Immunocompatibility and biocompatibility of cell delivery systems

Advanced Drug Delivery Reviews 42 (2000) 65–80

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Immunocompatibility and biocompatibility of cell delivery systems ˇ´ ´* Blanka Rıhova ´ ˇ ´ 1083, 142 20 Prague 4, Czech Republic Institute of Microbiology, AS CR, Vıdenska

Abstract Immunoisolation therapy overcomes important disadvantages of implanting free cells. By mechanically blocking immune attacks, synthetic membranes around grafted cells should obviate the need for immunosuppression. The membrane used for encapsulation must be biocompatible and immunocompatible to the recipient and also to the encapsulated graft. The ability of the host to accept the implanted graft depends not only on the material used for encapsulation, but also on the defense reaction of the recipient, which is very individual. Such a reaction usually starts as absorption of cell-adhesive proteins, immunoglobulins, complement components, growth factors and some other proteins on the surface of the device. The absorption of proteins is difficult to avoid, but the amount and specificity of absorbed proteins can be controlled to some extent by selection and modification of the device material. If the adsorption of proteins to the surface of the implanted material is reduced, the overgrowth of the device with fibroblast-like and macrophage-like cells is also reduced. Cell adhesion at the surface of the implanted device is, in addition to the selected polymeric material, greatly influenced by the device content. Xenografts trigger a more vigorous inflammatory reaction than allografts, most probably due to the release of antigenic products from encapsulated deteriorated and dying cells which diffuse through the membrane and activate adhering immune cells. There is an evident effect of autoimmune status on the fate of the encapsulated graft. While encapsulated xenogeneic islets readily reverse streptozotocin-induced diabetes in mice, the same xenografts are short-functioning in NOD autoimmune diabetes-prone mice. Autoantibodies, to which most devices are impermeable, are not involved. Among the cytotoxic factors which are responsible for the limited survival of the encapsulated graft the most important are cytokines and perhaps some other low-molecular-weight factors released by activated macrophages at the surface of the encapsulating membrane.  2000 Elsevier Science B.V. All rights reserved. Keywords: Immunocompatibility; Biocompatibility; Polymeric materials; Polymer implants; Immune response; Inflammatory response; Cytokines; Encapsulation; Transplantation; Immunoprotection; Immunoisolation; Cell adhesion; Autoimmunity; Complement; Antibody; Immunoglobulin

Contents 1. Introduction ............................................................................................................................................................................ 2. Biocompatibility and immunocompatibility of material used for encapsulation ............................................................................ 2.1. Adsorption of serum and tissue proteins, components of the complement system and other substances onto the surface of an encapsulating device ........................................................................................................................................................ 2.2. Cell adhesion at the surface of polymer material................................................................................................................. *Tel.: 1 420-2-475-2267; fax: 1 420-2-472-1143. ˇ´ ´ E-mail address: [email protected] (B. Rıhova). 0169-409X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-409X( 00 )00054-5

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3. Immunoisolation and immunoprotection ................................................................................................................................... 3.1. Penetration of antibodies and components of the complement system................................................................................... 3.2. Penetration of cytokines ................................................................................................................................................... Acknowledgements ...................................................................................................................................................................... References ..................................................................................................................................................................................

