Human Pancreatic Islet Transplantation

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pancreatic pancreatic of This diseaseiscausedbyautoimmunedestruction Arch Metab. Endocrinol 2015;59/2 periodsoftime,withsudden epi cillations over short glycemic os which is characterized by unpredictable “brittle T1DM”, number ofT1DMpatients present age(5).Moreover,ity inindividualsaproductive a associated withhighmorbidityandmortal which are complications, (3,4), thisdiseasestillleadstochronic decades ofT1DMhavebeenseeninrecent treatment (1,2).Althoughsignificantadvancesinthe to survive exogenous insulin require ficiency andfates patients to T INTRODUCTION Mirela J.Mirela Azevedo Division oftheHospitaldeClínicasPortoDivision Alegre –RioGrandedoSul,Brazil. processes, andtodescribetheestablishmentofahumanisletisolationlaboratoryatEndocrine tient qualityoflife. The goalofthispaperwas toreviewthehumanisletisolationandtransplantation decrease plasmaglucoseandHbA1clevels,theoccurrence ofseverehypoglycemia, andimprove pa insulin independent after a 3-year follow-up. Therefore, islet transplantation is able to successfully try (CITR) has revealed that approximately 50% of the patients who undergo islet transplantation are against hypoglycemic episodes.Recently, Islet datapublishedbytheCollaborative Transplant Regis showed permanentC-peptidesecretion,which facilitatedglycemiccontrolandprotectedpatients followingthetransplantation,but able declineintheinitialinsulinindependenceratesateightyears drugs displayed aconsider than one donor and were treated with steroid-free immunosuppressive tion. isletinfusionsfrommore The Edmontongroupdemonstratedthatmostpatientswhoreceived plantation maybeatherapeuticoptiontorestorebothinsulinsecretionandhypoglycemic percep have severehypoglycemia withoutadrenergicsymptoms(hypoglycemia unawareness), islettrans is stillassociatedwithanincreasedincidenceofhypoglycemia. Inpatientswith “brittle T1DM”, who of the development of chronicreduce the likelihood diabetes complications. However, this treatment insulintherapyhasbeenableto ity andmortality age.Intensive ratesinyoung adultsofproductive Type 1diabetesmellitus(T1DM)isassociatedwithchronic complicationsthatleadtohighmorbid ABSTRACT Jakeline Rheinheimer pancreatic isletisolationlaboratory oftheestablishmentdescription ofa transplantation: anupdate and Human pancreatic islet Fernanda S. deOliveira Human pancreaticislets;type1diabetesmellitus;isletisolation;transplantation Keywords 2015;59(2):161-70 Aline A. F. Estivalet TaísS. Assmann approximately 10%ofalldiabetescasesworldwide. approximately for ype 1diabetesmellitus(T1DM)isresponsible β cells,whichleadstocompleteinsulinde 1,2 , Jorge L. Gross 2 1 , Natália E. Lemos , Ana P. Bouças 1,2 , Andrea C. Bauer 1 , Lavínia A. Cruz 1,2 1,2 , B. Cristiane Leitão , Annelise R. Rosa 1,2 , Carlessi Rodrigo 1 , Letícia A. Brondani 1 , Sandra P. Silveiro - - - - 1 1,2 , BiancaM. deSouza kidney transplantorafter it(9,10).Although,pan with end-stagekidneydisease, atthesametimeas for T1DM patients complications, being recommended diabeticchronic andisableto prevent glycemic control optionforthesepatients(7,8). treatment an appealing of hypoglycemic symptoms and represents andtheawareness endogenousinsulin secretion restore orislettransplantationistheonlymannerto pancreas of condition. The replacement forthis no consensusindicatingtheoptimaltreatment is sents amajorchallengefortheclinicians,andthere stillrepre death (6).The“brittleT1DM”treatment mia, whichmayevolvetoconvulsions,coma,andeven sodes , DaisyCrispim 1,2 ,

Whole-pancreas transplantation results in adequate transplantation results Whole-pancreas 1,2 1,2 of hyperglycemia followed by severe hypoglyce of hyperglycemia followedbysevere , ,

Arch EndocrinolMetab. 1,2 1,2 , ------Porto Alegre, RS, Brazil Rio GrandedoSul(UFRGS), Universidade Federal do Medical Sciences:Endocrinology, 1 2 Porto Alegre, RS, Brazil de Porto Alegre (HCPA), deClínicas Division, Hospital Islet Biology;Endocrinology DOI: 10.1590/2359-3997000000030 Accepted onFeb/23/2015 Received onMay/6/2014 [email protected] –Porto90035-003 Alegre, RS, Brazil prédio 12, 4°andar Rua Ramiro Barcelos, 2350, Daisy Crispim Correspondence to: Laboratory ofHumanPancreatic Laboratory Post-Graduate Program in cells through whole- β cellsthrough review creas- 161 - -

