Copyright C Blackwell Munksgaard 2003American Journal of Transplantation 2003; 3: 53–63

Blackwell Munksgaard ISSN 1600-6135

Improved Islet Yields From Macaca Nemestrina andMarginal Human Pancreata After Two-Layer MethodPreservation and Endogenous Trypsin Inhibition

Shinichi Matsumotoa,b, Theodore H. Rigleya,Jo Anna Reemsa,c, Yoshikazu Kurodad andR. Brian Stevensa,b,e,*

a Puget Sound Blood Center/North-west Tissue Center,

Seattle, WAb Department of Surgery, Division of Transplantation,

University of Washington Medical Center, Seattle, WAc Department of Hematology, Division of Medicine,

University of Washington, Seattle, WAd Department of Gastroenterological Surgery, Graduate

School of Medical Sciences, Kobe University, Kobe, Japane University of Washington Diabetes Endocrinology

Research Center, Seattle, WA

*Correspondence: R. Brian Stevens, MD, PhD,

rbstevens@surgery.unmc.edu

We tested whether two-layer method (TLM) pancreaspreservation and trypsin inhibition (Pefabloc) duringprocessing allows longer preservation while retainingor improving viable islet recovery. Non-marginal pri-mate (Macaca nemestrina) and marginal human (is-chemic or preservation-injured) pancreata were pro-cessed with a research-oriented pan technique (Se-attle method). Organs were processed upon arrival (∫Pefabloc), or after TLM or University of Wisconsin solu-tion (UW) preservation (π Pefabloc). Islet yield, viabil-ity, and function were assessed.Pefabloc increased M. nemestrina islet yields from9696∫1749 IE/g to 15822∫1332 IE/g (p∞0.01). Two-layer method preservation (∞6h) further increasedyields, to 23769∫2773 IE/g (vs. π Pefabloc; p ∞0.01).Similarly, Pefabloc increased marginal human isletyields from 2473∫472 IE/g to 4723∫1006 IE/g(p∞0.04). This increase was maintained after lengthyTLM preservation (30h; 4801∫1066 IE/g).We also tested the applicability of TLM preservation(23.5∫3.2h) to the processing of marginal humanpancreata by the Edmonton/Immune Tolerance Net-work clinical protocol. Islet yield and function ap-proached published results of pancreata processed4.8∫0.8h after organ recovery (pΩ0.06).Pefabloc, and TLM vs. UW preservation, prolonged thetolerable interval between organ recovery and islet iso-lation. Islet yield, viability, and functionality improvedfrom both marginal and nonmarginal pancreata.

Key words: Primate islet isolation (Macaca nemestri-

na), human islet isolation, trypsin inhibition, Pefabloc,two-layer method

53

Received 21 May 2001, revised and accepted for publi-cation 31 January 2002

Introduction

A group at the University of Alberta at Edmonton, by emphas-izing donor quality and minimal delay between organ recov-ery and islet isolation, reported that islet allotransplantationcan achieve insulin-independence in persons with type I dia-betes (1,2). However, current isolation techniques usually re-cover fewer than half the islets from a given pancreas, neces-sitating the transplantation of islets from two or more donorsto establish euglycemia (3). In contrast, pancreas transplan-tation, although accompanied by significant morbidity, rou-tinely cures with a single donor (4).

In this study we examined two promising strategies for im-proving results of clinical islet isolation. Before islet isolation,we used two-layer method (TLM) preservation to oxygenatethe pancreas (5), and during isolation we inhibited trypsinactivity with the serine-protease inhibitor Pefabloc [AEBSF;4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride].Separately, both pancreas TLM preservation (6,7) and Pefab-loc have increased islet yields (8–12) in both animals and inhumans.

Two-layer method preservation has been shown to increasethe oxygen and adenosine triphosphate content of dogpancreata, and to maintain cell viability and integrity by sup-porting active metabolism and sodium/potassium pumping(13–15). We have preserved pancreata from the small pri-mate Macaca nemestrina with the TLM, after inflicting a

American Society of Transplantation (Fujisawa) Faculty DevelopmentAward (RBS); JDRFI grant .4–1999847.This article was partially presented at the American Society of Transplan-tation 2000 in Chicago (May 2000), the American Society of Transplan-tation 2001 in Chicago (May 2001), the Annual Meeting of the InternationalPancreas and Islet Transplantation Association (IPITA) at Innsbruck (June2001), and the American Transplant Congress in Washington DC (April2002).The Human Islet Transplantation in Seattle (HITS) Consortium is a jointprogram directed by Dr Paul Robertson of the Pacific North-west ResearchInstitute, and includes the Puget Sound Blood Center/North-west TissueCenter, the Fred Hutchinson Cancer Research Center, Virginia Mason Medi-cal and Research Center, Swedish Hospital Medical Center, and the Univer-sity of Washington Medical Center.

Matsumoto et al.

warm ischemic injury but before islet isolation, and have ob-served better islet yields and function than with preservationin UW solution (16). Similar observations have been made ina canine model (17).

Pefabloc used during pig islet isolation improved the poor isletyields that accompanied pancreas protease activation (8–10).We speculated that endogenous trypsin activity reduces isletyields by eroding the pancreatic ductules, creating leaks thatcompromise the crucial delivery of collagenase digestion solu-tion throughout the pancreas (18). The resulting incomplete di-gestion reduces islet yields by generating acinar-embeddedislets that resist being purified sufficiently for transplantation(18). We hypothesized that inhibiting trypsin could increaseyields by improving collagenase delivery throughout the pan-creas, and by reducing trypsin-mediated destruction of liber-ated islets. Others have corroborated the benefit of trypsin inhi-bition with Pefabloc, but in this and a previous report we haveadditionally quantified trypsin activity and dose-dependent Pe-fabloc trypsin inhibition during islet isolation (11,12).

