bioartificial pancreas: materials, devices, function, and limitations

DIABETES TECHNOLOGY & THERAPEUTICSVolume 3, Number 3, 2001Mary Ann Liebert, Inc.

Bioartificial Pancreas: Materials, Devices, Function, and Limitations

ALES PROKOP, D.Sc.

ABSTRACT

The term “bioartificial endocrine pancreas” (BEP) was introduced by Anthony Sun in 1980.1

It was in 1968, however, that Thomas Chang2 proposed the use of microencapsulated islets asartificial b-cells. By applying a semipermeable membrane on the top of microcapsules, a sys-tem can be produced that is impermeable to viable islet cells and large effector molecules ofthe immune system, thus providing a protection for transplanted islets against rejection. Sincethen, the term BEP has not often appeared in papers. Instead, the term “bioartificial pancreas”(BAP) has gained widespread use. In a broader sense, BAP would include an application ofsuitable endocrine cells and protective polymeric vehicles, but not necessarily providing a fil-tration barrier of precisely defined properties (e.g., cells injected into a gel of hyaluronate).

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IN THIS REVIEW , I outline which steps are nec-essary to procure and design BAP, review the

latest developments, point out the inadequa-cies, and indicate solutions to these problems.This discussion will mostly refer to the insulin-dependent diabetes, diabetes mellitus (IDDM)type of diabetes, although this type sharessome characteristics of loss of b-cell functionwith the non–insulin-dependent diabetes mel-litus (NIDDM) type. Thus, some b-cell re-placement strategies (pancreas and islet trans-plantation, islet microencapsulation) developedfor IDDM are applicable to this other majortype of diabetes.

SOURCES AND HANDLING OF ENDOCRINE CELLS

Sources of cells

Primary cell lines. Current efforts to treat di-abetes via islet transplantation are hampered

by the lack of available human organ (cells) suit-able for transplantation. Likewise, the expansionof primary human pancreatic tissue, despite nu-merous attempts, has met with limited success.This is due to limitations in proliferative capac-ity and maintenance of the differentiated phe-notype. Furthermore, use of human stem cellsfor transplantation remains largely unexplored,mainly due to difficulties in processing and ex-panding them and due to the current politicalcontroversy over use of human stem cells. Thus,the primary cells remain the main source of cellmaterial for transplantation (be it free or encap-sulated). Commonly used sources of primarypancreatic cells are human, pig, rat, mice, rabbit,and trout. The latter four are used in the exper-imental domain only.

Pigs are considered relatively safe as a donorcell source.3 Animal studies of porcine islet celltransplantation in diabetic animal models haveestablished that cross-species restoration ofphysiologic activity and regulated release of in-

Review

Chemical Engineering Department, Vanderbilt University, Nashville, Tennessee.

sulin in host tissues area feasible.4 However,the risk of transmitting endogenous porcineretroviruses (or of generating a new form ofretrovirus via recombination) embedded in thepig chromosome, although speculative, maypose a serious regulatory hurdle.5 The risk ofendogenous retrovirus infection is under dis-cussion.6,7 The widespread use of pig cells fortransplantation is further hampered by theirfragility at isolation and culturing.8 One way ofalleviating this problem is using pig islets ob-tained before the animal reaches the adult stageof development. However, fetal pig islets ex-hibit a poor insulin secretory response to glu-cose.9 Neonatal pig islets10 show more promise.Their insulin secretory capacity is higher andas such they can be grown in vitro (5–9 days).Neonatal pig islets are also believed to be lessimmunogenic compared to adult islets. Theyshow lower T cell reactivity,11 even thoughaGal epitope (a[1,3]-galactose) is quite abun-dant in neonatal islets.12 Recent studies suggestthat this epitope is not expressed in fully dif-ferentiated, freshly isolated adult pig islet en-docrine cells, but rather on intraislet ductal andendothelial cells.13 However, 7-day culture di-minishes their expression. Likewise, fetal pigislets also gradually lose this epitope in culturewhile the islets mature.14

Improving phenotype. Engineered cell lineshave several advantages as sources of endo-crine tissue: they can be grown at low cost un-der pathogen-free conditions and can maintainhigh quality. Several tumor (e.g., RIN, MIN-6)and transformed b-cell lines (bTC) have beendescribed. Tumor cell lines, however, displaya defective phenotype (with a secretagogue re-sponse in a nonphysiologic manner) and oftenlose their capacity to secrete insulin with higherpassage numbers; also, generally, their capac-ity to secrete insulin is low, amounting to 20%of the normal b-cells.15 bTC is an insulinomacell line derived from transgenic animals ex-pressing SV40-T antigen. This cell line is hy-persensitive to glucose and expresses insulin ina constitutive manner; in other words, it se-cretes insulin at or near maximal rates at verylow glucose concentrations compared to nor-mal islets, with no feedback control from glu-cose (unregulated gene expression).16 Both

sources will undoubtedly be genetically ma-nipulated further to improve their function toarrive at b-cell surrogates suitable for cell trans-plantation. The possible danger of escape ofsuch endocrine cell sources from a devicewould hopefully be minimized by natural im-mune mechanisms.

