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solation of Viable Porcine Islets by Selective Osmotic Shock Withoutnzymatic Digestion

. Atwater, M. Guajardo, P. Caviedes, S. Jeffs, D. Parrau, M. Valencia, C. Romero, C. Arriagada,. Caamaño, A. Salas, F. Olguin, M. Atlagich, R. Maas, D. Mears, and E. Rojas

ABSTRACT

Islet transplantation is a potential cure for type 1 diabetes, but clinical results have beendisappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which hassignificant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass andviability, poor reproducibility, high cost, a relatively low number of islets obtained per wholepancreas, and selection of islets for collagenase resistance rather than for glucose responsive-ness. In the present study we performed a series of experiments in a porcine model todemonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS)using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOSmethod can be carried out at room temperature or in the cold eliminating warm ischemia timewhich damages the islets. The SOS method does not depend on the texture of the pancreas soall pancreases can be processed identically and the process can be fully automated. The SOSmethod isolates all the islets of the pancreas regardless of size and shape allowing a greaternumber of islets to be harvested. The SOS method avoids exposure to toxins in collagenasesolutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters,representing the best glucose responding islets. The SOS method showed a comparablerecovery of islets from young pig pancreas and the islets showed improved viability. Weconclude that the selective osmotic shock (SOS) method of separating islets from the

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YPE 1 diabetes mellitus is a worldwide disease primar-ily among pediatric patients. It is characterized by the

elective immune-mediated destruction of insulin-producingeta cells by the pancreatic islets of Langerhans. Theatients require life-long treatment with exogenous insulin.espite intensive therapy, these patients are susceptible tovariety of complications that are responsible for increasedorbidity and mortality rates. In a subset of metabolically

nstable patients, acute hypoglycemia secondary to insulinherapy is a characteristic, devastating complication. Duringhe past decades, islet cell transplantation has been consid-red to be a viable option.4,32,43,45 Even though insulinndependence is rarely achieved for more than 2 years,atients who have received islet transplants are protectedgainst severe hypoglycemia and show improved metabolicarameters.32,43

One of the major challenges in islet transplantation is toutomate and improve the islet isolation method, seeking toeproducibly extract a large number of viable19,26 islets

2010 by Elsevier Inc. All rights reserved.60 Park Avenue South, New York, NY 10010-1710

ransplantation Proceedings, 42, 381–386 (2010)

or transplantation therapy is presently based upon enzy-atic digestion of the pancreas with subsequent semi-

utomated purification using a physical density gradient as

From the Neuro-endocrine Laboratory (I.A., M.G., P.C., S.J.,.P., M.V., C.A., A.S., M.A., R.M., D.M., E.R.), Human Geneticsrogram and Molecular and Clinical Pharmacology Program,

nstitute of Biomedical Sciences (ICBM), Faculty of Medicine,nd Department of Surgery (M.G., F.O.), Office for the Support oflinical Investigation (OAIC) (I.A.), and Endocrine Laboratory

C.R., E.C.), J.J. Aguirre Clinical Hospital, University of Chile,antiago Chile, and Health Sciences Faculty (D.P.), Diego Por-

ales University, Santiago, Chile.Supported in part by grants from National Science and

echnology Fund (FONDECYT), Chile #1030596 (I.A.), #1030611E.R.), #1030661 (C.R.), and #1050571 (D.M.) and from theuvenile Diabetes Research Foundation #5-2008-796 (I.A.).Address reprint requests to Illani Atwater, PhD, Neuro-endocrine

aboratory, Human Genetics Program, Institute of Biomedicalciences (ICBM), Faculty of Medicine, University of Chile, Inde-

