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Diabetes & Metabolism
Vol 24, N° 3  - juin 1998
p. 200
Doi : DM-06-1998-24-3-1262-3636-101019-ART50
ISOLATION, CULTURE AND FUNCTIONAL EVALUATION OF ISLETS OF LANGERHANS
 

N.J.M. LONDON, S.M. SWIFT, H.A. CLAYTON
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Isolement, culture et évaluation fonctionnelle des îlots de Langerhans.

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Abstract


Mots clés : transplantation, îlot, homme, porc, isolement, collagénase,purification, culture, viabilité


Review

The collagenase digestion phase of islet isolation is variable and unpredictable. Thisresults partly from the vagaries of collagenase itself but also from the complex effectsof organ retreival and storage on collagenase digestion. Improvements in the purificationof islets by density gradient centrifugation will not result from the production of newdensity gradient media, but rather from the continued modification of the biochemicalcomposition of the solvents in which established gradient media are dissolved. Thechallenge is to produce solutions that will minimise acinar tissue swelling withoutcompromising islet yield and viability. It is sobering to note the low recovery of isletsafter 48-h tissue culture (56 %), and it has to be concluded that present tissueculture techniques for human and porcine islets are inadequate. Part of the problem isthat the media and techniques used for islet tissue culture were designed for single cellculture and were not formulated specifically for the culture of insulin-producingmini-organs with a high metabolic rate. Cell viability is notoriously difficult toquantify, and this is no less true for isolated islets than for other tissues. The mainproblem is that although it is possible to determine the viability of isolated islets bymeasuring specific cellular functions such as glucose-stimulated insulin release, it isimpossible to compare this with the same function performed by islets within the nativepancreas. For this reason, it is not possible in absolute terms to determine the effect ofislet isolation on islet viability.

Key-words : islet transplantation, human, porcine, isolation, collagenase,purification, tissue culture, viability.

L'étape de digestion par la collagénase de l'isolement d'îlots est variable etimprévisible, du fait d'une part de la variabilité de la collagénase mais aussi deseffets complexes du prélèvement et du stockage du pancréas sur la digestion par lacollagénase. L'amélioration de la purification des îlots par la centrifugation surgradient ne résultera pas de la mise au point de nouveaux milieux pour le gradient dedensité, mais plutôt de modifications de la composition biochimique des solvants danslesquels sont dissous les milieux de gradient. Il conviendrait de produire des solutionsqui minimiseront la désintégration du tissus exocrine sans compromettre le rendement etla viabilité des îlots. En constatant la faible récupération d'îlots après48 heures de culture (56 %), on peut en conclure que les techniques de cultureactuelles des îlots humains et porcins sont imparfaites. Cela résulte en partie du faitque ces techniques actuelles ont été conçues pour des cultures de cellules isolées etpas pour celles de mini-organes insulinosecréteurs dont le métabolisme est élevé. Laviabilité cellulaire est notoirement difficile à quantifier aussi bien pour des îlotsisolés que pour d'autres tissus. Le principal problème est que, bien qu'il soit possiblede mesurer la fonction insulaire par l'insulinosecrétion stimulée par le glucose, il estimpossible de la comparer à la fonction des îlots au sein du pancréas entier. Pourcette raison, il n'est pas possible de déterminer, en valeur absolue, l'effet del'isolement d'îlots sur la viabilité insulaire.

mots-clés :

N.J.M. London, Department of Surgery, Clinical Sciences Building,Leicester Royal Infirmary, Leicester LE2 7LX, UK. Tel : 01162 523252. Fax :01162 523179.

lthough there is a wealth of experimental data concerning the isolation, culture andfunctional evaluation of rodent and small animal islets of Langerhans, much of thisinformation has not proven directly applicable to the large animal and human situation.This review will therefore focus on the problems posed by human and pig islet isolation,the former because human islet isolation is already a clinical reality, the latter becausepig islets are likliest to be used first for clinical xenotransplantation.