A novel strategy that could dramatically im-prove islet transplantation is conferring (via ge-netic engineering) immunoprotection through alocal cellular production of immunomodulatorycytokines. Ex vivo IL-4 and IL-10 modulation ofpancreatic islet cells with recombinant adeno-as-sociated virus vector, followed by normal cap-sule transplantation, demonstrated no interfer-ence with insulin secretion and expression inboth b- and non–b-cells.17On the other hand, tar-geting proinflammatory cytokines, including b-cell death by apoptosis, was attempted by Rabi-novitch et al.18 b-cells transfected with theproto-oncogene, anti-apoptotic bcl-2 gene werefully protected from impaired insulin secretionand destruction resulting from incubation withIL-1b, TNF-a, and INF-g.

Engineering somatic cells. Lately, several at-tempts to provide insulin production from ec-topic sites have been reported. These involveintroducing a copy of the human proinsulingene into somatic cells (other then pancreas)and linking them to a suitable promoter. Reg-ulated expression of human insulin under thecontrol of a liver-type pyruvate kinase pro-moter has been demonstrated in transgenicmice.19 A delivery of mature insulin was shownin BB rat stomach implanted with autologousrat vascular smooth muscle cells, transducedwith replication-defective retroviral vector,bearing the furin-cleavable proinsulin gene.20

Successful prevention of hyperglycemia in di-abetic rats was achieved via subcutaneous im-plant of rat pituitary cells stably transfectedwith a furin-cleavable human proinsulin cDNAlinked to the rat PRL promoter. Secretagogue-stimulated insulin secretion was observed invitro.21 In another report a transgenic mousewas generated bearing the human insulin genelinked to glucose-dependent GIP and wasspecifically targeted to K cells of gut epithe-lium.22 This ectopic insulin production pro-tected mice from developing diabetes and

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maintained glucose tolerance after destructionof the native insulin-producing b-cells (STZ).In another report, PDX-1, the pancreatic andduodenal homeobox gene, essential for regula-tion of pancreatic development and islet func-tion, was shown to have the capacity to repro-gram extra-pancreatic liver tissue toward ab-cell phenotype. By using adenovirus-medi-ated gene transfer of the PDX-1 gene into mice,several silent insulin genes and protohormoneconvertase were activated. Mature insulin, gen-erated in situ, ameliorated hyperglycemia in di-abetic mice treated with STZ.23 The advantageof all these approaches is that they do not re-quire an immune suppression or immunopro-tecting device. A problem with relying exclu-sively on transcriptional regulation of insulinsecretion is that such a response may have arelatively long lag time, not matching the rapidresponse of physiologically regulated sensingand secretion.

Engineering islet neogenesis. Islet neogenesis isthought to occur during partial pancreatec-tomy. This regenerative process leads to theformation of new islets—that is, the repetitionof embryonic development with respect to ductepithelium regeneration (this is why the term“neogenesis” is used). Several recent attemptsdemonstrated that this process could be dupli-cated in vitro,24 perhaps via sequential prolif-eration and differentiation processes. The adultduct cells of the pancreatic tissue have the po-tential to lose their specific duct phenotypewith rapid proliferation, reverting to multipo-tent cells that can differentiate into islet cells ifproper signals are provided. Recently, Bonner-Weir et al.25 demonstrated that human ductcells could be expanded via growth (in thepresence of keratinocyte growth factor) andlater triggered to differentiate (by means of cul-ture overlay with gelled Matrigel, containinglaminin and other growth factors) into glucoseresponsive islet tissue. Although immature intheir responses, such b-cells, once optimized,could generate plentiful human islet tissue fortransplantation. At this point, the numbers ofb-cells are not adequate, although the processyields 10- to 15-fold increased insulin contentwithin about 1 month of in vitro culturing, Cy-tokeratin 19 has been readily used as marker of

duct cells.26 On the other hand, Ramiya et al.27

was able to grow pancreatic duct cells on glu-cose-depleted medium with serum and, later,to switch them into differentiation in a long-term culture at low-glucose and low-serum lev-els without the addition of other differentiationfactors. Such cultures generated islets that re-sponded to glucose challenge and preventedinsulin-dependent diabetes after their implan-tation into NOD mouse. The b-cell expansionwas claimed to be 10,000-fold within a 3-yearperiod following serial transfer. Again, this ap-proach does not provide any practical solutionof a short-term expansion into b-cells. In addi-tion, none of the above methods preclude thepossibility of involvement of pancreatic stemcells. In principle, allogenic b-cells generatedvia neogenesis would not necessitate immuno-protection in a device. Recently, the very firstinitiation protein capable of specific inductionof proliferation of duct epithelium was identi-fied.28

Engineering islet growth and differentiation. Theadult b-cell population is kept in delicate bal-ance by processes of neogenesis, proliferation,and cell death by apoptosis and necrosis. Theb-cell mass is closely correlated with bodyweight and insulin demand. Proliferation anddifferentiation of islets are considered mutuallyexclusive events. Proliferation is often accom-panied by dedifferentiation in that expandedcells lose the expression of insulin, glucagon,and somatostatin.29 The question is how to in-duce an expanded population to restore thisfunction. Glucose, hepatocyte growth factor,nicotiamide, and prolactin have been differen-tially implicated in proliferation and differenti-ation. Our target, perhaps, should be the smallfraction (about 15%) of adult b-cells that appearto be present in units smaller than 20 mm, with-out associated glucagon, somatostatin, and PPcells, located in or along ductules,30 and alwaysdetected in high numbers at pregnancy. The ex-istence of a specific b-cell growth factor that in-duces the proliferation (and maintains thefunction) of terminally differentiated b-cells invitro (and in vivo) remains elusive.

