases by these inhibitors. Here we report the potency effect of these inhibitors on endogenous proteases, bacterial neutral proteases and islet function in vitro.…”

“…The addition of Pefabloc, Trasylol and UTI during the islet isolation process has been reported to improve islet yield, presumably by inhibiting pancreatic endogenous protease activity. [16][17][18][19][20][21][22][23][24][25][26][27][28] To confirm this, we tested the effect of these inhibitors on the activity of trypsin, chymotrypsin, elastase and switch samples. Pancreatic digestion switch samples usually consist of multiple enzymes (trypsin, chymotrypsin, elastase, thermolysin/neutral protease and collagenase).…”

“…15 It has been shown that addition of protease inhibitors, such as Pefabloc, Trasylol or Ulinastatin (UTI), during islet isolation, resulted in improved yield. [16][17][18][19][20][21][22][23] However, the effects of these protease inhibitors on endogenous proteases and enzymes essential for tissue dissociation, in particular neutral protease, have yet to be determined. This study investigated the effect of three protease inhibitors, Pefabloc, Trasylol and UTI, on pancreatic endogenous proteases, thermolysin/neutral protease and islet function.…”

Activation of c-Jun NH2-terminal Kinase during Islet Isolation

“…University of Wisconsin (UW) solution is usually used for pancreas preservation. Recent reports have shown that the two-layer method (TLM), which employs oxygenated perfluorochemical (PFC) and UW solution, is superior to simple cold storage in UW solution [55][56][57]. Matsuda et al showed caspase, JNK, and p38 activity in isolated islets after preservation of UW or TLM [58].…”