0041-1345/10/$–see front matterdoi:10.1016/j.transproceed.2009.11.030

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eveloped by Ricordi.27,37,42,47,53 The Ricordi method,ometimes with slight modifications, is used universally, butt has several significant pitfalls: collagenase-induced dam-ge to the islets, low reproducibility, relatively low numbersf islets per whole pancreas, and high cost. The collagenaseamage has been attributed to an inherent variability ofollagenases,41 even among purified Liberase prepara-ions,3 to contamination of Liberase preparations withndotoxins,17 to variable collagen structure in individualslets,22,23 and to warm ischemia during processing at 37°C toctivate the enzyme.11 One study has shown that islet cellsctually internalize exogenous enzymes, causing decreasedunction and subsequent apoptosis.2 Significant research ef-orts have focused on improving islet isolation techniquessing collagenase or Liberase.1,7,8,10,11,17,21,22,23,27,28,34,41,53

Furthermore, it is known that individual islets of Langer-ans show enormous variability in size, micro-architecture,nd function.9,35,52 With the collagenase method, the iso-ated islets are most likely selected for size, position in theancreas, and physical resistance to the digestion process,ot necessarily for responsiveness to glucose or for robust

nsulin secretion, making the process even less efficient.everal improvements have been developed, such as the usef a two-layer method with perfluorocarbon, or anotherethod for oxygenation, improved preservation with coldniversity of Wisconsin solution before islets isolation,21,46

ddition of nicotinamide to the processing medium,25 andse of a short-term culture period,33 sometimes in associa-ion with caspase inhibitors or other agents that inhibitpoptosis. However, islet yield continues to be a majortumbling block to the procedure.19,34 Thus, the relativelyow success in this procedure has kept islet transplantations an experimental procedure of limited clinical applica-ion.24,36

Pancreatic islet cells are endowed with glucose transport-rs (GLUT 2) in their cell membranes.12,31,49,51 Theseransporters equilibrate glucose from the external mediumith the cytoplasm in a matter of seconds, playing a central

ole in the extracellular glucose-sensing mechanism andeing a hallmark of “glucose-responding” islets.12,16,20,39,40

urthermore, these transporters are not saturated, even atigh glucose concentrations. Thus, glucose is not osmoti-ally active in cells from glucose-responding islets. Veryigh concentrations of glucose, added on top of osmoticallyalanced salt solutions or culture media, cause cells withoutLUT 2 transporters to shrink almost instantaneously

hrough a net water efflux. After several minutes, most cellsestore their initial volume5,18,38 by activation of ion14,29,30

nd urea fluxes in a process called regulated volumencrease, which leaves the cytoplasm relatively hypertonican increased concentration of internal ions balancing thexternal glucose concentration). When these cells are againxposed to physiological solutions, ie, media without glu-ose, the opposite occurs in terms of water fluxes, and theells without Glut 2 swell and burst (Fig 1). In this manner,n the pancreatic tissue exposed first to a solution enriched

olution, we expect to see the selective destruction ofancreatic acinar cells and other non– glucose-respondingells.

We performed a series of experiments in a porcineodel seeking to demonstrate the feasibility of a new

xperimental islet isolation and purification method, theelective Osmotic Shock (SOS) method (patent pend-

ng), based on the “selection” of glucose-responsive isletsy treatment with a high-glucose solution, followed by aero-glucose solution. We evaluated morphological andunctional parameters of the isolated islets in the culture,omparing islets isolated with the SOS method to thosesing the traditional collagenase method. We observedhat the SOS method was feasible, showing recovery ofbundant viable islets. We also noted that the process isaster and cheaper than the traditional method usingollagenase. It can be performed at room temperature orn the cold. Also, this new technique is potentially suitedo be fully automated.