ISLET ISOLATION

Islet isolation is critically affected by all stages that precede it, and this isparticularly true for the human pancreas. Warm ischaemia is especially [1] damaging andshould be avoided at all costs ; similarly, cold [2] ischaemia should be minimised.

Insitu

vascular perfusion is now an integral part of the organ donation operation inmany countries ; and, although vascular perfusion impairs porcine islet isolation[3], a prospective randomised study from Edmonton has shown for human islet isolation thatvascular perfusion with University of Wisconsin (UW) solution does not reducepost-purification islet yields as compared to no perfusion at all [4]. The question ofwhich vascular perfusate to use for the human pancreas has not been the subject ofprospective studies. However, EuroCollins solution appears to be detrimental to humanislet isolation [5] and is certainly not an effective vascular flush solution if coldischaemic time is greater than 6 h [6]. Although UW solution appears to providereasonable pancreas preservation for up to 18 h [7] prior to human islet isolation(based on International Islet Transplant Registry data [8]), it would seem inadvisable totransplant islets isolated from UW-perfused human pancreata with a cold ischaemic time ofmore than 12 h.

Intraductal distention with collagenase suspended in UW solution immediately afterpancreas excision allows effective storage of the pig [9] pancreas for up to 24 hprior to islet isolation. Unfortunately, this approach has provided very poor islet yieldsand viability in the human for both stored and non-stored pancreata [10], probably becauseUW solution contains inhibitors of collagenase [11] and is toxic to human tissues at37Ý C [12]. The challenge in the human therefore is to develop UW-based cold storagesolutions that optimise preservation when given intraductally but do not compromise isletyield or viability. It is convenient to subdivide the process of islet isolation intopancreas dissociation and islet purification.

Pancreas dissociation -

Current methods for human and pigpancreas dissociation are based on the principles described by Gray

et al.

[13] in1984. The technique consisted of the intraductal administration of collagenase [14],followed by incubation at 39Ý C, with gentle teasing and shaking, and then triturationthrough wide-bore needles. Although improvements in this manual method have been described[15], the description of an automated method by Ricordi

et al.

[16] is generallyagreed to represent a significant advance. Indeed, porcine studies [17] that have comparedmanual with automated methods have shown significantly increased islet yields (three timesas many IEQ) with the automated method. The principles behind the automated method areminimal physical trauma and continuous collagenase digestion of the pancreas during whichislets are continually removed from the injurious action of collagenase [18].

Regardless of the technique used, the collagenase digestion phase is criticallydependent upon the efficiency of the collagenase. A good enzyme will release intact isletsthat are entirely free of exocrine tissue (cleaved). Unfortunately, batch-to-batchvariation and deterioration of a good batch with storage are a common experience. Indeed,the vicissitudes of collagenase [19-21] have driven many investigators to despair, and fewwould disagree with Scharp [22] that collagenase is one of the major obstacles tosuccessful human islet transplantation. Crude collagenase is derived from cultures of

Clostridiumhistolyticum

and may contain up to 11 different collagenases, each with differentspecificities towards different collagens [23]. In addition, crude collagenase containsnumerous other enzymes [21]. Highly purified collagenase is ineffective for isletisolation [24], and some of the other components of crude collagenase are thereforenecessary. Recent rat studies have investigated the role of the various types ofcollagenase present in crude collagenase on islet isolation and have shown that pancreasdissociation can be manipulated by using defined mixtures of collagenases. These studiesneed to be repeated in the human because it has been shown that there is a marked speciesvariation in both total pancreatic collagen content [25] and collagen type anddistribution [26]. Thus, unfortunately, a collagenase batch that works well in the pig ordog may not work in the human. The recent development of a collagenase blend called"Liberase" may represent an advance [27] ; prospective randomised trialsare however still awaited.