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 162 Figure 1. Infusion ofhuman pancreatic isletsintotheportalvein of arecipientwithdiabetes mellitus type1. theinfusionofhealthyislets. through diabetic rodents hyperglycemia in and cols.(13)managedtoreverse tempted in1893(12),itwasonly1972thatLacy islettransplantationwasfirstat Although pancreatic HISTORICAL ASPECTS transplantation. kidney plantation inT1DMpatientswhounderwent tobeginislettrans equipped andtechnicallyprepared Brazil). Currently, is Alegre, Porto this laboratory (HCPA; Alegre of the Hospital de Clínicas de Porto isletisolationintheEndocrineDivision pancreatic forhuman described thedevelopmentofalaboratory wealso healthscenario.Furthermore, international inthe statusofthisprocedure as evaluatethecurrent isolation andtransplantationinT1DMpatientsaswell islet pancreatic regarding literature view the current transplantation(11). whole-pancreas after let infusionis20timeslowerthanthatobserved complicationsassociatedwithis risk ofperi-procedure (CITR),the the CollaborativeIsletTransplant Registry to radiologytechniques(7).According interventional 1) using vein (Figure the portal into the liver through infuseddirectly tions. Thisoccursbecausetheisletsare which isnotassociated with major complica cedure, T1DM” patients,asitconsistsofacellinfusionpro optionfor“brittle plantation hasbecomeaninteresting (7,9,10). Inthiscontext,allogeneicislettrans cedure high as those associated with other major surgical pro as ratesare plantation, sinceitsmorbidityandmortality akidneytrans to T1DMpatientswhodonotrequire isusuallynotrecommended thisprocedure survival, transplanted patientsshowhighratesoflong-term Human pancreatic islettransplantation Therefore, the aim of the present studywastore theaimofpresent Therefore, Donor Pancreas Isolated islets ------Infusion bag In the1980s,useofautologousislettransplanta to achieve more promising results: aninsulininde results: promising to achievemore that islet transplantation began Edmonton protocol, ofthe it wasonlyintheyear2000,aftercreation ledbyDr.group inMiami,USA(19), CamilloRicordi, islet isolationwasmadepossiblein1989bytheresearch only 2%after5years(18).Althoughsemi-automated 22% after2years,11%36%4yearsand the transplant:66%after1month,40%year, rates ofinsulinindependenceoverthetimefollowing between theyears1893and2000,showingdecreasing worldwide performed 493 islettransplantationswere thatatotalof (ITR)revealed let Transplant Registry the Is rates of only 10% (16,17). Data obtained from leading to insulin independence results, promising islettransplantationinhumansdidnotproduce creatic forthiscondition. who soughtapossiblecure of T1DM has been investigated by several researchers, lished, theuseofislettransplantationfortreatment firstpub the transplant(15).Sincethesedatawere tain insulinindependenceforupto13yearsfollowing abletomain (14).Thesepatientswere was reported pancreatitis ofchronic painasaresult for untreatable totalpancreatectomy tion inpatientswhounderwent lished the results ofafive-yearfollow-up65patients lished theresults pub transplanted islets(20).In2005,thesamegroup themassof to increase donors,inorder different from and multipleisletinfusions a lowdoseoftacrolimus), and with daclizumabandmaintenancesirolimus (induction protocols immunosuppressive corticoid-free wasattributedtothefollowing factors:useof protocol in achieving insulin-independence with this provement transplant insevenpatientswithT1DM(20).Theim pendence rateof100%wasachievedafter1yearpost Nevertheless, thefirstattemptsatallogeneicpan Nevertheless, Recipient Arch Metab. Endocrinol 2015;59/2 Liver Portal vein ------Arch Metab. Endocrinol 2015;59/2 andAustralian locations. Atotalof76cen European Americaaswellinsome inNorth tion performed allislettransplanta and publicationofdataregarding analysis the registration, through in such procedures safety greater transplantation technology and toensure ofislet inan attempttomonitortheprogress created the world(http://citregistry.org). TheCITRwasalso transplantationcentersaround tablishment offurther centers, andmotivatedthees in allexistingresearch