Engineering expansion of stem cells. Identifica-tion of pluripotent stem cells may enable the

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custom design of organs, and solve the prob-lems of donor organ scarcity as well as the needfor immunocompatibility and immunosup-pression to avoid graft rejection. The major ad-vantage of stem cell transplantation is that thetherapy is applied just once; in addition, thereexists the potential for an unlimited supply.Such an opportunity will only be realized ifstem cells can be expanded in vitro, differenti-ated (avoiding dedifferentiation), and maturedinto functional cells for implantation. Stem cellscould then be delivered to the desired site, andbe allowed to populate and regenerate thefunction, while bypassing immunorejection.The delivery of these cells in the end would stillrequire some kind of polymeric scaffold, a de-vice similar to BAP. Two major drawbacks pre-venting their use in diabetes treatment are,first, our inability to locate progenitor (stem)cells; and second, we have not identified whatspecific combination and sequence of molecu-lar signals is needed to reactivate quiescentstem cells to allow repopulation and repair. Thesearch for the elusive pancreatic stem cell iseven more problematic in embryonic pancreas;although it provides tissue-type restriction, itdoes not yet provide a b-cell–specific pheno-type. The main drawback is a lack of suitableearly embryonic cell markers and the satisfac-tory number of cells for experimentation.15 Atpresent, adult stem cells are the focus of atten-tion. Ramiya et al.27 apparently isolated suchprecursors from pancreatic duct tissue. Re-cently, Zulewsky et al.31 were able to isolatesuch cells from pancreatic islets from both ratand human tissues, using the neural stemcell–specific marker nestin. The procedure,lasting up to 8 months for both species, is basedon the commonly accepted belief that neuraland islet cells share many phenotypic proper-ties during early embryonic development. Thewhole procedure is remarkably similar to thatof Ramiya et al.27 in the sense of having a dis-tinct proliferative phase with a high-glucoseconcentration and two growth hormones (FGFband EGF), followed by a differentiation-specificmorphogen growth factor phase, featuring lowglucose and the presence of nicotinamide. Theonly difference in the outcome is the fact thatthe expanded phenotype is distinct from ductepithelial cells. The insulin expansion was

moderate. The maturation phase, when in-sulin-containing cells fully gain the ability tosecrete insulin in the normal biphasic mode in response to glucose, remains to be addressed. The cell encapsulation technologywould provide a possibility of in vivo differen-tiation and maturation enhanced by the pres-ence of encapsulating agents.

Another major technical problem remainsthe significant apoptosis associated with stemcell in vitro expansion (and neogenesis). Thesame remains unsolved for in vivo expansion.The development of functional responsivenessin vivo and full maturity may take a long time,especially for cell material initiated at stem cell,fetal, and neonatal levels. Korbutt et al.10 re-ported that several months were needed for thedevelopment of functionality of microencapsu-lated neonatal islets in mice. In addition, suchimplants are exposed to the danger of oxygendeprivation during handling and the earlyphases of implantation, and hence viabilitymay be lost.

And last, but not least, an important aspectof securing an adequate source of endocrinecells is the generation of therapeutically usefuland phenotypically stable cells.

Immunomodulating cell sources

One way of circumventing islet cell allograftrejection is a modification of the islet tissue tobe transplanted. Culture conditions, such astemperature, may provide a simple handle. Ex-tended culture (up to 50 days) at 37°C of neona-tal rat and adult pig islets results in reductionof immunogenicity, perhaps by downregu-lating the MHC class I antigen present onislets.32,33 This process is apparently potenti-ated at lower temperatures. Cultivating adultrat islets at 22°C (for up to 2 weeks) leads to re-duced immunogenicity, as demonstrated byLacy et al.34 some time ago (recently revisited,for example, by Brandhorst et al.33). This treat-ment, followed by islet encapsulation, resultedin markedly prolonged allograft survival. Ashort-term high-temperature treatment (heatshock for 60 min at 42°C) of adult rat and hu-man islets leads to the total inhibition of IL-1–stimulated inducible nitride oxide synthase(iNOS) expression, presumably by heat-shock

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proteins.35 iNOS can inhibit the islet function.None of these culture treatments found theirway to clinical trials, however.

Another unique strategy for preventing thedevelopment of autoimmune type I diabetes inanimal models is islet pretreatment with TGF-b1. Such incubation significantly reduced thesusceptibility to lysis by diabetic spleen cells,and susceptibility of islet cells to autoimmunedestruction.36 The protective effect of a localparacrine TGF-b1 supply on autoimmune dia-betes is directed against CD41 and CD81 effec-tor lymphocytes and their balance. The expres-sion of IL-4 (and IL-10) can generate apermanent state of tolerance in a similar man-ner.37 Likewise, pretreatment with the IL-1b an-tagonist (N(G)-mono-methyl-L-arginine) pre-vents cytokine-induced (NO) islet damage.38

Coencapsulation of such cytokines with isletswould provide a necessary initial trigger. Mori-tani et al.39 generated in situ TGF-b1 productionin transgenic b-cells in NOD mice by placingTGF-b1 cDNA under the rat glucagon promoter.