1. Introduction To create hybrid bioartificial organs by immunoprotecting the living cells or tissues in polymeric membranes was the idea of Chang [1]. The immunoprotection of transplanted cells offers transplantation without the need of immunosuppression of the host. Moreover, immunoisolation of a xenograft, for example a transplant of cells from non-human species, has been suggested as a way of overcoming the limited supply of human organs for transplantation [2–6]. Many polymeric devices have been developed for the purpose of immunoisolation of transplanted cells secreting hormones, neurotransmitters, growth factors or other bioactive cellular secretory products. Such allogeneic or xenogeneic cells are enclosed in a semipermeable membrane which should allow the free exchange of molecules important for cell survival and function such as nutrients, oxygen, essential metabolites, transport proteins and toxic products of cell metabolism. To control the synthesis and secretion of a desired cellular product the membrane also has to be permeable to physiological signals such as glucose or insulin. In order to meet the requirement of immunoisolation and to minimize both the humoral and cellular immune response of the host, the device wall should restrict direct contact with cells of the immune system, and exclude the penetration of cytotoxic molecules into the capsule. Some of the most dangerous molecules are allogeneic and xenogeneic antibodies, autoantibodies, components of the complement system, some cytokines and other cytotoxic substances. The encapsulated cells are expected to remain functional for several months to more than 1 year and to continue the synthesis of the desired product. Potential applications of encapsulation technology include functional replacement of major organs such as pancreas or liver as well as transplantation of engineered cells for gene therapy [7–11]. A variety

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of cell types have been tested for encapsulation, among them islets of Langerhans as an alternative to insulin treatment [3,12–25], hepatocytes in the case of acute liver failure to provide adequate temporary metabolic support to allow spontaneous liver regeneration or, in the case of irreversible liver lesions, as a bridge before orthopic liver transplantation [10,11,26–30], parathyroid cells [31], dopaminergic neurones [32], dopamine-secreting cell lines [33–35] and genetically engineered cell lines for the delivery of neurotrophic factors to the central nervous system [36–40] and for the supply of the recipient with other biotherapeutic proteins [41–43]. The advantage of transplantation under an immunoprotective barrier has led to the development of spherical microcapsules [18], microcapsules with controllable biodegradability [44], tubular diffusion chambers [19], hollow fibers [21,45,46], and other immunoisolating devices [6] including those anastomosed to the vascular system as arteriovenous shunts [44].

2. Biocompatibility and immunocompatibility of material used for encapsulation The membrane used for encapsulation of living cells must be biocompatible and immunocompatible to the recipient and to the cells it encloses. Compatibility of medical material reflects a complex of characteristics which influences the fate of the material in its biological host. However, the ability of the host to accept the material depends not only on the material itself but also on the host, its genetic dispositions and physical status [47,48]. Patients must always be prepared to be exposed for a long time or repeatedly to a rather large quantity of foreign material. Ideally, the device used for encapsulation should evoke no or only minimal fibrous tissue reaction, macrophage activation, and cytokine and cytotoxic agent release [49,50]. Along with maintaining these

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cellular requirements, the material for the immunoprotecting device must have suitable permeability characteristics and should be chemically stable and physically durable in order to withstand the new in vivo environment [3,22,51], and that for immunoprotecting capsules must have controllable biodegradability which allows the breakdown and absorption of the implants when encapsulated cells die or become functionally inactive [44]. Most cell encapsulation technologies use a modification of the procedure originated by Lim and Sun [18], who were the first to describe the coating of living cells by a semipermeable biocompatible membrane to avoid immunodestruction by the host immune system. Their polymer system, which used alginate / PLL / alginate capsules for transplantation of pancreatic islets, has been employed by a number of research groups [15,16,52]. Other authors use encapsulation in Ba 21 cross-linked alginate, which seems to be at least as good [17,25,53–55]. Lanza et al. [44] reported long-lasting transplantation of discordant xenogeneic islets of Langerhans in ‘composite’ microcapsules fabricated from alginate and lowrelative-mass poly-L-lysine with controlled biodegradability. A multilayer protamine–heparin membrane (protein membrane formed by protamine and heparin) represents another rather well tolerated material impermeable to the main cellular and humoral components of the immune system [56]. While alginate / PLL technology does not offer the option of independently adjusting the mechanical strength and permeability of the microcapsules [6,23,57], capsules made from hydroxyethyl methacrylate–methyl methacrylate (HEMA–MMA) can be tailored to particular morphological and diffusive properties using different acrylate monomers in different proportions to prepare the polymers [6,58,59]. The independent modification of parameters critical for immunoisolation and cell survival such as capsule size, membrane thickness, mechanical strength and membrane permeability is also offered by the multicomponent entrapper system [2,6,60–62] which is formed by a polyelectrolyte complexation of sodium alginate (SA) and cellulose sulphate (CS) with poly(methylene-co-guinidine) (PMCG) hydrochloride in the presence of Ca 21 and Na 1 . Some materials such as acrylonitrile membrane