inthenumberofislettransplantationsperformed crease year follow-upstudy(21). units/kg/day, P=0.001),asdemonstratedinafive- period (0.34 than in the pre-transplant cantly lowerdosesofinsulinfollowingthetransplant signifi (HbA1c: 6.7vs.9.0%,P=0.025)andrequired with total graft loss (absence of C-peptide secretion) muchbetter thanthose tained theirglycemiccontrol tion (definedbyC-peptidelevels>0.5ng/mL)main graft func successful. Individuals with partial partially isconsidered inlowdoses,theprocedure still required hypoglycemic episodes,evenifexogenousinsulinuseis orelimination of severe and tothe reduction control ever, glycemic whenthetransplantleadstoimproved successful whenever this outcome is achieved. How completely considered are procedures and, therefore, goal ofislettransplantationisinsulinindependence comes associatedwithislettransplantation.Themain ings ledtoanewdefinitionoftheobjectivesandout insulin independenceafteroneyear(22).Thesefind 80%ofpatientsmaintaining withapproximately results, obtainedthebest experienceinthearea with greater cess ratesvariedwidelybetweencenters,sothatthose and significantlowerratesofhypoglycemia(22).Suc inmetaboliccontrol had asubstantialimprovement graftfunction although thosewhomaintainedpartial after2years, insulin-free 13.8% ofpatientsremained (22).Only ninecentersintheUSAandEurope from ance Network,whichinvolved36patientswithT1DM multicenter studyorganized bytheImmuneToler number ofhypoglycemicepisodes. inthe andtothereduction ment ofglycemiccontrol contributingtotheimprove able C-peptidesecretion, graftfunction,withdetect 80% ofpatientshadpartial than 5years(21).However,tonotethat itisimportant formore insulin-free only 10%ofpatientsremaining duration ofinsulin independence was 15 months, with who hadundergone islettransplantation.Themean These encouraging results ledtoasignificantin These encouragingresults ina waslaterreplicated The Edmontonprotocol vs. 0.66 insulin ------the outcomesof677patientswhohadundergone is assessed CITRreport years.Arecent overrecent results istration (FDA). Admin being evaluated by the Food and Drug rently the endofalarge multicenterclinicaltrial,whichiscur ment option for “brittle T1DM” in 2015, following asatreat transplantation isexpectedtobeapproved tal basis(phase3clinicaltrials)(27).IntheUSA,islet onanexperimen let transplantationisstillperformed However, inmanyothercountries,includingUSA,is (25,26). by public and/or private health services ered is cov T1DM”. In these countries, such procedure optionforpatientswith“brittle acceptable treatment considerislettransplantationtobean Belgium, already Canada, Switzerland, Australia, Japan,Englandand (24).Anumberofcountries,suchas such procedure (23) and,untilMay2011,730patientshadundergone ters conductedislettransplantationintheyear2005 obtained for graft survival at5yearsafterislettrans obtained forgraftsurvival (11).Thelatestdata characteristics andrequirements knowledgeandunderstandingofβcell and increased patient selection careful more regimens, nosuppressive ofimmu techniques, aswellto the improvement ofisletisolationand isletculture to theimprovement may be attributed the transplant (11,24).Thisresult yearsafter sulin independencerateofalmost50%three had significantlybetteroutcomes,andachievedanin transplantationsinthe“RecentEra” who underwent hypoglycemia(11).Patients ofsevere and frequency levels, stablefastingglycemia, insulin independence inHbA1c decrease ofC-peptidesecretion, presence assessed: Era” (2007-2010).Fivemainoutcomeswere (1999-2002), “MidEra” (2003-2006) and “Recent “EarlyEra” performed: the periodtheirsurgeries were to according groups dividedintothree Patients were let transplantationsbetweentheyears1999and2010. plantation, the outcomesofencapsulated islettrans followingallogeneicislettrans drugs munosuppressive These studies include investigations of im procedure. patients forsuch recruiting currently studies whichare “islettransplantation”,identified34 using thekeyword 2014 (www.clinicaltrials.gov), inJanuary performed transplantaloneinthesameperiod(29). with pancreas similartothoseachieved so far(28).Thesenumbersare dependence hasbeenachievedin7yearsoffollow-up, independence rate.Insomestudies,70%ofinsulinin ininsulin plantation haveshownasignificantincrease Islet transplantation has led to progressively better Islet transplantationhasledtoprogressively A search foropen trialsontheClinicalTrialsA search website Human pancreatic islettransplantation 163 ------