The efficiency of the collagenase digestion phase may be affected by a number offactors in addition to the collagenase batch. The pancreas should be uniformly distendedbecause islets are not isolated from undistended segments [28]. The question as to whetherthe collagenase solution should be perfused or loaded into the pancreatic duct has beeninvestigated by Warnock

et al.

[6] who showed, although not conclusively, that theductal perfusion technique improved islet yields. A further issue is the optimum timing ofcollagenase injection. There is inevitably a period of cold storage between pancreasexcision and the commencement of islet isolation. Ohzato

et al.

[29] have shown inthe rat that cold storage leads to loss of ductal integrity, so that during ductaldistention the pressures achieved were lower, pancreas distention was suboptimal, andcollagenase was more likely to enter the islets themselves. It was also shown that theyields of rat islets were increased if collagenase was given intraductally in Hanks'balanced salt solution (HBSS) at the time of pancreas excision prior to cold storage for4 or 6 h. It has subsequently been shown that for young human organ donors(< 30 yr) intraductal injection of collagenase in HBSS prior to, rather thanafter, 3-h cold ischaemia, significantly improves islet yields [30]. It has however beenshown for donors of all ages that immediate intraductal injection of collagenase improvesislet survival in low temperature culture.

It can be deduced from the foregoing discussion that the collagenase digestion phase ofhuman islet isolation remains variable and unpredictable. Indeed, it has been shown thatthe proportion of pancreatic insulin recovered in the pancreatic digest after collagenasedispersion of the human pancreas ranges from 7 to 91 % [15]. Much of thisvariability relates to the effects of cold storage and the vagaries of collagenase itself,and these issues must be considered as research priorities.

Purification -

Although a large number of islet purificationtechniques, including differential sedimentation at unit gravity [31], filtration [32],the use of Velcro to remove acinar tissue [33], centrifugal elutriation [34],cryopreservation [35], gamma irradiation [36], anti-acinar cytotoxic antibodies [37],tissue culture [38], the selective destruction of acinar cells by laser energy [39] orhypotonic lysis [40], magnetic microspheres coated with anti-acinar cell monoclonalantibodies [41], and fluorescence-activated cell sorting [42] have been used in otherspecies, density gradient centrifugation is the only technique that has been successfullyused for large-scale porcine and human islet purification. The technical aspects of thediscussion will therefore focus on density gradient purification. Human islet purificationhas proven particularly difficult because both the density and the diameter of acinartissue, and to a lesser extent of islets, changes from one preparation to another [43].Islets normally vary in diameter from 15-500 µm and, in addition, the diameter ofislets and acinar tissue is critically dependent upon the collagenase digestion stage ofthe isolation process. This intrinsic variation in islet diameter, combined with theeffect of collagenase digestion, means that we have no control over endocrine and acinartissue size distribution. Because of these highly variable and overlapping tissuediameters, it is not possible to purify islets from acinar tissue using velocitysedimentation, and we are forced to use isopycnic centrifugation.

There are a number of possible causes for variable acinar tissue density. First, it ispossible that acinar tissue density depends on the secretory status of the acinar cells.Second, the density of acinar cells may be affected by the size of the aggregates formed[44] by the collagenase digestion of the pancreas, and finally, acinar tissue density maybe reduced by cellular swelling and oedema. Recent studies [45] have shown that the singlemost important factor affecting the density of acinar tissue during islet isolation isacinar tissue swelling and oedema, acinar tissue degranulation being relativelyunimportant. Acinar cell swelling can be provoked by a number of insults, includingmechanical trauma [46] and hypothermia, and collagenase digestion has been shown toinfluence cell membrane permeability and to cause cell swelling [47].