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AN69 do not activate macrophages. The opposite was reported for alginate poly-L-lysine, which is mitogenic [31], stimulates the release of TNF-a and interleukins [31], triggers the activation of human macrophages in vitro, a fibrotic reaction around the implanted microcapsules in vivo [63] and multinucleated giant cell reaction in peritoneum of rats 9 months after their implantation [44]. It was suggested that the mannuronic rather than the guluronic acid content of the alginate outer layer of the microcapsules stimulates antibody and fibrotic response in the recipient organism [64,65]. Alginate rich in mannuronic acid (high M) evokes an inflammatory response by stimulating monocytes to produce proinflammatory cytokines, such as TNF, IL-1, and IL-6 [66], and humoral immune response. Antibodies against high-M alginate capsules but not against high-G alginate were detected in the sera of mice implanted with this type of capsule. They were not detected in the sera of Wistar rats [64]. It is accepted that pancreatic islet cell transplantation into the peritoneal cavity and the renal subcapsular space possesses some advantages compared with the liver and subcutaneous sites [23,53,67,68]. However, the site of implantation influences the biocompatibility of the implanted material. The peritoneal cavity has certain restrictions in terms of functional activity of the microencapsulated cells because of peritoneal macrophage reactions and the increased number of transplanted cells required compared to the alternative implantation sites such as kidney, liver and spleen [23]. Tissue reaction against implanted microbeads composed of agarose and poly(styrene sulfonic acid) (PSSa) mixed gel differs markedly depending on the concentration of PSSa. Five percent agarose / 5% PSSa microbeads were found floating in the peritoneal cavity, while those containing 10% PSSa were surrounded by adipose tissue [24]. Neither fibrosis nor inflammatory response or aseptic peritonitis was reported at the cellulose sulphate (CS) capsule implantation site during observation periods as long as 10 months. Capsules containing antibody-producing cells and implanted into the peritoneal cavity remained mobile and non-vascularized, while capsules implanted under the skin formed neo-organs which became vascularized within days [69]. An effect of the local environment on the stability of multicomponent

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capsules was reported by Wang [60], who observed that the multicomponent capsules inside normal C57BL mice were not too sensitive to minor polycation concentration changes, while even small changes in polycation concentration influenced the fate of capsules inside autoimmune-prone NOD mice.

2.1. Adsorption of serum and tissue proteins, components of the complement system and other substances onto the surface of an encapsulating device An absolutely inert biomaterial probably does not exist [70]. Defense reaction against implanted material may considerably affect the fate of transplanted encapsulated cells. It usually starts as adsorption of cell-adhesive proteins, immunoglobulins, complement components, growth factors and other tissue fluid proteins on the surface of the device. If HEMA–MMA (75% HEMA) microcapsules were maintained for 1 week in PBS, only a pluronic surfactant from a precipitation bath was adsorbed on their surface. On the other hand, calcium deposits, which could act as nucleation sites for calcification of the polymeric material in vivo, and traces of serum proteins were found on the surface of the same capsules maintained for 1 week in a medium containing fetal bovine serum [71]. Wang et al. [6] reported remarkable differences among multicomponent capsules of similar physical appearance, mechanical strength and permeability in their ability to induce fibrosis after intraperitoneal transplantation. The reported differences suggest that even minor variations in the capsules may have a strong impact on the in vivo fate of encapsulated cells. Using direct radioimmunoassay, Sawhney and Hubbell [9] demonstrated a considerable decrease in the adsorption of complement components by grafting the highly charged alginate / PLL microcapsules with PEG. The iC3b binding was the least on microcapsules made from protein-repellent pentalayered alginate / PLL-g-MPEG compared to microspheres made of alginate, bilayered alginate / PLL or trilayered alginate / PLL / alginate. Babensee et al. [71] characterized the human and rat proteins associated with hydroxyethyl methacrylate–methyl methacrylate (HEMA–MMA) copolymer