The next strategy involves protection of isletsto oxidative environment typical for diabetes.The incubation of islets with liposomally en-capsulated SOD, catalase, or glutathione perox-idase revealed modest protection against the oxidative stress.40,41 Engineering of SOD expression in cell lines as surrogates for trans-plantation cell therapy would be a solution. Spe-cific nitride-oxide-synthase inhibitor (NG-methyl-L-arginine) microencapsulated togetherwith islets prevented cytotoxic destruction of ratislets.42 Similarly, coencapsulation of autolo-gous erythrocytes provided an effective protec-tion from macrophage-mediated lysis.42 Chae etal.43 documented facilitated oxygen delivery tothe microencapsulated islets by means of coen-capsulation of PEG-conjugated hemoglobin. Fi-nally, cotransplantation of allogenic islets withallogenic Sertoli cells allows for long-term graftsurvival without immunosuppression.44 Theprecise mechanism by which this occurs remainsto be elucidated. It is possible that the immuneregulatory protein Fas ligand likely plays a role,perhaps by conferring an immunity privilege toadjacent allogenic islets.

Among other methods of modifying donorcells is the enzymatic treatment of pig islets bya-galactosidase to reduce a-galactosyl epitope

on intact cells.45 However, such treatment isnot a permanent solution. There is a reappear-ance of the epitope at the cell surface 48 h af-ter this treatment. This result is in agreementwith partial abolishment of the reactivity ofporcine endothelial cells with serum, althoughthe treatment was remarkably effective in pro-tecting the islets from antibody and comple-ment-dependent killing. Another way of atten-uating hyperacute rejection is by humanizingpig cell epitopes.5,46 If the antigenic epitopes oftransgenic xenografts are sufficiently altered toavoid evoking the destructive force of innateimmunity, this could solve the acceptance ofporcine tissues. A knockout of the pig gene thatencodes galactosyl transferase or expression ofhuman complement–regulating protein wouldeliminate the main cause of heyperacute rejec-tion.46 Such technologies, however, are not yetavailable.

Selective modification of islet cells is quite anattractive approach and is less problematicthan direct modification of the immune system.

Immunomodulating host’s system

Selective modification may seek an immensenumber of target cells and could be enormouslycomplex; thus, it may raise a possibility of un-desirable systemic effects. I will only mention afew undesirable effects. The blockage of the costimulatory molecule, B7, with the soluble fusion protein, CTLA4-1g, prolongs survival ofmicroencapsulated neonatal porcine xenograftsin NOD mice47 and induces nonresponsive-ness. However, neither microencapsulation norCTLA4-Ig alone prevents NOD destruction ofneonatal pig islets; there is apparently a synergyinvolved. Another example of immunomodulat-ing host’s system is that of transient expressionof IL-4 (by biolistic-mediated gene delivery intoskin) in NOD mice, avoiding repeated injectionof this cytokine.48 Such expression resulted in aTh2-type response and significant protectionfrom the onset of diabetes. This report showshow the immune system could be modified rel-atively easily. What is, perhaps, needed is an ini-tial stimulus to enable a shift from an unwantedcell type to a more advantageous one. Other pos-sible modulations of the immune system arelisted in the article by Weber et al.47


Cell banking

There is a need for reliable methods of isletstorage, preservation, and quality control. Cry-opreservation emerged as an attractive alter-native for islet banking.49 An improved methodwas introduced by the same group, which in-volved the substitution of ethylene glycol forthe standard cryopreservant, DMSO.50 This ap-proach yielded a simplified protocol and im-proved the survival and function of rat pan-creatic islets. This procedure was also adaptedfor pig islets. Enhanced a-cell survival (glucagon)after cryopreservation can explain the im-proved in vivo function of such grafts. Furtherimprovement was achieved by applying mi-croencapsulation prior to islet freezing. Suchislets exhibit increased functional viabilityupon thawing, since the capsule environmentprotects them from freezing damage.51,52 An-other improvement is worth mentioning: co-culture of islets prior to freezing with allogenicSertoli cells significantly improves both theyield and functional capacity of islets follow-ing cryopreservation.53

In vitro islet function and quality control

Table 1 presents a comprehensive list of teststhat are useful for evaluation of the graft func-tion prior to their implantation. The most im-portant single test is that of islet perifusion, andit deserves an extended discussion.

Success of BAP of any design is contingenton the rapid transfer of the glycemic signal

across a semipermeable membrane and of in-sulin from the BAP to the transplanted tissue.The transplanted tissue is used both for glucosesensing and insulin delivery. Besides the dy-namics of sensing and secretion, the site oftransplantation is also important.