Broadly speaking, techniques for optimising density gradient purification can bedivided into physical and chemical ones. We will first consider physical parameters. Forcell separation to be optimised, it is essential that certain experimental conditions becontrolled. Temperature may affect the results of density gradient purification [48], andit has been shown that the results of human islet purification using BSA are the same at 4ÝC and 22 ÝC, whereas porcine islet purification is more efficient at 4 ÝC [49].Continuous gradients offer many theoretical advantages over discontinuous gradients [50],and it has been demonstrated that large-scale continuous density gradients can beestablished on the COBE 2991 processor [51, 52]. This therefore is the preferredtechnique for large-scale human islet purification. It has been shown that, compared todiscontinuous gradients on the COBE 2991, continuous gradients improve human islet yieldby 26 % and also improve islet viability.

Chemical methods for improving the purification of human islets by isopycniccentrifugation aim to increase or maintain acinar tissue density whilst leaving isletdensity relatively unaltered. One approach has been described by van der Burg

et al.

[53] who greatly improved the results of canine islet purification by collecting andwashing the pancreatic digest in UW solution prior to density gradient centrifugation.These findings have been confirmed in the human [54], and for some isolations the resultsof density gradient purification are further improved by storing the digest in UW solution(at 4 ÝC) for 1 h prior to centrifugation. The beneficial action of UW storageresults largely from the presence of the extracellular impermeant anion lactobionate [45]and the colloid hydroxyethyl starch [55].

The most commonly used gradient media for human islet isolation are Ficoll [6],Euroficoll [56], Ficoll-Diatrizoic acid [1] and hyperosmolar bovine serum albumin (BSA)[57, 58]. Further improvements in the purification of islets by density gradientcentrifugation will not result from the production of new density gradient media, butrather from the continued modification of the biochemical composition of the solvents inwhich established gradient media are dissolved. These modifications may however produce anunphysiological environment [59], and consequently their effect on islet viability must bedetermined. In the human, the challenge is to produce solutions that will minimise acinartissue swelling without compromising islet yield and viability. It has been shown thathyperosmolar BSA density gradients improve human [43] (500 mOsm/kg/H

2

O)and porcine [49] (400 mOsm/kg/H

2

O) islet purification. This phenomenon hasbeen reported for other cell types [60] and in the case of islet purification results fromthe hypertonic environment

differentially

increasing the density of acinar tissuemore than endocrine tissue [61]. A further promising approach to minimise acinar tissueswelling is the construction of density gradients by the addition of extra hydroxyethylstarch or Percoll to University of Wisconsin solution [62].

ISLET CULTURE

The culture of islets poses several unique problems. First, due to the size of theislets, the central core can become necrotic, probably as a result of inadequate oxygensupply. Second, unlike many single cell types, islets do not proliferate in culture, andthis makes the assessment and comparison of different culture techniques difficult.Usually, the proliferation rate of a cell line is the determinant of the effect of achange in the culture medium composition. Finally, islets from different species may haveunique requirements or behaviour in tissue culture. For example, pig islets tend tofragment very easily in culture, probably due to the lack of a defined capsule [63].

It is pertinent to begin by asking "is it necessary to culture islets ?"First, there is the need to store islets prior to transplantation. Second, a period of lowtemperature may be used to immunomodulate islets to abrogate rejection of the transplantedtissue. Third, Weber

et al.

[64] have demonstrated that a period in culture can beused to purify islet preparations. Although there are many positive reasons for culturingislets, the potential risk of infection should also be considered. One recent report hashighlighted the problem of infected islets leading to episodes of bacteremia in islettransplant patients [65]. Although the source of this infection was determined to becryopreserved islets, it is apparent that any increase in the number of proceduresundertaken on islets will increase the risk of infection occurring. Other reportsassessing the sterility of islet preparations or the incidence of infection in patientsfollowing islet transplantation have tended to demonstrate factors other than the cultureof islets as having the greatest risk. For example, Scharp

et al.

[66] found thatthe greatest infection source was from the pancreas transport fluid, whilst the Ficolldensity gradient medium was the major source of contamination introduced duringprocessing.