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 164 and forthemonitoringofdigestedislets; 2 Figure 2. Pancreaticisletisolationandtransplantation fromthedonortorecipient. Adapted fromreference31. 1 ducts,enzymatic perfusion, cannulation ofpancreatic and thesame:cleaningpancreas remain procedure thebasicstepsof transplantationgroups, of different totheexperience recent years,according modified over has beenslightly Although thisisletisolationprotocol in1989(19). gestion developedbyCamilloRicordi di pancreatic a semi-automatedmethodofcontrolled attributabletotheuseof atleastpartly are in thisarea decades(7) and advances phisticated overthepast three so Islet isolationmethodshavebecomeincreasingly andfunctionalintegrity(30). theirstructural serving whilepre thepancreas, cellsfrom insulin-producing of islet isolation involvestheextractionof The process ISOLATION OFHUMANPANCREATIC ISLETS (TRIMECO, http://clinicaltrials.gov). forT1DM clinicaltreatment with thoseonstandard theoutcomes of islet transplantation trial to compare ble, France, is conducting the first randomized clinical Islet Transplant attheUniversityofGreno Program South America.Atthetimeofwritingthispaper, the beingconductedin currently of theseinvestigationsare transplantation incasesoftotalpancreatectomy. None plantations andtheapplicabilityofautologousislet Human pancreatic islettransplantation and collagenasesolution; 4 Flask containingpancreatictissue 2 and preservation solution and preservation Agitator =peristalticpump toensurethecirculationofsolutions inthesystem;5 Infusion bag 5 =chambertemperaturemonitoring probe (37°C);3 Digestion 3 4 1 Recirculation - - - - Purified isletcultures islet dilution, purification and culture (Figure 2)(31). (Figure islet dilution,purificationandculture followedby distension anddigestionofthepancreas, tissue and are notover-digested,tissue andare large volumesofdilu theexocrine separatedfrom agitation. Whenisletsare the digestionphase,whichisfacilitatedbymechanical silicone beadsandfilledwith collagenase solution for Chamber(19)containing7-9 toaRicordi transferred isthencutinto8to10piecesand tissue. Thepancreas ofthepancreatic duct, leadingtothedisaggregation chains (collagenase)isinfusedintothemainpancreatic upcollagen whichanenzymebreaks through distension and digestion, a process sists of pancreatic duodenum andanyunderlyingfat.Thenextstepcon consists of in the organspleen, dissection and removal which isthecleaningofpancreas, of theprocess (QC)team.Thefirststage byaqualitycontrol research oftheisolatedisletsfortransplantationor the release ceived andassessedbyanisolationteam,endswith fy the islets that will be used in the transplant, throwing fy theisletsthatwillbeusedintransplant,throwing COBE 2991centrifuge.Thegoalofthisstepistopuri continuous ordiscontinuousdensitygradientsusinga theexocrineandductal tissues through separated from Inthepurificationstep, theisletsare digestion process. thenaddedtothetissuestop tion solutionare The process starts whenthedonatedorgan isre starts The process Dilution =RicordiChamberwithsiliconebeads, pancreatictissue Digested islets =heatingcoilina 50°Cwaterbath. = sampling location for islet collection = samplinglocationforisletcollection Arch Metab. Endocrinol 2015;59/2 Exocrine tissue Purified islets - - - - Arch Metab. Endocrinol 2015;59/2 optionforpatients with treatment safe andeffective to bea islettransplantationshave proved Pancreatic STATUS OFTHISPROCEDUREINBRAZIL ISLET TRANSPLANTATION ANDTHECURRENT tests (16,34). as well as mycoplasma and endotoxin bacterial cultures and anaerobic aerobic The sterility is assessed through low glucose(20).FunctionalisletsmusthaveaSI>1. sulin concentrationoftheisletsamplestimulatedwith the isletsamplestimulatedwithhighglucosebyin ulation index(SI)bydividinginsulinconcentrationof concentrations ofglucoseand,then,calculatingastim after incubation with low and high insulin secreted Islet functionisassessedbymeasuringtheamountof adequate