A dynamic profile of glucose-mediated in-sulin release is usually assessed in a perifusionexperiment using microencapsulated islets.The assumption here is that b-cells can keeptrack of levels of small nutrient molecules, par-ticularly of glucose, amino acids, and fattyacids. Glucose sensing by b-cells is compre-hended through the fact that intracellular andextracellular islet glucose levels rapidly equili-brate due to effective facilitated membranetransport.61 Although all the details of b-cellglucose sensor are not known, it is clear thatglucokinase is the key to sensing. The insulinrelease is coupled to glucose metabolismthrough an intricate mechanism involvingATP-regulated potassium channels in mito-chondria and voltage-sensitive calcium chan-nels on islet periphery.62 The other two secret-agogues, amino and fatty acids, act asstimulants and modifiers of the glucose-trig-gered insulin release. The thresholds for thesethree different secretagogues are different, de-pending on the ingestion of a meal (postpran-dially) or fasting. Apparently, b-cells distin-guish between these different nutritionalsituations and release insulin in response to themeal, but not the fast. In addition, feedback ho-meostasis is provided by insulin receptors on

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TABLE 1. GRAFT QUALITY CRITERIA: IN VITRO FUNCTION

Criterion Description/comments Reference

Islet morphology Microscopically evaluate islet size/shape 55

Islet number Count and measure islet number and size and 55

determine the islet size equivalents (IEQ)Islet purity Microscopically detect the contaminating tissue 55

Islet viability Measure islet viability with fluorescent dyes; carry 55–57

image analysisInsulin storage Stain islets with dithizone or immunochemically 58

Static insulin Measure insulin release after islet exposure to a high 55

release test glucose concentrationPerifusion test Measure insulin release in a continuous flow of 55,59

perfusate following change in glucose concentration;islets are immobilized in a microporous membrane

C-peptide test Measure concentration of C-peptide released into 60

culture medium

Adapted from Hunkeler et al.54

liver, muscle, and adipose tissues, thereby con-trolling removal of nutrients from the bloodand peripheral organs.

None of these complex requirements are re-flected in a standard islet perifusion experiment.It also does not evaluate possible effects ofneural and endocrine hormones. The lack ofthese responses is due to damage incurred during their isolation (loss of inervation). Someother hormones (e.g., glucagon, catecholamines)can, in contrast, inhibit glucose-stimulated in-sulin release. Collectively, the above leads to anotion that isolated b-cells are not completelycompetent secretory cells, equivalent to in vivosituation. A comprehensive in vitro test of isletfunction, however, does not exist.

What is the role of non-b-cells in the inte-grated function of the islet? It appears that asmooth modulation of concentration/responsecurve and long-term metabolic control may re-quire the presence of non–b-cells in the isletpreparations,15 particularly of a-cells contain-ing machinery for glucagon synthesis and of d-cells for somatostatin. Hsieh et al.63 and Cher-rington64 noted that a rapid feedback is estab-lished via glucagon in dogs, secretion of whichchanges by three fold while glucose concentra-tion decreased by 20%. The islet isolationmethod (and cultivation and storage) shouldthus allow for retainment and viability ofnon–b-cell population. Likewise, geneticallymodified b-cells should also include some a-and d-cell functions to generate a perfect sur-rogate. In fact, the flow of blood through thepancreas is from the islet core (b-cells) to itsmantle (a to d), while b-cells control the a-cellfunction. This control is via inhibition ofglucagon (a potent insulin secretagogue) ofglucose-induced insulin secretion.15 Recently,Lou et al.65 reported on a-cell deficiency of iso-lated human islets upon prolonged enzyme digestion. Such cells exhibited poor insulin re-sponse to glucose stimulation and did not ef-fectively correct hypoglycemia in diabetic mice.Approaches to reconstruct the spatial and func-tional islet organization are yet to be devel-oped.

Although the standard perifusion test evalu-ates the ability of cells to turn-off the insulinsecretion after the glycemic challenge is inter-rupted, again, dynamics of that process is more

complicated because of nonendocrine cells anda possibility of autocrine and paracrine regula-tion within the islet itself. For most frequentlyused peritoneal implantation, it has been dem-onstrated that the peripheral glucose reflectsblood glucose at basal state and during varia-tions of glycemia66 and that BAP can respondappropriately to an increase in blood glucoseconcentration, without overshoot hypogly-cemia and with a time lag compatible withnormal physiologic insulin delivery.67–70 Forsuch delivery, including other peripheral sites(and injections), only 10% glucose enters theliver and 56% muscle tissue (liver accessed bythe portal vein) and can lead to hyperglycemiaand b-cell desensitization.64 Cherrington notedthat “if we want a cure, we will have to deliverinsulin portally because only this way glucoseis distributed more uniformly: one-third to theliver, one-third to the muscles and one-thirdelsewhere.”

Among other quality control methods, aquantitative method was suggested to assessthe viability of frozen-thawed islets using dou-ble fluorescence confocal microscopy (acridineorange/propidium iodide, AO/PI) and imageanalysis. This method is also applicable to non-frozen islets. When used as a supravital stain,AO accumulates in acid organelles such aslysosomes and insulin-containing secretorygranules. The viable cells fluoresce green withpale green nuclei and orange lysosomes. PIstains non-viable cells as brightly red fluores-cence. At higher cooling rates the damage isconcentrated around the central region.56

In conclusion, islet function and qualitywithin the context of complex spatial organi-zation of islets has not yet been addressed in aquality program. Additional issue is that oftransplant localization.