Islets were isolated using these innovations by a research-oriented pan technique (Seattle method) (19,20) from bothnonmarginal M. nemestrina and marginal human (ischemicor preservation-injured) pancreata. Pancreata from M. neme-

strina were used to model the effect of immediate TLM pres-ervation with minimally injured pancreata, for which equiva-lent human organs were not available. However, the humanmarginal-pancreas experiments are of particular clinical im-portance, as marginal donors and organs with preservationtimes in excess of 12h account for a large percentage of thecadaver pool available for clinical transplantation (21). Unex-pectedly, we discovered that the benefits of TLM preser-vation on marginal human pancreata develop over as long as24h or more of storage (Stevens, in preparation).

We also processed TLM-preserved marginal human pancrea-ta according to the Edmonton/Immune Tolerance Network(ITN) clinical islet isolation protocol (1,2). The clinical trial ofthe FDA-approved Edmonton/ITN protocol at the time ofthese experiments specified that islet isolation be completedwithin 12h of organ recovery, frequently precluding the useof organs procured nonlocally (1,2). We processed TLM-pre-served organs with this protocol to determine whetherpancreata transported from remote recovery sites, and thusnot processed within 12h, could provide islet yields compar-able to those published by the Edmonton group (1).

Our experiments show that TLM preservation, regardless ofisolation method, and trypsin inhibition with Pefabloc improveislet yields and prolong the tolerable interval between organrecovery and islet transplantation.

Materials and Methods

Macaca nemestrina (groups 1–4) and human (groups 5–9) pancreatawere distributed into experimental groups (Tables1–3). Control organs

54 American Journal of Transplantation 2003; 3: 53–63

(groups 1 and 5) were processed to recover islets according to a basicprotocol, as soon as possible after they arrived in our laboratory. The pro-cessing of groups 2 and 6 (π Pefabloc) differed only by the inclusion ofthe trypsin inhibitor Pefabloc in the collagenase solution.

Pancreata were stored with TLM preservation either at organ recovery(group 3) or after an initial interval (13.0∫2.5h) (group 7) in UW solution(ViaSpanA, DuPont Pharma, Wilmington, DE). Groups 4 and 8 were main-tained in UW storage from the time of organ recovery. After storage,groups 3, 4, 7, and 8 were processed with Pefabloc, the same as groups2 and 6. Two-layer method preservation consists of floating the pancreasat the interface formed between a highly oxygenated water-immiscibleliquid perfluorocarbon (perfluorodecalin, C10F18, F2 Chemicals Ltd, Preston,UK) and a layer of UW solution (5–7).

Group 7 (TLM π Pefabloc) and group 8 (UW π Pefabloc) were created bydividing human pancreata into head and tail halves, distributing the headsand tails alternately and equally between the groups in order to averageout the effect of the nonuniform distribution of islets between the pancreashead and tail. This approach provided efficient use of costly researchpancreata and minimized the effect of donor variability. We also processedsix nonsplit TLM-preserved marginal pancreata by using the Edmonton/ITN islet isolation protocol (group 9, Table3) (1,2).

Donor and pancreas digestion characteristics were similar within the M.

nemestrina (groups 1–4) and human (groups 5–8) experiments (Tables1and 2). Donor age and gender were identical for human groups 7 and 8because the pancreata were split. Among all human experiments (groups5–9), there were no differences in donor factors known to influence isletisolation outcome (e.g. age, body mass index, hypotension/vasopressortherapy, organ procurement by local team, etc.). The differences amongthe groups were derived from the experimental design and duration ofpreservation (Tables2 and 3).

In the M. nemestrina experiments warm ischemic time was defined asthe interval between asystole and placement of the pancreas into chilledmedium. Cold ischemic time was defined as the interval between place-ment of M. nemestrina pancreata into chilled medium and infusion of col-lagenase solution (Table1).

In the human marginal-pancreas experiments (groups 5–9), cold ischemictime was defined as the interval between cross-clamping the cadaveraorta and the infusion of collagenase solution into the pancreas. Humanpancreata of groups 5 (control) and 6 (π Pefabloc) were preserved 12h,while the pancreata of groups 7 (TLM π Pefabloc) and 8 (UW π Pefabloc)were stored 32h (Table2). Group 9 (Edmonton/ITN protocol) pancreatawere preserved for 23.5∫3.2h (Table3).

All pancreas’ weights (groups 1–9) were determined after complete ex-cision of extraneous tissues (duodenum, spleen, fat, and associated ves-sels and mesentery) (Tables1–3).

Animal tissue and organ processing, and the use of live animals, wereperformed in compliance with the guidelines of the University of Wash-ington Animal Care and Use Committee. The Institutional Review Board ofthe University of Washington approved our use of human organs.

Non-human primate model

Pancreas procurement and preservation

Adult M. nemestrina pancreata were obtained from the Regional PrimateResearch Center at the University of Washington (Seattle, WA; supportedby NIH grant RR00166) (Table1). Pancreata were obtained at the time ofeuthanasia and immediately placed into ice-cold IMDM tissue culture me-dium (Iscove’s Modified Dulbecco’s Medium, Gibco Life Technologies,

Improved Islet Yields From TLM and Trypsin Inhibition

Gaithersburg, MD). We either transported the organs on ice to our labora-tory and isolated islets, or immediately transferred the pancreata into 250-mL Nalgene jars and preserved them before islet isolation for approximate-ly 5h, under refrigeration at 4 æC in either the TLM or conventional UWsolution (Table1).