fortransplantationifitsviabilityisover80%. (35).Theisletbatchisconsidered microscope rescent inafluo andexaminingtheresults damaged cellsred), iodide,whichstains andpropidium viable cellsgreen, diacetate, whichstains two typesofdye(fluorescein brane. Thisisassessedbystainingtissuesampleswith bytheintegrityofcellmem cells) isdetermined ber oflivecells/totalnumbercells,includingdead volume of5-10mL(34).Theviabilityislets(num body weight, ina maximum IEQ/kg oftherecipient’s Transplantation isonlypossibleiftheIEQ>5000 bytheestimateddiameterofislets. IEQ isdetermined fortransplant.The with purity>50%canbeapproved Only thesamples stages ofisletisolationandculture. the in theisolatedmaterial)isassessedthroughout and sterility(34). viability,150 μmdiameterorgreater), cellfunctionality tooneisletwith lents (IEQ–oneIEQcorresponds consists ofQCtestsforpurity, numberofisletequiva thelaststepinisolationprocess (33). Therefore, be abletogenerateconsistentlyhigh-qualityproducts to mustbedefined,validated,andproved tion processes able fortransplantation,theisletisolationandpurifica suit islets mustbeassessed.Fortoconsidered T1DM, thesafety, purity, ofthe potencyandefficacy optionfor asatreatment transplantation isapproved days(32). up tothree for cultured (16).Afterisolation,isletsare thrombosis and veinhypertension theriskofportal vein, reducing tissue (10mLmaximum)willbeinfusedintotheportal cells,sothatonlyasmallvolumeof away theremaining Purity (the proportion ofisletstoothertissuetypes Purity (theproportion allogeneicislet The FDAhasstatedthat,before ------in developing methods for isolating the greatest num in developingmethodsforisolatingthegreatest isasignificantinterest insulin independence(37),there rates of to improve donors in order two to three from isletsisolated each transplantpatientgenerallyrequires ofdonororgans (37).Since (34,36) andthescarcity thehighcost the complexityofisletisolationprocesses, islet transplantationinmanyhealthcentersislimitedby “brittle T1DM”.However, theclinicalapplicabilityof established this variable as a potential pancreas alloca established this variableasapotentialpancreas a definingparameterforsuccessful isletisolationand ofthedonorBMIas theimportance (53) reinforced function adequately(50,51). ThestudybySáandcols. number of islets, these islets do not always a greater effective collagenase used in pancreatic digestion(Lib collagenaseused in pancreatic effective asthemost intoisletisolationresearch, was introduced able islets.Inthepast6years,anadditionalobstacle lead tofragmentationanddamageoftheβ, might bythe isletsduringtheisolationprocess fered ence isletquantityandquality(38). and anextensiveknowledgeofthefactorswhichinflu considerable experience with cell isolation procedures However, whichrequires thisisachallengingprocess, ofeachdonor. thepancreas ber ofviableisletsfrom 25 kg/m Although organs donated by individuals with BMI > quality andquantityoftheisolatedislets(30,50,51). tent, durationofcoldischemia)mayalsoinfluencethe characteristics(size,fatcon sor agents)andpancreas body massindex[BMI],causeofdeath,usevasopres with LiberaseMTF. SI as compared seem to be associated withanimproved NB1enzymes viability); however, andServa Vitacyte andquality (purityand islet quantity(IEQ/gpancreas) enzymesleadto similarlevelsof that allaforementioned (49) found group analysis conductedbyour research results (40,42,45-48).A meta- conflicting produced digestion have be themostadequateforpancreatic studies thathavesoughttoassesswhichenzymewould forisletisolation(44).However, alternatives promising cyte (CIzyme HA, Indianapolis, USA) (37,41-43) are Liberase MTF (Roche, Indianapolis, USA) and Vita Heidelberg, Germany), NB1(Serva, such asServa encephalopathy transmission(40).Othercollagenases, riskofspongiform tween theenzymeandanincreased ofanassociationbe the marketduetodiscovery from wasremoved erase HI,RochePharmaceutics) cells (39),influencingthequantityandqualityofavail The physical,chemicalandmechanicalimpactssuf Donor characteristics(age,gender, medicalhistory, 2 and/or age over 50 years tend to produce and/orageover50years tend toproduce Human pancreatic islettransplantation α, δ andPP 165 ------