Commercially available media -

One of the earliest studiescomparing the culture of islets in different commercially available media was undertakenusing rat islets in 1978 by Andersson [67]. The study compared the effectiveness of TCM199, RPMI 1640, CMRL 1066, MEM and Ham's F10. The results demonstrated that the islets inF10 had the highest insulin content, but that the highest insulin biosynthesis ratewas in the islets cultured in RPMI. Andersson therefore concluded that RPMI was the mediumof choice for islets, possibly due to the nicotinamide content and the 11mM glucoseconcentration. Davalli

et al.

[68] reported the results of culturing porcine isletsin CMRL 1066, RPMI 1640 without glucose, RPMI 1640 with 11mM glucose, Ham'sF12 or TCM 199. All the media were supplemented with 10 % FCS, and the isletswere tested by insulin release. Only TCM 199 gave an increase in glucose-stimulatedinsulin release, whilst islets cultured in either CMRL or RPMI (both with or withoutglucose) showed a decrease in release. The authors commented that TCM 199 is the onlyone of these media which contains adenosine phosphates and xanthine, and that these couldtherefore be of importance in islet culture. They also suggested that the relatively highglucose concentrations of Ham's F12 (9.9mM) and RPMI could be toxic to the islets.

The results of the above studies suggest that islets isolated from different speciesmay have different requirements in culture. This is supported by the results of Holmes

etal.

[69] who cultured rat, porcine and human islets for a week in ten differentcommercially available media, providing a more comprehensive comparison than hadpreviously been undertaken. They concluded that Iscove's MEM was the best medium for ratislets, Ham's F12 nutrient mixture was best for porcine islets, and CMRL1066 was best for human islets. These results do not concur with the above studies,although this could be due to Iscove's medium not having been previously tested. Moreover,the length of time the islets were in culture was greater than that used for porcineislets by Davalli

et al.

[68] (48 h compared with a week), which could haveaffected the results. A recent report from Rastellini

et al.

[70] examined theeffect of adding pyruvate (7mM) to CMRL-1066 on the viability of cultured humanislets. After 60 days in culture, 67 % of pyruvate-cultured islets could berecovered compared to 0 % in the control group. This promising approach certainlymerits further investigation.

Serum-free media -

Foetal calf serum (FCS) or newborn calfserum (NBCS) is routinely added to culture medium for a wide range of cells and tissues.The reason for this is because the serum contains many components which are beneficial forcell survival and growth. Unfortunately, however, there can be a considerable variationbetween batches of serum, some of which may even be toxic to islets. Much of the workundertaken on the effect of serum on islet culture has assessed whether it is possible forislets to survive without the presence of serum if other constituents are added to themedium. A major reason for trying to omit serum from islet culture medium is to move awayfrom the use of animal products when tissue is to be used for human transplantation. Tai

etal.

[71] undertook a study on porcine islets, assessing the use of serum replacementproducts. They found that they were not suitable for long-term culture and suggested thatthis could partially be due to variation between batches of these products. These studiesdemonstrate that more developmental work is required before islets can be routinelycultured in serum-free medium.

Oxygenation -

Dionne

et al.

[72] assessed the effect ofoxygen concentrations on insulin release from canine islets, comparing normoxia(142 mmHg) with a range of hypoxic conditions (minimum of 5 mmHg). They foundthat the second phase of insulin release was decreased due to hypoxia. The authorscommented that this was thought to be due to pO

2

gradients outside and insidethe islets. This results in the islets being exposed to low pO

2

decreasingradially from the periphery to the core, possibly leading to depletion of the energystores from the beta cells. It has been shown that liver cells are in oxygen debt within afew hours of establishing a primary culture if the medium is 3 mm deep. It isprobable that such an effect would occur with islets under similar circumstances, and thishighlights the speed at which less than optimal culture conditions can affect theviability of the tissue. It is clear, therefore, that methods of improving oxygen supplyto the islets in culture, and diffusion into the centre of the islets, are required. Onefactor, already highlighted above, is the depth of the medium. This should be kept to aminimum to allow rapid diffusion of oxygen to the islets, and a depth of 2 mm hasbeen recommended for cells with a high oxygen requirement.