ATERIALS AND METHODSslet Isolation, Purification, and Culture Conditions

oung Landrace swine of 26–35 kg underwent total pancreatec-omy under general anesthesia, without perfusion using a coldreservation solution. Immediately after extraction, the pancreashich was cleaned under sterile conditions, was weighed, sub-erged in povidone- iodine solution, and washed with 2 L of sterile

aline solution, all at room temperature (approximately 18°C).Under a sterile laminar flow hood, the pancreas was chopped

nely and divided into sterile 40-mL conical tubes, according to thexperimental protocol. After weighing the content, the islets weresolated by enzymatic digestion with Liberase CI (Roche Cat. No.1814435001, Basel, Switzerland) for 20 to 23 minutes at 37°C. Theecovery rate was more than 60% of the islets as free islets. Isletssolated by SOS were obtained as follows: samples of choppedissue were exposed to RPMI 1640 media supplemnted with either00 mmol/L of glucose or 600 mmol/L of glucose for 20 or 40inutes at room temperature (approximately 22°C). All samplesere then centrifuged for 2 minutes at 1000 rpm and the solution

apidly replaced by a zero-glucose RPMI medium. The tissue wasently mixed, centrifuged, and washed again with fresh zero-lucose medium; the washing procedure was again repeated. Theesultant mixture was either placed in culture or further purified asollows. Each of the portions of tissue was then processed by gentleechanical disruption by suction and expulsion from a sterile

yringe without a needle for approximately 15 minutes. Theesulting mixture was centrifuged through a density gradient ofistopaque 1077 (Sigma-Aldrich no. 1077, St. Louis, Mo). The

slet-enriched fraction was placed in RPMI 1640 culture mediaithout bicarbonate, but with 5.6 mmol/L glucose, 20 mmol/LEPES, 1% of streptomycin, 1% of penicillin, and 20% of

nactivated horse serum at 37°C in a 5% CO2 incubator. Theulture media were replaced the first day after isolation andhereafter every 48 hours. The same purification and cultureonditions were used for the Liberase-treated samples.

slet Quantification and Purity

o evaluate islet recovery after isolation, we counted the total

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f 50 �L and multiplied it by the total volume. The procedure waserformed on day 4 of culture after purification to evaluate theumber of intact islets. The number of islets per gram pancreaticissue was also calculated.

Samples of the islet preparations were photographed using anlympus (Center Valley, Pa) digital camera and a Wild (Colorado

prings, Colo) dissecting microscope. Photographs were processedsing ImageJ (Wayne Rasband, National Institutes of Health,ethesda, Md, United States).

ssessment of Islet Functionslet viability and function were evaluated using insulin secretionests on day 4 after the isolation. Washed islets isolated underarious experimental conditions were exposed to RPMI 1640ulture media with 5.6 mmol/L glucose and 20% horse serum for 90

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inutes at 37°C in a 5% CO2 atmosphere. Then the samples wereentrifuged at 2000 rpm for 15 minutes and the supernates wereollected and frozen at �20°C. The islets were resuspended inedia as above but with 16.7 mmol/L glucose and cultured for a

urther 90 minutes. The supernate was analyzed using an immulite/mmunlite 1000 insulin determination assay (Diagnostic Productsorporation, Los Angeles, Calif) with results expressed as �IU/L. Finally, insulin secretion was normalized to basal secretion

alues and also to the number of islets in each dish. All protocolsere approved by the Animal Ethics Committee of the Universityf Chile.

ESULTSslet Recovery and Purity

fter SOS treatment, the number of isolated islets wasighest using 600 mmol/L glucose for 20 minutes, asounted after 4 days in culture. In a representative experi-ent, 4089 islets per gram of tissue were isolated from the

ortion of pancreas exposed to a 300 mmol/L glucoseolution, whereas 13,423 islets per gram of tissue werebtained from the portion exposed to 600 mmol/L glucose.rom the same pncreas, Liberase isolation yielded 8543

slets per gram of tissue.Figure 2 compares the purity and quality of the islets

solated by 600 mmol/L glucose (Fig 2B, 2D and 2F) withhose isolated with Liberase (Fig 2A, 2C and 2E). It can beppreciated from the photographs that both the purity of thereparation (Compare Figs 2A and 2C with Fig 2B and 2D)nd the capsular integrity of islets (Compare Figs 2E with FigF) isolated by SOS were superior to islets isolated by Lib-rase in the traditional fashion.