Macaca nemestrina islet isolation with the Seattle open-pan research

method (groups 1–4)

Macaca nemestrina islet isolation was performed using the Seattlemethod as previously described (19,20). Pancreata were distended by theintraductal infusion of 35mL of a room-temperature solution of HBSS con-taining 1.5mg/mL of purified collagenase mixture (Liberase HI, Roche Mol-ecular Biochemicals, Indianapolis, IN) (22,23), 10U/mL of heparin (Elkins-

Table1: Donor and digestion characteristics of primate pancreata

Age Pancreas Warm Cold Digestion Embedded(y) weight ischemic ischemic period islets

(g) time time (min) (%)(min) (min)

Group 1(No Pefabloc) (nΩ10) 9.7∫0.8 11.8∫1.0 4.4∫3.2 109∫15 13.7∫2.4 6.2∫1.8

Group 2(π Pefabloc) (nΩ10) 8.9∫0.8 11.2∫1.1 3.2∫1.9 103∫10 12.3∫1.1 1.1∫0.7

Group 3(TLM π Pefabloc) (nΩ5) 9.4∫1.7 7.5∫1.2 4.6∫2.0 275∫162 10.3∫1.4 5.7∫3.1

Group 4(UW π Pefabloc) (nΩ5) 9.8∫0.8 8.5∫1.8 9.0∫3.1 285∫412 13.8∫2.4 8.3∫3.6

p-value1 0.91 0.07 0.42 0.000013 0.57 0.09

UWΩUniversity of Wisconsin solution, TLMΩ two-layer method.1Four groups were statistically compared using ANOVA. Fisher’s protected least significant difference (PLSD) post-hoc test was used to detectdifferences among the groups.2Group 3 and 4 pancreata were placed into TLM or UW preservation after brief immersion in ice-cold Iscove’s Modified Dulbecco’s Medium(IMDM) (10min).3Consistent with experimental design, groups 3 and 4 had significantly longer cold ischemic times than groups 1 and 2. Groups 1 vs. 2 and3 vs. 4 were not significantly different.

Table2: Summary of donor and digestion characteristics of marginal human pancreata processed according to the Seattle research isletisolation method (groups 5–8)

Gender Age Pancreas Cold Digestion Embedded(F/M) (y) weight ischemia period islets

(g) (h) (min) (%)

Group 5(No Pefabloc) (nΩ9) 3/6 38.3∫5.1 64.4∫10.4 7.6∫1.4 19.9∫3.1 44.4∫11.23

Group 6(Pefabloc) (nΩ8) 3/5 46.7∫7.8 62.0∫7.5 10.4∫2.2 21.2∫2.9 25.6∫7.5

Group 7(Pefabloc π TLM) (nΩ8) 3/5 43.5∫5.1 42.3∫4.1 32.6∫3.6 16.2∫2.6 18.1∫4.3

Group 8(Pefabloc π UW) (nΩ8) 3/5 43.5∫5.1 41.8∫5.6 32.3∫3.0 16.0∫2.3 15.6∫3.8

p-value1 0.77 0.07 0.00012 0.54 0.04

UWΩUniversity of Wisconsin solution, TLMΩ two-layer method.1Four groups were statistically compared using ANOVA. Fisher’s PLSD post-hoc test was used to detect differences among the groups.2Consistent with experimental design, groups 7 and 8 had prolonged cold ischemic time compared with groups 5 and 6. (p0.0001).Groups 5 vs. 6 and 7 vs. 8 were not significantly different. Group 7 TLM hoursΩ19.6∫??num?1.9.3Group 5 had significantly more embedded islets than groups 7 (p0.02) and 8 (p0.01).

55American Journal of Transplantation 2003; 3: 53–63

Sinn, Inc, Cherry Hill, NJ), and 6.7 Kunitz units/mL of DNase (Roche Mol-ecular Biochemicals, Indianapolis, IN). The protease inhibitor Pefabloc (Ro-che Molecular Biochemicals, Indianapolis, IN) was used in groups 2, 3,and 4 (Pefabloc, TLM π Pefabloc, and UW π Pefabloc, respectively). ThePefabloc concentration (4.0mmol) was based on published reports (8–10)and the recommendation of the manufacturer.

Four different lots of Liberase (84648720, 85142320, 85204720,85210020) were used for M. nemestrina islet isolations. The islet yields fromM. nemestrina pancreata did not vary with the Liberase lots used (pΩ0.30).

Pancreata were perfused with collagenase and digested in a 22¿30¿5-cm temperature-regulated processing pan (custom fabrication, Rigley). We

Matsumoto et al.

began to distend the pancreas while maintaining the pan temperature at4 æC. When the pancreas was distended, the processing pan temperaturewas raised to 37 æC, a stainless-steel sieve (450mm mesh) was placed intothe pan, to which the pancreas was transferred. The collagenase solutionin the pan was then diluted 1 : 1 with plain HBSS.

When the pancreas was approximately half digested, chilled HBSS with5% neonatal calf serum (Gibco BRL, Rockville, MD) was added and thepan temperature was reduced to 4 æC. Digestion time was defined as theinterval between completing the enzyme infusion and adding chilled HBSS(Table1). The digestate was collected and washed three times using chilledHBSS with 5% neonatal calf serum.