Alternative methods may include the addition of oxygen carriers to the medium,constantly bubbling oxygen through the medium, a rolling culture system, or a stirredculture system in which the islets are maintained in suspension by a magnetic stirring barsuspended from the top of the bottle. The potential problem with any system designed tokeep the islets constantly moving is the fragility of the islets, especially porcineislets. Tests would be required to determine whether this type of culture system issuitable for islets, and whether it can be applied to all species of islets.

The role of exocrine tissue -

A study undertaken by Metrakos

etal.

[73] assessed the effect of interactions between different cell types on thesurvival of islets

in vitro

. After preparing ductal tissue from the pancreas andsetting up cultures of the islets together with the ducts, they found an increase inproliferation of beta cells in the presence of the ducts compared with the culture ofislets on their own. They concluded that there is an interaction between the ducts and theislets, and that this leads to an increase in proliferation, possibly as a result of therelease of factors from the ductal tissue.

Culture temperature -

Culture temperature has been investigatedwith respect to its effect both on islet immunomodulation and recovery. In 1991, Lacy andFinke [74] reported their findings on the effect of culturing rat islets at 24 ÝC ratherthan at 37 ÝC which had been the standard culture temperature. They found that at thelower temperature they achieved almost complete removal of the intraislet lymphoid cells,as long as CMRL 1066 was used as the culture medium. When RPMI 1640 was used,there was a considerable reduction in the number of lymphoid cells remaining, althoughthere were still residual cells detectable. Interestingly, RPMI was originally developedfor the culture of lymphocytes [75], whereas CMRL 1066 was originally developed forthe culture of fibroblasts and kidney epithelial cells [75]. Recently, Dabbs

et al.

[76] have shown that 7-day low-temperature (24 ÝC) culture can slightly prolong isletallograft survival in CsA-treated dogs.

Lakey

et al.

[77] assessed human islet recovery after 24-h culture as part of astudy on cryopreserved islets. They reported a mean recovery rate of 72.5 % forislets cultured at 22 ÝC compared with 54.8 % at 37 ÝC. This study indicates aconsiderable benefit for islets cultured at 22 ÝC as compared to 37 ÝC.

Culture matrix -

One factor which also needs consideration iswhether islets should be cultured in a three-dimensional matrix or be maintained as afree-floating culture. Ohgawara

et al.

[78] cultured foetal porcine pancreas in acollagen gel matrix. They found that nicotinamide had to be added to the culture toprevent the degradation of the collagen gel. However, the authors noted that thenicotinamide had the added benefit of preventing the growth of fibroblast like cells.Brendel

et al.

[79] compared the culture of human islets in standard suspensionculture and in matrices of either agarose or alginate. They found that the number ofislets surviving over the period of study was greater when the matrices were used, andthat agarose provided better results compared with alginate. The insulin release resultswere also better in agarose, although the ratio of total insulin release to total DNA wasnot significantly different between the groups. All the islets had good perifusionresponses, but the best transplantation results were obtained with the islets which hadbeen cultured in the agarose matrix. The authors commented the ease with which the isletscould be retrieved from the gel when they were required for transplantation, a problemwhich can result in the loss of a lot of islets when other matrices are used.The two majorproblems associated with the culture of islets in three-dimensional matrices are the lossof islet tissue when they are retrieved from the matrix, and the diffusion of nutrientsand waste products across the matrix. The latter study suggests that these problems mayhave been overcome. If this is the case, this system of culture may be of use for routineculture of islets. However, before this can happen, the other aspects of the isletphysiology and viability require study, for example, the supply of oxygen to the islet andthe problems of central necrosis.