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 166 isolation wasthe mostexpensivephaseof theprocess, plant itselfanditsfollow-up. Thestudyfoundthatislet untilthetrans the timeoforgan removal tion from study evaluatedthecostofislettransplanta French In 2004, a on this process. lation still place a burden transplant in2005(60). Foundation,whichconductedone and thePro-Kidney (Paraná), associated with the PUC-Paraná University inCuritiba transplant in2002(59);andalaboratory itsfirst University ofSãoPaulo(USP)andperformed lar Therapy)inSãoPaulo,whichisassociatedwiththe transplants: theNUCEL(NucleusofCellandMolecu outislet which,inthepast,hadalsocarried research, twolaboratoriesinvolvedinisletisolation are there of organs forislettransplantationinBrazil.InBrazil, islet isolationpurposes,indicatinganadequatesupply organs couldhavebeenusedfor (58). Theremaining usedinthefirstsemester of2013 ed (n=1273)were donat 6.3% (n=80)ofthetotalnumberpancreases Brazilian Organ Transplant Association notedthatonly issuedbythe city ofBDdonors(8).InBrazil,areport bymostislettransplantationcentersisascar ported causes(51). died ofheart-related tothose whose donors of lowerqualitywhencompared islets also foundthattheorgans ofBDdonorsproduce studyhas TNF levelsintheorgan (57).Anotherrecent in isolation,asevidencedby an increase long before appears tobedamaged case ofBDdonors,thepancreas foundthat,inthe group conducted byourresearch also influence the quality of the donated organ. A study longed coldischemia(56). incasesofpro mostpronounced were these results (54,55),although preservation pancreatic to improved method)led (two-layerperfluorocarbon ygen affinity use ofUWsolutionandothersubstanceswithhighox However, somestudieshavefoundthatthecombined other substancesthatcontributetocellintegrity(30). and solution,whichcontainselectrolytes of Wisconsin) involvetheuseofUW(University tion procedures (30).Mostisola ontransplant results effect important ofhypoxiamayhavean thedeleteriouseffects crease andde thepancreas type ofsolutionusedtopreserve the tissue.Therefore, time maydamagethepancreatic coldischemia sinceincreased the isletisolationprocess, to zil. The duration of cold ischemia is also relevant tion criterion for islettransplantationinBra pancreatic Human pancreatic islettransplantation Nevertheless, thehighcostsinvolvedinisletiso Nevertheless, cited,oneofthemainlimitations re As previously may ofbraindeath(BD)onthepancreas The effect ------to rise even further (61). to riseevenfurther mayleadcosts isolationreagents onimported reliance the inBrazil, where similar situationhasbeenreported accounting for30%oftotaltransplantcosts(36).A (“Good Practice in Drug Manufacturing”, August4 (“Good PracticeinDrug mustfollowRDCResolutions Numbers210 process the isolationlaboratoriesaswellislet transplantation. However, of boththeconstruction regulate islet is still no specific legislationto zil, there Act(62).InBra FDA andthePublicHealthService issuedbythe regulations andbiologicalproduct drug must follow the In the USA, the islet isolation process BRAZILIAN LEGISLATION (37,57,60). lated islets and insulin independenceinthelong term ofthequantityandqualityiso interms tive results posi likelytoproduce more for transplantationandare adequate the selectionoforgans thatmightbemore ent methodsforisletisolationand,whenpossible, tion centers have focused on the development of differ and kidneytransplantations (66). islet simultaneous 3) with end-stage kidney disease; and transplantation followingkidney transplants in patients patients;2)islet 1) islettransplantationinnon-uremic waysdependingonpatientcharacteristics: one ofthree in allogeneic islettransplantationtendtobeperformed symptoms(6,65).Inthesepatients, without adrenergic hypoglycemia ofsevere of lifeand/ortheoccurrence inquality which mightleadtosignificantimpairment glycemia,typicallyhigherthan200mg/dL, in capillary ofsignificantdailyvariations is definedbythepresence ment optionforpatientswith“brittleT1DM”,which asatreat Islet transplantationisgenerallyrecommended “BRITTLE T1DM” ISLET TRANSPLANTATION INPATIENTS WITH group. would be classified into the latter tation procedures treatment”, March 14 March treatment”, Technology and Centersinvolvedinclinicalresearch 