Purified M. nemestrina islets were obtained by isopycnic sedimentation ofwashed pancreatic digestate, as previously described (24). Isolations wereinitially purified in a COBE 2991 cell washer (Gambro BCT, Denver, CO),but subsequently, because of the small tissue volumes, M. nemestrina

islets were purified by using the same gradients in 250-mL centrifugebottles (24). There was no statistical difference in postpurification recoveryrates between these methods (recovery rate 80% for both).

With both purification methods, a two-chamber gradient mixer was used(custom fabrication, Rigley) to establish a gradient continuous for bothdensity and osmolality, using a fractionation medium of histidine lactobion-ate (HL) solution, and iodixanol (Optiprep, Nycomed Pharm, Oslo, Nor-way), as previously described (24). The osmolar gradient enhanced thedensity difference between the endocrine and exocrine particles, increas-ing the ability of the gradient to separate islets (25). Varying amounts ofisotonic and high-osmolality HL solution were combined with iodixanol tomake solutions of four different densities, and was used to produce threedistinct layers. High-osmolality HL solution was made by adding 10-mmol/L of lactobionic acid and 210-mmol/L of histidine to standard HL solution.For the most dense or bottom layer we combined high-osmolality HL andOptiprep, which was added to the washed pancreatic digestate (1.114g/cm3, 500mOsm/kg). The intermediate layer, the continuous density andosmolar gradient, was generated by mixing low-density (1.060g/cm3,330mOsm/kg) and high-density (1.110g/cm3, 500mOsm/kg) solutionswith the gradient mixer. The least dense or capping layer consisted of onlyHL solution, without Optiprep (1.030g/cm3, 330mOsm/kg).

We bottom-layered the mixture of digested tissue and bottom solution intoeither the COBE 2991 or the 250-mL centrifuge bottles and overlaid itwith the continuous gradient solution, beginning with high osmolality anddensity and ending with low. These layers were then capped with the HLsolution that lacked iodixanol. The tissue was allowed to stratify during5min of centrifugation at 200G, and then sequential fractions were col-lected into conical centrifugal tubes prefilled with chilled aliquots of CMRL1066 culture medium (Mediatech Inc., Herndon, VA). Fractions that werefound to contain purified islets were combined and washed three timeswith the CMRL medium.

Purified islets were cultured at a concentration of 1500 IE/mL inCMRL1066 with 0.002% ciprofloxacin (Bayer Corporation, West Haven,CT) and 10% FBS (Gibco BRL) at room temperature without CO2 for 24hin suspension-culture T75 flasks (Sarstedt Inc., Newton, NC).

Measurement of trypsin activity

We collected and snap-froze 1-mL aliquots of supernatant at the com-pletion of both M. nemestrina pancreas distension and disaggregation.Trypsin activity was measured by absorption spectrophotometry (l253nm;Ultrospec 2000, Pharmacia Biotech Ltd, Cambridge, UK) according to themethod of Schwert and Takenaka, using the reagent BAEE (N-benzoyl-L-arginine ethyl ester; Worthington Biochemical, Lakewood, NJ) for the tryp-sin substrate (26). The assay was calibrated by using dilutions of a stock

56 American Journal of Transplantation 2003; 3: 53–63

solution of 1mg of trypsin (.LS003708, Worthington Biochemical) in25mL of 0.001 N HCl to generate a standard curve. All samples wereassayed in triplicate. Absorbance was measured at 1, 2, 3, 4, 5, and 6min.A BAEE unit was defined as a D optical density of 0.001 per min.

Macaca nemestrina islet evaluation

Islets were identified by dithizone staining, and sized and counted as pre-viously described (27). Embedded islets, defined as islets with 50% oftheir surface covered with intact nonendocrine tissue, were counted. Isletviability was evaluated before and after culture based on acridine orange(10mmol/L) and propidium iodide (15mmol/L) (AO/PI) fluorescent stains(27–31). The ratio of living to dead cells was determined for 50 randomlyselected islets, and was then used to calculate average islet viability.

The in vitro function of cultured islets was evaluated by measuring insulinreleased after glucose stimulation. Aliquots containing 100 IE were incu-bated for 2h at 37 æC with 5% CO2 in RPMI 1640 culture medium (Bi-oWhittaker, Walkersville, MD) containing either 2.8mmol or 20mmol ofglucose. Insulin was measured with a human insulin ELISA kit (ALPCOInsulin ELISA kit, Windham, NH). Insulin concentrations for the peak stimu-lation portion (20mmol) of testing were divided by the insulin concen-trations obtained for the basal time period (2.8mmol), and were reportedas a stimulation index: SIΩ [insulin] 20mmol/[insulin] 2.8 mmol (1,32).

Macaca nemestrina islet transplantation into diabetic nude mice

In vivo islet function was tested by renal subcapsular transplantation of2000 IE from groups 1 (control, nΩ5), 2 (π Pefabloc, nΩ5), and 3 (TLMπ Pefabloc, nΩ4) into diabetic nude mice. Nude mice were previouslymade diabetic by tail vein injection of 220mg/kg of streptozotocin(Pharmacia & Upjohn, Northpeapack, NJ) (32,33). Because of low yieldand viability, islets from group 4 (UW π Pefabloc) were not transplantedin these experiments. Mice were deemed diabetic when two sequentialblood glucose levels, on separate days, were greater than 250mg/dL. Theactual mean blood glucose level before transplantation was 321∫11mg/dL. Higher streptozotocin doses produced more severe diabetes (bloodglucose 400mg/dL), but these mice usually died immediately or failedto survive the transplantation procedure.