nsulin Secretion

e evaluated insulin secretion from islets isolated by SOSn response to a culture medium supplemented with 5.6

mol/L glucose or 16.7 mmol/L glucose after 4 days inulture. Table 1 shows the results of islets isolated after 20

ig 1. Representation of selective osmotic shock hypothesis. Cellsn left side of illustrations represent islet-cells containing Glut 2ransporters in the membrane; cells on the right side illustrate pancre-tic acinar cells. In A, both cell types are exposed to physiologicalolutions, small dots representing sodium ions, mostly in the extracel-ular space, and large dots representing potassium ions, mostly in thentracellular space. In B, both cell types are exposed to very highlucose concentrations, approximately tripling the osmotic strength ofhe external solution. Large stars represent glucose. Islet-cells take uphe glucose via Glut 2 transporters in the membrane in order toquilibrate the osmotic strength of the external solution whereas acinarells rapidly loose water in order to equilibrate internal and externalolutions, and this causes the cells to shrink. In C, acinar cells take upotassium ions to recover cell volume and to adapt to the extracellularsmotic strength. In D, the very high glucose solution is removed andhe cells are again exposed to physiological solutions. Islet cells loosehe extra glucose, again through Glut 2 transporters in the membrane.owever, acinar cells rapidly take up water to dilute the hypertonic

ntracellular solution, and this causes the cells to swell. In E, the acinar

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384 ATWATER, GUAJARDO, JEFFS ET AL

inutes of exposure to high, 300 or 600 mmol/L, glucose.fter 4 days in culture, islets responded to a physiological

lucose stimulus showing an increment in insulin secretionf about 3-fold over basal values.Islets isolated by exposure to high glucose for 40 minutes

howed lower levels of basal insulin secretion and re-ponded poorly to glucose (results not shown). Islets iso-ated with Liberase showed no detectable insulin secretion,lthough the number of purified islets was similar.

ig 2. Light microscopy photo-raphs comparing islets purifiedy SOS to enzymatic digestion.igures A, C, and E representypical islets isolated with Lib-rase. Figures B, D, and F repre-ent islets isolated with the SOSethod. Scale bars: A,B: 500 �m.,D: 200 �m. E,F: 50 �m.

Table 1. Insulin Secretion from Islets Isolated by SOS

Experimental Condition

Baseline Insulin Secretion/gof PancreasAverage (SD)

Stimulated InsulinSecretion/gof PancreasAverage (SD)

00 mmol/L glucose 39.59 (10.54) 97.46 (47.19)00 mmol/L glucose 173.4 (33.2) 384.9 (83.44)

Note: Insulin secretion in response to the culture media supplemented with

i.6 mmol/L glucose (baseline) and 16.7 mmol/L glucose (stimulated) after 4ays in culture.

ISCUSSION

he SOS technique to isolate islets of Langerhans repre-ents a novel approach in the field of islet cell transplanta-ion. The elimination of enzymes from the islet isolationrocedure has several advantages: enzymatic damage to

slets is avoided, preparation purity is improved, and pro-essing can be performed at cooler temperatures, is lessostly and, most importantly, has the potential for fullutomation.

In recent years, there has been an explosion of interest inurified collagenase and Liberase preparations. Differentreparations have been advocated for each type of pancreaseeking to leave insular capsule undigested and preferen-ially digest the connective tissue of the acinar panc-eas,1,7,8,10,11,21,22,23,27,34,53 thereby reducing endotoxins,17

nd minimizing variability among enzyme batches,41 andven within a particular lot.24,28 Some of these concerns are

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oncerns has made the ultra-pure Liberase preparationsxpensive.