MATERIALS, DEVICES, PERFORMANCE,AND LIMITATIONS

Polymers and materials

Table 2 provides a list of polymers and ma-terials so far tested for islet encapsulation. Al-though many different polymer constructshave been reported,89 only few were tested orare presently used successfully for islet encap-


sulation. The most common is the A/P/A cap-sule, in spite of its fragility. Multipolymericcapsules, providing better mechanical stability,are gaining the use.70,79

Effect of polymers on islet function in vitro

The effect of polymers and materials used fordevice manufacturing on in vitro function ofislets is listed in Table 3. Such tests are intendedto help eliminating unsatisfactory polymers,not suitable for BAP design, not necessarilyguaranteeing in vivo success.

Devices

There are three categories of devices used forislet cell encapsulation, encompassing differentshapes and configurations:

� Microencapsulation: microcapsules1,97,98; con-focal (coherent) capsules99,100; and thinsheets101 (Islet Sheet Medical, 0.15-mm-thickalginate gel reinforced with polyester mesh)

� Extravascular (extracorporeal) devices: flatdiffusion chambers and tubular mem-branes102–104; hollow fibers105–108 (Orsetti etal.105 in 1978 first reported BAP); mesh-reinforced hydrogel tubes109 and othershapes110,111

� Intravascular diffusion chambers112–115

It appears that the interest in extravasculardevices is fading. It would be difficult to pre-dict what kind of device will ultimately be usedin future. Lacy in 1994116 predicted that hollowfibers would be applied in humans after ex-periments in animals prove to be successful. Interms of encapsulation, he advocated use of hu-man islets first, then pig islets and finally ge-netically engineered b-cell lines. Cherrington in200063 speculated that the route to the BAPwould likely be preceded by the replacementof whole organ pancreas with naked islet trans-plantation, followed by encapsulation.

Mechanical and transport properties andengineering of devices in vitro

The suggested mechanical and transportproperties of devices that should be tested invitro are summarized in Table 4. Besides the

mechanical stability of devices, the most im-portant design property is that of diffusioncharacteristics. The device permeability affectsits performance as it may impose limitation ontransfer of substances out and in the device.

Engineering approaches, particularly theo-retical mechanistic modeling, allow delineat-ing physical limitations of devices, guiding ex-perimentation and the optimization of theirdesign. Such approaches were mostly used inthe early period of implementation of the BAPconcept, much less in recent times. Analysis ofcontributions of convective and diffusivetransport to insulin release lead to the realiza-tion that the membrane hydraulic blood per-meability of diffusion chambers is the key formaintaining proper insulin secretion.123–128

Likewise, a comprehensive nutrient and meta-bolic model was designed for bT3 cell line en-capsulated in microcapsules.16 Such modelscould be extended for following the fate of au-toantigens released from the donor tissue andcytokines generated outside the device bymacrophages.

Some useful engineering experimental dataare also available. Hirotani and Ohgawara129

measured complement permeability for a dif-fusion chamber fitted with internal agarose/collagen gel and external Nucleopore mem-brane with a pore size of 0.1–0.2 mm. The com-plement (C3) became inactivated upon the passage through the mixed matrix with Nucle-opore pore size of 0.1 mm. Whalen et al.130 mea-sured in vitro and in vivo oxygen transport characteristics of a planar immunoisolatingmembrane. The conclusion of this work is theadjacent fibrotic tissue does not present any ex-tra resistance to oxygen transport, an importantfinding. Based on these data, one can predictthe maximal islet area loading for such device.Another study reported an in situ oxygen gen-erator, which decomposes water electrochemi-cally, to provide oxygen to a planar diffusionchamber.131 Experimental data were supple-mented by a theoretical model of oxygen dif-fusion for a device loaded with bT3 cell line.

In vivo device function

The in vivo performance criteria are summa-rized in Table 5. They should be viewed all to-

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TABLE 2. POLYMERS USED TO MANUFACTURE IMMUNOPROTECTIVE DEVICES FOR ISLET ENCAPSULATION

Device system Polyanion (internal phase) Cation Comment Reference

A/P/A Alginate (A) Poly-L-lysine (P); Alginate coat (A); 1

calcium chloride or calcium chelated 71

calcum lactateA/A/A Alginate (A) Poly-L-arginine (A) Alginate coat (A); 72

calcium chelatedA/B/A Alginate (A) Barium chloride Alginate coat (A); 73

(B) alginate beads (nochelation)

A/P/AA Alginate (A) Poly-L-lysine (P); Double alginate coat 74

calcium chloride (AA); calcium chelatedA/Ca Alginate (A) Calcium chloride Uncoated calcium 75

alginate gelledcapsules

AH/PS-PEI Alginate (A); Protamine sulfate (PS); Alginate ionically 76

heparin (H) polyethyleneimine precast;(PEI) heparine/protamine

complexA/P/PEG-A Alginate (A) Poly-L-lysine (P); Polyethylene 77

calcium chloride glycolamine (PEG-A)coat

A/C/C Alginate (A); (and Chitosan (C) Crosslinked (C) by 78

polyethylene glycol carbodiimide (EDC) orPEG) glutaraldehyde (GA)