Pre-transplantation blood glucose levels were not significantly different be-tween the groups. Blood glucose levels were measured on post-transplan-tation days 3, 7, and 14. The stability and severity of the diabetic state inthis model were confirmed by persistently elevated blood glucose levelsin a sham-transplant group (nΩ5; mean of all nonfasting blood glucoselevelsΩ359∫19mg/dL, rangeΩ276–525mg/dL). Sham-transplant micecontinuously lost weight and none survived to postoperative day 21.

Human model

Pancreas procurement and preservation

Cadaver pancreata for these experiments were obtained between February17, 1999, and December 3, 2000, through LifeCenter North-west (UNOSregion 6, Seattle, WA), the National Disease Research Interchange (Phila-delphia, PA) and the International Institute for the Advancement of Medi-cine (Jessup, PA).

Human islet isolation with the Seattle open-pan research method (groups

5–8)

The human isolation method differed slightly from the M. nemestrina

method, in that we used a roller-pump rather than a 60-mL syringe toinfuse Liberase into the pancreatic duct (18,19). Three different lots ofLiberase (84648720, 85142320, 85147720) were used for human isletisolations. The islet yields from marginal human pancreata did not differamong the Liberase lots (pΩ0.22).

Human islets were purified using continuous density gradient centrifuga-

Improved Islet Yields From TLM and Trypsin Inhibition

tion in the COBE 2991 cell processor as described in the M. nemestrina

section, but the recovery of purified human islets was maximized by ad-justing the density of the bottom and intermediate layers of the fraction-ation medium to optimally match the tissue particle density in each di-gestate (7,24). The density of endocrine and exocrine tissue particles inthe washed digestate from each pancreas was revealed by the pattern ofsedimentation present after centrifugation of small volumes of digestedtissue in six aliquots of solution adjusted with Optiprep to different testdensities. As described in the M. nemestrina procedure, purified isletswere washed and assessed for yield, viability, and in vitro function.

Human islet isolation with the Edmonton method (group 9)

As specified in the Edmonton/ITN protocol, we digested the pancreata in aRicordi chamber without Pefabloc, and used Ficoll to generate the densitygradient used in islet purification (1).

Statistics

Values for the data collected are means ∫ SE. Experimental groups 1–4and 5–8 were compared using ANOVA followed by Fisher’s PLSD post-hoctest. Group 9 and the data published by Edmonton were compared usingthe Wilcoxon rank-sum test. We considered p-values less than 0.05 to besignificant.

Results

Macaca nemestrina experiments, groups 1–4 (Seattle

open-pan research islet isolation method)

Two-layer method preservation and Pefabloc additively

inhibit trypsin activity during M. nemestrina islet isolation

At the end of distension, Pefabloc in groups 2 (π Pefabloc),3 (TLM π Pefabloc), and 4 (UW π Pefabloc) reduced trypsinactivity in comparison with group 1 (control) (Figure 1). Atthe end of digestion, when trypsin activity is greatest, Pefab-loc reduced trypsin activity by 78% (group 2 vs. 1, p 0.001).The combination of Pefabloc and TLM preservation (group 3)suppressed trypsin activity below detectability (group 3 vs. 4,0.0∫0.0 vs. 44∫15 BAEE units; p 0.04). These obser-vations show that passive oxygenation of pancreata beforeislet isolation, and use of Pefabloc during islet isolation, re-duce trypsin activity associated with pancreas digestion andpreservation injury.

Trypsin inhibition with Pefabloc increases M. nemestrina islet

yield without affecting viability or function

Islet yields per gram of M. nemestrina pancreas tend to besignificantly larger than those from human pancreata. Evenwhen isolation involves the use of crude collagenase and in-efficient techniques for mechanical disruption and purifi-cation, yields from M. nemestrina of 10000 IE/g have beenreported (34). In our experiments, the addition of Pefabloc tocollagenase during pancreas digestion increased the post-purification islet yield by 63% (group 2 vs. 1, p 0.01) (Fig-ure 2A). There was no difference in the viability of islets fromgroups 1 and 2, either postpurification (pΩ0.31) or postcul-ture (pΩ0.92) (Figure 2B). Glucose-stimulated insulin re-lease assays revealed no difference between groups 1 and2 (pΩ0.40) (Figure 3A). These findings demonstrate that

57American Journal of Transplantation 2003; 3: 53–63

Figure1: Impact of Pefabloc and pancreas two-layer method(TLM) preservation on trypsin activity during Macaca neme-

strina pancreas distention and disaggregation. Triplicatesamples of supernatant were collected and analyzed spectrophoto-metrically for the action of pancreatic trypsin on the substrate N-benzoyl-L-arginine ethyl ester (BAEE). A BAEE unit corresponds toa change of optical density of 0.001/min (23). UWΩUniversity ofWisconsin solution

Pefabloc increases M. nemestrina islet yields without impair-ing islet function.