2003) and9(“GuidelinesforthefunctioningofCell use andallogeneiccellmanipulation.Islettransplan totheirpurpose:autologous two categoriesaccording number9dividescelltherapy methodsinto resolution Sanitária(Anvisa)(63,64).RDC Nacional deVigilância In lightofthesefacts,anumberislettransplanta th , 2011)issuedbytheAgência Arch Metab. Endocrinol 2015;59/2 th ------, protocols havehadthefollowingcharacteristics:agebe protocols Arch Metab. Endocrinol 2015;59/2 willbegina study ofhuman islet transplantation future, conducted 28humanislet isolations and,inthenear tion inpatientswith“brittle T1DM”.Thiscenterhas islettransplanta and3)performing logical features; physiologicalandpatho cells inpatientswithdifferent and quantityofisolatedislets; 2)studyingfunctionalβ the quality mental studiestoassesswaysofincreasing the goalofisolatingisletsfor:1)developingexperi established attheEndocrineDivisionofHCPA with forhumanisletisolationwas In 2010,anewlaboratory LABORATORY INTHEHCPA DEVELOPMENT OFAHUMANISLETISOLATION damage(70). environmental the isletsfrom whilesparing lation, whichallowsforrevascularization engraftment oftheislets.Oneexampleisencapsu abetter an attempttodeveloptechniquesthatensure other infusionsitesandmethodshavebeenstudiedin andisletviability (71). Currently,on insulin secretion drugs ofimmunosuppressive and thedamagingeffect transplant, giventheuniquecharacteristicsofliver intheperiodimmediatelyfollowing destroyed are cell infusion(70).Itisestimatedthat50-70%ofislets vein,whichisnotthemostadequateplacefor portal isthe infusion usedbymoststudiesintheliterature (68,69). riskofearlygraft loss atgreater transplant periodare levelsinthepre- andhigherlipidserum sulin resistance suggestiveofin higher HbA1clevels,clinicalprofiles influence transplantoutcome. Younger patientswith (67).However,failure patientcharacteristicsmayalso comes and may bea key contributing factor to graft disease,BMI>26kg/m arterial untreated mune Tolerance of Networkstudy(22),thepresence complications associatedwithdiabetes(30).IntheIm ofchronic hypoglycemiaandprogression recurrent vere ofse detectable C-peptideconcentrations,occurrence tween 18and65years,T1DMforover5yearswithun has been found to be correlated withworseclinicalout has beenfoundtobecorrelated ofanti-isletantibo the persistenceorreappearance exclusioncriteriafortransplantations. considered were ofinfectionsorpsychiatricdiseases well asthepresence > 1.5mg/dLand/oralbuminuria300mg/24h,as for insulindoses>0.7UI/kg,HbA1c12%,creatinine To date,patientsincludedinislettransplantation It is also important tonotethatthesiteofislet It isalsoimportant In islet transplantations, as in pancreas transplants, In islettransplantations,asinpancreas 2 , the need , theneed dies ------63.6 m transplantation. undergone kidney in T1DMpatientswhohadalready contains a dressing room for changing intosterilegar room contains adressing der ofthelabiskeptatClass10.000.Thelaboratory perm orgreater 0.5 microns withasizeof Class 100.000(uptoparticles is kept at cleanliness room The dressing rooms. gery forsur thesameasthoserecommended are laboratory attheisletisolation accesspoliciesineffect restricted air(HEPA) particulated filters,and with highefficiency Institute, UniversityofMiami(EUA). the islettransplantationteamatDiabetesResearch by provided Anvisa (63),aswellrecommendations (numbers134and210)issuedby in RDCregulations followedtheguidelinesestablished process struction wasbuiltbetween2008and2010,itscon oratory project entitled “Establishment of a Human Pancreatic entitled“Establishment ofaHumanPancreatic project agencies. Aresearch support national research ships from and apost-doctoralfellow, scholar allofwhomreceived assistant masters’ andPhDstudents, a technicalsupport islet isolation team isassisted by undergraduate students, ty of Miami (Florida, EUA) for a period of one year. The InstituteoftheUniversi tients attheDiabetesResearch oftransplantedpa as wellmonitoringandtreatment docrinologist, whowastrainedinhumanisletisolation Belgium) foroneyear. Theteamalsoincludesanen (Brussels, University of Brussels Medicine of the Free β while thebiologistwastrainedinratisletisolationand versity ofMinnesota,Minneapolis,EUA)fortwoyears, transplantation at the Schulze Diabetes