ACS/G/A Alginate (A); Poly-methylene- Alginate coat (A), 70

cellulose sulfate co-guanidine (G); calcium chelated(CS) calcium chloride

AGPSS/PB/C Agarose (A), Polybrene (PB) Agarose thermally 79

polystyrene precast; PSS/PB formsulfonate (PSS) complex, CM-cellulose

(C) coatingR-PEG Reactive-PEG — R-PEG covalently 80

isocyanate reacts with isletsurface amino acids,modifyingimmunogenicity

PEG-DA Polyethylene glycol — PEG-DA-conformal 81

diacrylate (PEG- coat byDA) photopolymerization

(hydrogel)PAA-SH Thiol derivative of — Hydrogel via radical 82

polyacrylamide polymerizationSi1 Silica — Sol-gel process 83

Si2 Methyl-modif-silica — Conformal coating by 84

silica gelNiPAM NiPAM-base — Thermoreversible gel 85

polymerPAN-PVC Polyacrylnitrile- — Hydrogel fibers 86

polyvinyl chloride extruded from(PAN-PVC) solvent/nonsolvent

processPAN-MAS Polyacrylnitrile- — Hydrogel fibers 87

methallyl sulfonate extruded from(PAN-MAS) solvent/nonsolvent

processHM-PSU Hydro-methyl- — Modified commercial 88

modif-Polysulfone hollow fibers(HM-PSU)

gether as a comprehensive assessment of de-vice performance.

Postexplant criteria

The postexplant in vivo criteria are listed inTable 6. The polymer biocompatibility and im-plant vascularization are expanded below.

The successful, long-term implantation of ar-tificial organs such as the BAP depends on thetissue compatibility of the device and its stablevascularization. The islets themselves arelargely devoid of vascular system upon theirisolation and must thus depend mostly onblood vessels arising from the host tissue.142

The technological goal of permanent engraft-ment is hampered by tissue reaction to the im-plant device, which must support the growthand function of the islet while maintaining thegraft in an immunologically privileged state.Viability of the graft requires nutrient and oxy-gen supply and metabolite removal. The sup-

port material should be assessed within thecontext of its in vivo response. Three require-ments are envisioned: (1) encouragement ofgranulation tissue with a network of blood cap-illaries; (2) minimization of fibrotic growtharound the implant as a terminal stage in thesequence of events; and (3) maintenance (orrestitution) of physiological response of im-planted islets via externally encouraged angio-genesis, resulting in permeable vascular fi-brotic growth. While the first two requirementsare largely dependent on the material and tex-ture of implant, the second one will depend onan angiogenic response of the implant mater-ial and/or on a dose–response relationship be-tween externally available angiogenic factors,eventually added, and the degree of mainte-nance of blood supply in the vicinity of im-plant, where pancreatic islets would reside.

Polymer membrane–driven neovasculariza-tion has been tested as a means of providingbetter environment for islets.143–145 Microfiltra-

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TABLE 3. EFFECT OF POLYMERS ON ISLET IN VITRO FUNCTION


Cytotoxicity Incubate islets in presence of individual polymers and 89

polyelectrolyte complex (membrane); measure isletviability (LDH release, fluorescence)

Induction of Incubate monocytes with polymers; assay cytokines in 90,91

cytokine production supernatantStatic insulin Repeat static test (Table 1) in presence of polymers and 92

release polyelectrolyte complex (membrane); or with encapsulated(or otherwise entrapped) islets

Perifusion test Repeat perifusion test (Table 1) in presence of polymers 89

and polyelectrolyte complex (membrane)Mitogen test Incubate fibroblasts in presence of polymers and 93–95

polyelectrolute complex (membrane); carry mitogenic teston a suitable cell line

Endotoxin test Detect endotoxin levels in polymers by gelclot assay 95,96


TABLE 4. MECHANICAL AND TRANSPORT PROPERTIES OF DEVICES IN VITRO


Permeability test Apply inverse size-exclusion chromatography with a standard 117,118

molecule (e.g., dextran)Antibody permeability test Measure the amount of entrapped antibody, following protein-A– 118,119

Sepharose encapsulation and incubation with labeled antibody 120

Dry bursting test Measure the applied force to break the capsuleOsmotic stability Measure volume change following media, buffer or saline addition 121,122


tion membranes of certain porosity (pore sizeabout 60–100 mm) are considered the best toachieve growth of blood capillaries into the im-plant and provide the implant’s access to nu-trients and oxygen.141,146 Some prefabricatedpolymers are known to induce angiogenesisthemselves. A good example is an expandedpolytetrafluoroethylene (ePTFE, Goretex; W.L.Gore, Flagstaff, AZ). Some water-soluble polymers (e.g., hyaluronic acid, heparin, andlaminin147) are known to stimulate angiogene-sis, albeit moderately when compared to an-giogenic factors delivered externally. However,such vascular structures are resorbed overtime.148 At a molecular level, capillary regres-sion is explained on the basis of growth factordeprivation, leading to a programmed apopto-sis of endothelial cells.149 Initially, of course, atransient angiogenic response is observed forsome limited time upon the implant introduc-tion, as a result of normal reaction to woundhealing at implant introduction.