Two-layer method preservation further increases M. nemes-trina islet yields, and improves or maintains M. nemestrinaislet viability and in vitro function

Purified islet yields from group 3 (TLM π Pefabloc,23769∫2773 IE/g) were larger than group 2 (π Pefabloc,15822∫1331; p 0.01) and group 4 (UW π Pefabloc,9287∫570 IE/g; p 0.0003) (Figure 2A). Purified isletviability of group 3 was 5.4% better than that of group 4(p0.05) (Figure 2B). After 24h in culture, group 3 viabilityhad become 18% greater than group 4 (97∫1% vs.82∫6%, respectively; p 0.02). Furthermore, the postcul-ture islet viability of group 4 (82∫6%) was lower than thatof group 1 (93∫4%; pΩ0.06) and group 2 (94∫2%; p0.05) (Figure 2B). Group 3 islets released significantlymore insulin than the islets from group 4 during static glu-cose challenge assays (stimulation indexesΩ3.2∫0.3 and1.6∫0.2, respectively; p 0.01) (Figure 3A). The stimulationindex results for group 3 are in keeping with values reportedby others for immediately processed pancreata in anothernonhuman primate model (32).

These results show that, in comparison with UW preser-vation, TLM preservation improves the yield, viability, andfunctionality of islets isolated from M. nemestrina pancreata

Matsumoto et al.

Figure2: Effect on islet yield and viability of two-layermethod (TLM) preservation and Pefabloc use during pan-creas distention and disaggregation. (A) Islet yields were tabu-lated on the basis of ‘islet equivalents.’ Islets under the microscopewere identified by bright red/orange color after staining with dithi-zone, and then their diameters were individually measured with acalibrated eyepiece reticle. The count was normalized to the num-ber of islets that would be present if they were all 150 mm in diam-eter. (B) Islet viability was assessed under the fluorescent micro-scope by estimating the fraction of cells in each of 50 randomlyselected islets that turned red when incubated with propidium iod-ide. Healthy cells with intact membranes are able to exclude thisdye, and appear green from the action of the acridine orangecounterstain we used. Culture recovery rate was measured after24h of culture

(Figures 2 and 3A). Our results also demonstrate that TLMpancreas preservation (group 3) before isolation improves is-let yield and maintains viability and function similar to thatfrom pancreata processed immediately (groups 1 and 2)(Figures 2 and 3A).

Macaca nemestrina islet function in vivo

After transplantation with the islets from groups 1 (control),2 (π Pefabloc), and 3 (TLM π Pefabloc), the diabetic nudemouse mean blood glucose levels were significantly lower

58 American Journal of Transplantation 2003; 3: 53–63

Figure3: In vitro and in vivo function of Macaca nemestrina

islets. (A) In vitro function of M. nemestrina islets was measuredby islet insulin release in response to low vs. high glucose concen-trations. Stimulation index, SIΩ [insulin] 20mmol glucose/[insulin]2.8mmol glucose. (B) In vivo function of M. nemestrina islets wasassessed by transplanting islets isolated without Pefabloc (group 1;nΩ5), with Pefabloc (group 2; nΩ5), and with Pefabloc after two-layer method (TLM) preservation (group 3; nΩ4), into diabeticnude mice. A control group (nΩ5) received a sham operation only.The average time-to-cure was 14days for group 1 and 3days forgroups 2 and 3. Islets from pancreata isolated with Pefabloc afterUniversity of Wisconsin solution (UW) preservation (group 4) werenot transplanted because of low viable-islet yields

than in the sham-operation group (Figure 3B). By post-trans-plant day 3, diabetic nude mice that had received islets fromgroups 2 and 3 had stable mean blood glucose levels of150mg/dL. In contrast, nude mice receiving islets fromgroup 1 required 2weeks to achieve similar blood glucoselevels. By post-transplantation day 14, the survival rates forgroups 1, 2, 3, and the sham-operated mice were 60% (3/5),60% (3/5), 100% (5/5), and 80% (4/5), respectively. Theseobservations indicate that Pefabloc and TLM preservation donot impair islet function, and when combined (group 3) may

Improved Islet Yields From TLM and Trypsin Inhibition

Figure4: Human islet yields, before and after purification, asaffected by Pefabloc use and type of pancreas preservation.(A) The effect on yield of adding Pefabloc during digestion ofpancreata with moderate University of Wisconsin solution (UW)preservation times (group 5, control, 7.6h; group 6 π Pefabloc,10.4h). (B) Comparison of effect on yield of lengthy UW vs. two-layer method (TLM) pancreas preservation. Organs were divided inhalf for this experiment, and each group received an equal numberof head and tail halves. The mean pancreas weight of group 7 (TLMπ Pefabloc) was 42.3∫4.1g and group 8 (UW π Pefabloc) was41.8∫5.6g

improve the function of islets transplanted from preservation-injured pancreata.

Human experiments, groups 5–8 (Seattle open-pan re-

search islet isolation method)

Pefabloc increases islet yield and purity from marginal hu-

man pancreata without impairing viability or function

In comparison with group 5 (control), group 6 (π Pefabloc)islet yields increased by 61% in the digestate (4007∫417 IE/g vs. 6454∫861 IE/g; p 0.02) and by 91% after purifi-cation (2474∫472 IE/g vs. 4723∫1006 IE/g; p 0.04)(Figure 4A). Islet purity was 54% higher in the Pefabloc group(82∫6% vs. 53∫12%; p 0.04). This difference in puritypartially resulted from a differential prevalence of embeddedislets between these two groups (Table2). We observed nosignificant differences between groups 5 and 6 for viability

59American Journal of Transplantation 2003; 3: 53–63

of islets postpurification, recovery rate after culture, andviability of islets postculture. Glucose-stimulated insulin re-lease assays revealed satisfactory islet function (SI values of2) irrespective of the presence of Pefabloc (pΩ0.15). Ourfindings in humans and M. nemestrina indicate that Pefablocincreases islet yields without islet toxicity or dysfunction.