Collagenase digestion of young pig pancreata yieldsslet-like cell clusters, which tend to fall apart upon cultureith few large or robust islets.6 In the experiments reportederein, we confirmed that islets isolated from 30-kg pigs byhe Liberase method lacked integrity; however, those iso-ated by the SOS method contained intact capsules. Fur-hermore, with the SOS method, a greater number of isletsere isolated per gram of tissue (13,423) than that reported

rom adult pigs (4210)7 or (12,36353) or from humanadaveric donors (2279).47 We propose that the collagenheath of the young porcine islets may be particularlyensitive to collagenase treatment and that for this reasonoung pigs have not been recommended for islet isolations.6

ith the present method, however, young pigs are satisfac-ory islet donors.

Most researchers believe that cooling the pancreas andhe islets of Langerhans during isolation and preparation ismportant to maintain long-term islet function.7,48,50 Be-ause collagenase digestion requires physiological temper-tures of about 37°C for enzyme activation, islet tissue isxposed to a period of warm ischemia, with potential foramage. The SOS method was successful at cooler temper-tures, thus protecting islets from damage.

The islets of Langerhans are known to be variable in size,hape, and function within a given pancreas.9,35 Using theraditional method of islet isolation by collagenase diges-ion of the pancreas, some or many islets are damaged,34

hich probably relates to the type of collagen surroundingach islet, the size of the islet (larger ones may be destroyedhile the smallest ones remain trapped in acinar tissue),nd the particular state of the pancreatic tissue surroundinghe islets. None of these parameters have been correlatedith islet function. The density of GLUT 2 glucose trans-orters has, however, been identified as a positive index of

slet function and responsiveness to glucose.12,16,20,39,40 Theethod presented herein to isolate islets depends onLUT 2; thus, it positively selects functional islets.There have been several reports that the traditionalethod of islet isolation causes cells swelling. We propose

hat this is due to the use of glucose as an osmotic substituteor ions in the preservation or isolation media, thus causingells with Glut 2 transporters to be exposed to effectivelyypotonic solutions. This observation is compatible withur hypothesis that islet cells containing functional GLUT 2ransporters are osmotically blind to glucose in the media.lternatively, there is evidence showing that a moderatelyigh extracellular glucose medium induces swelling in alphand beta cells, a phenomena abolished by using a GLUTnhibitor, phloretin, or a nonmetabolized sugar such as-O-methyl glucose, indicating that swelling by moderateoncentrations of glucose may depend on metabolism, anday also indicate functional islets.13 With the SOS method,

lucose metabolism is inhibited by the cooler temperatures

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Finally, the SOS method does not depend on the size of theslets or the texture of the pancreas from which they are to bextracted. Thus, the method is appropriate for full automationf human pancreata to obtain islets for transplantation. Au-omation of islet retrieval from donor pancreata may achievereater success in islet transplantation because islets could besolated on site immediately after pancreas procurement with-ut the requirement for a specialized isolation team. Further-ore, isolated islets have been shown to remain viable for

everal days and can be easily transported to a transplantationite at room temperature.

In summary, we have described the development of a newslet isolation method without the use of enzymatic digestiony collagenase or Liberase. This SOS method is based on theddition of very high concentrations of glucose to the extra-ellular medium for a brief period to separate cells containingLUT 2 transporters from others, thus selecting islets with

lucose sensitivity. The method is faster and cheaper than theollagenase technique and avoids damage to the islets byxcessive enzymatic digestion, by endotoxins, and by warmschemia. Furthermore, the method can be fully automated.

CKNOWLEDGMENTS

he authors wish to acknowledge the generous participation ofeterinarian students Gail Alfsen, Bernardita Silva, and Rodrigoalenzuela for assistance during the surgical procedures. We also

hank the valuable cooperation of Pedro Martinez, Juan Carlos Soto,nd Juan Carlos Soto, Jr. for their excellent technical support.

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386 ATWATER, GUAJARDO, JEFFS ET AL

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