The generation of more stable vascularizedbed requires a supply of angiogenic growth fac-tors.150,151 The rationale for applying an exter-nal angiogenic growth factor is obvious. Sincethe half-life period of therapeutic proteins inthe body is generally very short to effectivelyexert the biological activity when they are in-

jected in the free form (a continuous infusionwas demonstrated145), a controlled deliveryprovides one possible method to enhance theiran in vivo efficacy. An immobilization in a hy-drogel can be used to provide a slow-releaseregimen.150,152–153

Non-resorbable polymer meshes with suit-able opening can be used to retain microcap-sules with encapsulated islets and allow for bi-directional perfusion of nutrients and products.The prototype design is a biocompatible meshbag coated with angiogenic substances, loadedwith microencapsulated islets and eventuallyimplanted into peritoneum150 (Fig. 1). Alterna-tively, the device can be implanted for someperiod of time to allow for vascularization priorthe islet introduction.154–156 These studies sug-gest that it might be beneficial to induce vascu-larization in diabetic recipient before the implantation of islets because the neovascular-ization is difficult in diabetic recipients. The em-ployment of a coencapsulation of islets togetherwith an angiogenic factor (within the capsules)could be beneficial only for subcutaneous im-plants.157 We have observed extended vasculo-genesis for implants loaded with fibroblastgrowth factor-b (bFGF) even in peritoneum.150

The advantage of some of these constructs is thatsuch medical devices (vascularized bioartificial


TABLE 5. IN VITRO GRAFT FUNCTION


Glycemia Measure blood glucose levels after transplantation into diabetic animal 1

Glucose tolerance Measure blood glucose levels following IV, IP, or oral injection of glucose 55

Body weight Monitor animal body weight posttransplantation 132

Glucosylated hemoglobin Measure glycosylated hemoglobin following transplantation into diabetic 133

animalC-peptide Measure plasma levels over period of time 134


TABLE 6. POSTEXPLANT GRAFT CHARACTERISTICS


Foreign-body reaction Determine fibrotic layer thickness 132

Device permeability Determine permeability through the fibrotic layer 135

Cellular immunoreactions Determine quantity and quality of cells in infiltrate 136

Humoral immunoreactions Determine levels of anti-xenograft antibody in serum 137,138

Complement activation Determine the level of complement activation 139,140

Implant vascularization Determine the number of blood capillaries per area of sectioned tissue 141


pancreas, VBAP) can be retrieved from a patientin case of serious problems. Retrievable im-plants are in the focus of medical industry (e.g.,a recent NIH conference158).

Trends in bioartificial pancreas design

Immunoisolation versus immunoprotection. Im-munoisolation strategies have had increasinglyencouraging results in recent years, but thelarge volume of donor material is required. Forthat reason, initially, emphasis was given to ex-tracorporeal devices, later to microencapsula-tion technologies. When Sun reviewed thestate-of-the-art of microencapsulation of pan-creatic b-cells in 1988159, he stated: “durablecapsules containing viable cells can be pro-

duced, thus providing a total (underlined bythis author) protection to transplanted isletsagainst rejection.” We should now drop termimmuprotection.160 The release of donor anti-gens from xenografts, activating host’s den-dritic cells, cannot be eliminated by the presenttechnology. Such antigens and peptide frag-ments could be as small as few amino acids.The membrane technology (engineering of ma-terials) has reached some plateau. The molec-ular weight cut-off cannot be made tighterwithout compromising the health of implants(e.g., a supply of transferring). The key is en-try of some molecules essential for nutrition ofislets. Further progress might likely be madealong advances in molecular biology, geneticsand, primarily, immunology. I have reviewed

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FIG. 1. Design of in vitro experiment.

some possible changes which could be appliedfor modification of donor cells, as well as thehost’s environment. The blocking of autoanti-gens remains a challenge.

Allograft versus xenograft. Allogenic b-cellshave fewer problems with immunological re-jection (and viral infection), but are limited insupply. The human fetal tissue and in vitro ex-pansion of human b-cells as well as b-cellgrowth and differentiation from b-cell precur-sors will likely generate unlimited quantity ofallografts. These may, however, take some timeto develop. Pig b-cells derived from pathogen-free (and virus-free) animals will also be usedin parallel through animal farming. Im-munosolation technologies will be valuable device to enable efficient application of xeno-grafts. Levine and Leibowitz in 1999 pre-dicted161: “In the short term, because of thecomplexity of the b-cell, it is likely that b-celltherapy will be based on the genetic engineer-ing of a source of b-cells grown in vitro.Whether that cell will be derived from a cellline or primary tissue, and whether it will befrom humans or from another species, is un-clear.”

Regulatory issues. Besides a possibility ofretroviral infection by means of pig xenografts,another drawback is limited replacement ca-pability of present implantable BAPs in a clin-ical setting. There is at present a strong em-phasis on design of retrieval procedures toallow tissue or device to be removed from thebody in case of device or patient complicationsin mid-procedure. In fact, FDA normally doesnot allow a procedure which would not havesuch possibility build-in the system.162

ACKNOWLEDGMENTS

The support of the American Diabetes Asso-ciation is greatly acknowledged.

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Address reprint requests to:Dr. Ales Prokop

Chemical Engineering DepartmentVanderbilt UniversityNashville, TN 37235

E-mail: [email protected]


bioartificial pancreas: materials, devices, function, and limitations

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