Two-layer method preservation increases human islet yields

from marginal pancreata, and maintains or improves viabil-

ity and function

Human groups 7 and 8 tested whether TLM preservation,compared with UW storage, would improve islet yields fromhemodynamically unstable pancreas donors or those sub-jected to vasopressor therapy, and from organs that have sus-tained cold ischemic injury while in storage. Group 7 (TLM πPefabloc) islet yields were 145% higher than group 8 (UW πPefabloc) (4801∫1066 IE/g vs. 1959∫360 IE/g; p 0.03)(Figure 4B). The viability of the group 7 and 8 islets did notdiffer significantly immediately postpurification; however,after 24h of culture, the group 7 islets retained substantiallyhigher viability than those in group 8 (84∫5% and 59∫5%;p 0.003). The stimulation indexes from donors .2 and .5of group 7 were 3.44 and 2.76, respectively. The stimulationindexes from donors .2 and .5 of group 8 were 2.04 and1.27, respectively. In both representative cases, islets from theTLM group showed approximately 1.5-fold higher insulin re-sponsiveness to glucose.

Human islet morphology and size improved with TLM pres-

ervation (Figure 5)

The average prepurification diameter of the group 7 islets(TLM π Pefabloc) was 37% greater than in group 8 (UW πPefabloc) (168∫13mm vs. 123∫15mm; p 0.02). Assuminga spherical geometry, this islet diameter increase corre-sponds to an islet volume increase of 156%. The averagepostpurification islet diameter of group 7 was 83% largerthan that of group 8 (240∫29mm vs. 131∫18mm; p0.002), corresponding to an islet volume increase of 510%.This striking difference in islet volumes may be overstated,because of our simplifying assumption that islets are spheres.Figure5 includes representative views of islets from groups 7and 8, in which typical size differences can be seen. Thedensity of tissue particles in the pancreatic digestate of group7 (TLM π Pefabloc) was greater than in group 8 (UW πPefabloc) (1.109∫0.002g/cm3 vs. 1.099∫0.002g/cm3; p0.01). Elevated pancreatic tissue-particle density is knownto be associated with superior postpurification islet yield,viability, and purity (35).

In these human and nonhuman primate experiments a non-clinical, but cost-effective and simple, open-pan research is-let isolation method was used to test the hypotheses thatpancreas TLM preservation, and trypsin inhibition with Pefab-loc during processing, allow longer preservation while retain-ing or improving islet yield and viability. Our results, combin-ing several critical parameters of islet isolation success (isletyield, viability pre and postculture, in vitro and in vivo func-tion, size, and density) together support the advantages of

Matsumoto et al.

Table3: Summary of two-layer method-preserved marginal1 pancreata processed according to the Edmonton/immune tolerance networkisolation method (group 9)

Age (y), Total CIT TLM time Digestate Purified Purity Viability SIM/F (h) (h) islet count islet count (%) (%)

(IE/g) (IE/g)

47, F 27 14 5624 3596 60 94 4.634, F 30 18 10994 4961 30 97 2.835, M 16 12 6210 2410 50 89 2.258, F 33 21 2172 1313 70 96 3.534, F 22 18 4193 1199 40 91 4.348, M 13 5 2833 2215 80 97 2.243∫4 23.5∫3.2 14.7∫2.1 5338∫1297 2616∫588 55∫7.6 94.0∫1.4 3.3∫0.4

CITΩcold ischemic time, TLMΩ two-layer method.Data presented as mean ∫??num? SE.1Four pancreata, procured nonlocally, had University of Wisconsin solution preservation time 8h (11.3∫??num?0.9h avg.); one donor hadpancreatitis, and one donor (cause of death anoxia) had prolonged downtime with elevated creatinine.

these processing innovations. Our findings are in agreementwith the work of others in suggesting a resuscitative capacityof TLM preservation when applied to ischemically injured or-gans (16,36,37).

Human experiments, group 9 (Edmonton/ITN islet iso-

lation method)

Pancreata in the next experiments were preserved for23.5∫3.2h, the majority of time in the TLM (14.7∫2.1h),then processed with the Edmonton islet isolation method.The islet isolation results from group 9 are not statisticallydifferent from those of group 5 (control), processed after7.6∫1.4h of UW preservation (Table3 and Figure 4A). Theislet isolation results from group 9 are superior to those re-ported by others, with pancreata preserved in UW solutionfor more than 12h (37,38). In comparison with the isletstransplanted by the University of Alberta group, the numericaldifferences in yield (Edmonton vs. group 9,357336∫27260 IE vs. 248520∫40152 IE; pΩ0.06) andstimulation index (SI) (Edmonton vs. group 9, 6.5∫1.25 vs.3.3∫0.4; pΩ0.06) achieved marginally significant statisticaldifferences (1).

Discussion

The cadaver pancreas incurs injury from donor brain death(39,40), hypotension, and vasopressor therapy, and fromwarm ischemia after donor cross-clamping and cold ische-mic storage in chilled UW solution (38). There is a clear re-lationship between these insults and reduced success of sub-sequent islet isolation (16,17,21,41,42). The improvement in is-let yield that results from Pefabloc use during islet isolationsuggests that trypsin is directly destructive of islets, and re-veals the large role of trypsin-mediated pancreas injury. Also,inhibiting trypsin during human pancreas digestion reducesthe fraction of embedded islets, suggesting that trypsin maydegrade the ductules, reducing the delivery of collagenasesolution to the immediate neighborhood of the islets.