Although they did not use matrices for islet culture, Hober

et al.

[80] assessedthe use of wellplates with inserts for the culture of islets. They found that the insertsdid not allow cell attachment and therefore prevented fibroblast growth. The isletsmaintained good three-dimensional form and good viability. The authors also listed severaladvantages of this system : the endocrine tissue was of high purity ; it waseasier to change the medium ; they could perform tests by moving just the inserts,preventing the loss of any islets ; and the collection of islets was improved withoutcausing them any damage.

Conclusions -

The media currently used for islet culture wereoriginally designed for proliferating cell lines and were not specifically designed forthe culture of highly specialised metabolically active "mini-organelles". Theoptimum conditions for islet culture need to be determined, both in terms of the nutrientsutilised by the cells and the ability to ensure their supply to core cells at the centreof the islets. Furthermore, the results of published studies suggest that the culturerequirements of islets may be species-specific, and new media must be developed with thisin mind. In conclusion, at the present time islets are cultured in ‘adopted'media, and there is clearly an urgent need to develop specific culture media for islets ofLangerhans. Only then will islet culture be by design rather than by proxy !

ISLET VIABILITY

Islet viability may be adversely affected by innumerable factors including eventsoccurring before and during organ donation, during the period of cold ischaemia prior toislet isolation, the isolation process itself, and the period of storage (tissue cultureand/or cryopreservation) prior to transplantation. After transplantation, islet viabilitymay suffer due to impaired revascularisation or the action of islet-toxic substances. Allof these factors will of course increase the number of islets required to producenormoglycaemia. The impact of impaired islet viability on the outcome of human islettransplantation can be deduced from the observation that in the period 1990-1992 no isletpreparations prepared from pancreata with a cold ischaemic time greater than 12 hproduced insulin independence [8].

Unfortunately, cell viability is notoriously difficult to quantify [81], and this is noless true for isolated islets than for other tissues. The main problems are that, althoughit is possible to determine the viability of isolated islets by measuring specificcellular functions (e.g. glucose-stimulated insulin release), it is impossible to comparethis with the same function performed by islets within the native pancreas, so thatinsulin release reflects the function of only a single metabolic pathway within islets andchanges in insulin secretion in response to alterations in glucose concentration may notreflect changes in global islet function or viability. For these reasons, it is notpossible in absolute terms to determine the effect of islet isolation on islet viability.It is possible however to estimate the effect of various procedures (e.g. differentdensity gradient media or cryopreservation) on the viability of islets after isolation.The

in vitro

methods available include light [82] and electron microscopicmorphology [83], fluorometric membrane integrity assays [84-87], colorimetric tests ofmitochondrial function [88, 89] and glucose-stimulated insulin release [90, 91]. Theresults of islet perifusions should be interpreted with caution. Thus, cryopreservedcanine islets that failed to secrete insulin during perifusion consistently inducednormoglycaemia after autotransplantation [92], and it has been reported that a poorresponse from human islets during perifusion did not predict their

in vivo

functionafter transplantation [93]. Undoubtedly, the best index of viability is the ability oftransplanted islets to reverse diabetes [94], which in the case of human islets can beachieved by transplantation into the diabetic immunodeficient mouse [95, 96] or rat [97].The problems with these latter methods are the difficulties of maintaining immunodeficientrodents and that, although reversal of diabetes shows that the islets are viable, it doesnot show

how

viable.

In summary, although islet viability is a critical factor that determines the outcomeafter transplantation, there is no currently available method for standardising viabilityassessment. It would however seem sensible to at least confirm that human islets are notdead prior to transplantation, and membrane integrity assays or perifusions are the mostconvenient way of doing this at the present time.

© Masson, Paris, 1998 Brandhorst H, Klitscher D, Hering BJ, Federlin K, Bretzel RG. Influence of organprocurement on human islet isolation. Horm Metab Res, 1993, 25, 51-52.

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