Institute (Uni of human, pig and mouse islets as well as in human islet heldhertraining inisolation centers. Thenephrologist re training in international ployees who received HCPA andabiologist,bothofwhom are phrologist em reader.immunosorbent assay)micro-plate and an ELISA (enzyme-linked microscope fluorescent the QCcenterforisolatedislets,whichcontainsa ology Laboratory, and sterilizationroom, aglassware by a Cell and Molecular Bi is surrounded laboratory contamination.The to facilitatecleaningandprevent ismadeofstainlesssteel inthelaboratory All furniture exitchamber. acoldchamberandlaboratory room, storage areagent tion forisletisolationandculture, loca aseparateinternal area, ments, ahand-rubbing cell functionality at the Laboratory of Experimental ofExperimental cellfunctionalityattheLaboratory This islet isolation laboratory takes up an area of takesupanarea This isletisolationlaboratory Positive internal pressure, use of air-conditioning pressure, Positive internal The core oftheisletisolation team consists of a ne The core 2 in the Endocrine Division (Figure 3).Thelab intheEndocrineDivision(Figure Human pancreatic islettransplantation 3 ofair),whiletheremain ference ference 167 ------

Copyright© AE&M all rights reserved. Copyright© AE&M all rights reserved. 168 glass window; 10= Window forexiting of materials;11 = Window forenteringofmaterials. cabinets; Figure 3. ofHumanPancreaticIsletBiology–EndocrineDivisionHospital deClínicasPorto FloorplanoftheLaboratory Alegre. 1=Biologicalsafety notusedforwhole-pancreas donated organs whichare Center ofthestateRioGrandedoSul,Brazil.Any HCPA number08-131)andbytheTransplant (project Ethics Committee of by the Research ect was approved andorganizing anorgan donationsystem.Thisproj ry thelaborato specifically forthepurposeofconstructing attheHCPA” wasdeveloped Islet IsolationLaboratory Human pancreatic islettransplantation 2 =Incubators;3Freezer-86°C; 4 =Refrigerator;5Freezer-20°C;6 Water bath;7=Microscope; 8 =COBE2991;9Doorwith Antechamber 80 x 210 room Changing 80 x 210 98 x 110 x 100 10 9 7 4 80 x210 5 9 room Rubbing 9

120 x 210 1 2 2 6 Main area - - 80 x 210 3 of Human Pancreatic Islet Biology.of Human Pancreatic The surgical team re bytheLaboratory provided consentform an informed for isletisolationpurposesoncethedonor’sfamilysigns used transplantationare transplants orkidney-pancreas of Health–RioGrandedoSul. Organizations) oftheDepartment (Organ Procurement isassociatedwiththeOPOS sponsible fororgan removal Store room 80 x110100 90 x210 11 1 Arch Metab. Endocrinol 2015;59/2 Cold room 1 8 - Arch Metab. Endocrinol 2015;59/2 7. 6. 5. 4. 3. 2. 1. REFERENCES was reported. relevant tothisarticle nopotentialconflictofinterest Disclosure: ofCNPq-PQscholarships. recipients soal deNívelSuperior(Capes).MJB,SPS,JLG,CBLandDCare (HCPA)dePes Alegre deAperfeiçoamento andCoordenação à PesquisaeEventos(Fipe)attheHospitaldeClínicasPorto mento Científicoe Tecnológico (CNPq),theFundodeIncentivo Grande doSul(Fapergs), theConselhoNacionaldeDesenvolvi àPesquisadoEstadoRio theFundaçãodeAmparo from bygrants supported Acknowlegments: Thisstudywaspartially inBrazil. for islettransplantationandβcellresearch center becomeareference poses and,inthenearfuture, HCPA pur humanisletsforresearch aims toprovide IsletBiologyattheEndocrine Division ofthe creatic ofHumanPan financial investment.TheLaboratory significant whichrequires slow andcomplexprocess, clinicalpractice. ofroutine part islet transplantation will be eventually implemented as that to ensure as post-transplant follow-up procedures, islet isolationandtransplantationtechniques,aswell of the effectiveness increasing must be made toward efforts initiatives.Therefore, of experimentalresearch outonlyaspart isstillcarried Brazil, thisprocedure forT1DM.However,treatment incountriessuchas used as many countries, islet transplantation is already inpatientswith“brittleT1DM”.In glycemic control hypoglycemic episodes and improving of severe rence theoccur optionfordecreasing a usefultreatment islet transplantationshould be considered Pancreatic CONCLUSIONS The establishmentofisletisolationlaboratoriesisa Hepato-Biliary-Pan. 2009;16(2):101-12. 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