60 American Journal of Transplantation 2003; 3: 53–63

In this paper we have demonstrated that Pefabloc and TLMpreservation combine to eliminate measurable trypsin activityduring pancreas digestion. However, it is unlikely that thebenefit of TLM pancreas preservation can be attributed solelyto trypsin inhibition. During TLM preservation, pancreas ATPlevels increase and RNA and protein are synthesized(14,15,43). Heat-shock protein levels increase (HSP 60, 70,and 32) (15), which may effect cell repair, neutralize free rad-icals, and inhibit apoptosis, resulting in higher yields of moreviable islets (33,44–49).

The improvement in islet recovery that results from TLM pres-ervation shows that hypoxia damages the pancreas duringcold storage in UW solution. Two-layer method preservationsupports active metabolism at low temperatures, and prob-ably reduces trypsin activity associated with preservation in-jury during conventional UW storage. Hering and associatesrecently reported on three diabetic patients, each of whomreceived transplant islets from only one pancreas that hadbeen placed immediately into TLM preservation at the pro-curement site (50). Their average islet yield after processingby a Ricordi-based isolation method was approximatelydouble that reported by the Edmonton group, consistent withour experience of processing TLM-preserved nonmarginal M.

nemestrina pancreata.

We also tested whether marginal human pancreata could beTLM-preserved and processed successfully with the Edmon-ton/ITN protocol (Table3). In group 9, we isolated islets fromTLM-preserved human pancreata in compliance with anFDA-approved clinical processing protocol that excluded Pe-fabloc from the islet isolation procedure. This difference pre-cludes strict comparison with the previously described ex-periments, but we are reporting our observations because oftheir clinical relevance and because they can be evaluatedagainst the Edmonton group published results of transplant-quality pancreata processed 4.8∫0.8h after organ recovery(1). Two-layer method preservation allowed the islets isolatedfrom our Edmonton/ITN group (Table3), after approximately

Improved Islet Yields From TLM and Trypsin Inhibition

Figure5: Morphology of human islets isolated from pancreata after lengthy preservation. (A) Dithizone-stained islets in digestatefrom a pancreas after two-layer method (TLM) preservation (group 7) compared with (B) islets obtained from University of Wisconsinsolution(UW)-preserved pancreata (group 8). Typical appearance of AO/PI-stained islets (C) from TLM-preserved organs, compared to (D)that of islets from pancreata after UW storage

24h of cold ischemic storage, to compare surprisingly wellto those reported by the Edmonton group (1). Unlike earlierfindings with the pancreata selected by Edmonton for islettransplantation, our results were obtained from an unselectedseries of processing events with marginal, research pancrea-ta. According to ITN/Edmonton criteria, four of the six pancre-ata we processed were unsuitable for clinical islet transplan-tation because of prolonged UW storage or hypoxic injury.On the basis of our previous experience, and that of the Uni-versity of Alberta group itself, we would expect that preserv-ing these pancreata with the TLM and processing them with

61American Journal of Transplantation 2003; 3: 53–63

Pefabloc would produce a substantial increase in islet yield,with no loss in viability (Figures 2 and 4).

In a different group of marginal human pancreata, we haveobserved that TLM preservation, after less than 8h of UWstorage (minimal preservation injury), improved islet yields by77% (UWπTLM, 7763∫940 IE/g, nΩ4; UW alone,4378∫999 IE/g, nΩ6; p 0.05). In another case, we di-vided a pancreas into equal halves and compared immediateprocessing with islet isolation after 17 additional hours ofTLM preservation. The yield was 3290 IE/g from the half pro-

Matsumoto et al.

cessed immediately and 6306 IE/g from the TLM-preservedhalf, implying that TLM preservation may resuscitate pancrea-ta during cold storage, as has been reported in canine wholeorgan and islet transplantation models (16,36,37,51). We arecurrently determining the parameters for TLM resuscitationof human pancreata (Stevens, in preparation).

It must be recognized that islet quality is ultimately revealedby islet survival and engraftment in vivo, and may not bereflected accurately by standard in vitro tests of viability andfunction (52,53). Although viability determinations based onconventional microscopy of fluorescently stained islets areimperfect, as the islet interior cannot be visualized, it is note-worthy that significant correlations between islet viabilitystain assays and both glucose-stimulated insulin release andthe viability of the individual cells of disaggregated islets havebeen reported (28,30,31). Minimal islet transplantationmodels, with a higher sensitivity for identifying modulators ofearly post-transplantation islet dysfunction, may better revealthe impact of Pefabloc and TLM preservation on islet quality(33).

The uncertainties of in vitro islet quality assays have prompt-ed us to evaluate supplanting them with monitored continu-ous-flow perifusion. With use of this technique, islets main-tained in a slow current of nonrecirculated medium may bemonitored for the oxygen consumption rate, glucose-stimu-lated insulin secretion, and other parameters of tissue healthand metabolic capacity. When stimulated with glucose in thisassay system, our islets displayed robust, biphasic insulin se-cretion (20,54). We now intend to investigate large-scalecontinuous-flow culture of isolated islets as an optimalmeans of both monitoring and maintaining islet health (3).

Pancreas TLM preservation and the use of Pefabloc allow or-gans destined for islet transplantation to be shared betweengeographically distant sites. The improved viable islet yieldthat results from these innovations means that transplantableislets can be recovered from marginal pancreata, and maymake it possible to cure a patient with islets from just onedonor (3).

Acknowledgements

We are grateful to Sabrina Qualley and Shilpa Goel for their technical helpand to the many members of the HITS program who gave their support.

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