microcapsules following 1 week of in vitro incubation in a medium containing human serum, or after transplantation into rat peritoneum. Cell-adhesive proteins, proteins of blood coagulation, components of the complement system and some other proteins were detected on the surface of the microcapsules. Human proteins associated with capsules included fibronectin, plasminogen, IgG, vitronectin, Factor B, Factor H and Factor I. Complement activation fragments, however, were formed in the medium containing human serum independently of capsule presence and therefore reflect the biochemical processes in human serum maintained for several days at 378C. On the surface of HEMA–MMA capsules implanted into rat peritoneum were found rat fibrinogen, IgG, complement 3 fragments, antithrombin III, transferrin, a 1 -antitrypsin, fibronectin, albumin, a 2 macroglobulin, vitronectin, a 2 -microglobulin, Factor B and Factor I. All proteins were detected using antibodies against human analogues. There are a few other studies which have compared protein adsorption from biological fluids in vivo and in vitro [72–74]. The same proteins were adsorbed to polystyrene microspheres cultured in vitro in peritoneal fluid or implanted into the peritoneal cavity. Differences were found in the amount of adsorbed proteins [73] and in the proteins which were adsorbed at the surface of polystyrene microspheres incubated with peritoneal fluid or with plasma [73].

2.2. Cell adhesion at the surface of polymer material The acute inflammatory response is characterized by the presence of eosinophils and polymorphs, while the chronic form is characterized by the presence of macrophages and fibroblasts and by the foreign body multinucleate giant cell reaction. The implantation of biomaterials commonly leads to a stronger or weaker inflammatory reaction which is chiefly manifested as an overgrowth of the material by fibroblast-like and macrophage-like cells [9]. One of the mechanisms of inflammatory and immune cell adhesion to a synthetic surface is the interaction of their cell surface receptors, including FcRs and complement receptors (CRs), with proteins adsorbed to the surface of the material used for encapsulation. The proteins commonly found on the surface of

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synthetic material are vitronectin, laminin, thrombospondin, fibronectin, immunoglobulins, complement components, fibrinogen, albumin and some others [75]. If the adsorption of cell adhesion proteins to the surface of the implanted material is reduced, then the surfaces show less tendency to promote cell attachment and growth [76]. Some time after transplantation, fibrous capsula may be observed around the implant. Such reaction still does not mean that the material is incompatible. Signs of non-biocompatibility are calcification, granulomatous reaction and eventually tumorigenesis [77,78]. However, cell adhesion to the surface followed by a fibrotic reaction around the microcapsule membrane is highly undesirable as it often causes necrosis of the encapsulated cells and graft failure. It was found that the immunoprotective effectiveness of a device correlates with the thickness of the fibrotic layer surrounding the microcapsules [31]. A thick fibrotic layer does not only serve as a diffusion barrier which leads to an insufficient supply of oxygen and nutrients, but fibroblasts themselves will compete for this nourishment [79–82]. Alginate / PLL microcapsules tend to clump and stick together when implanted into the peritoneal cavity of a recipient. Weber et al. [83] observed that alginate / PLL microcapsules containing canine islets implanted into non-obese diabetic mice (NOD) functioned on average for only 11.5 days. As the microcapsules were not permeable to immunoglobulins, the loss of xenogeneic graft was most probably due to soluble cytotoxic factors released by cells layering the implanted microcapsules. Histologic examination of the microcapsule surface at the time of rejection revealed the presence of multiple layers of pro-inflammatory, cytokine-secreting cells such as macrophages, multinucleate giant cells, granulocytes and lymphocytes. It is a general opinion that the most important cellular elements in fibrosis formation around the foreign synthetic material are collagen-secreting fibroblasts and activated macrophages releasing growth regulatory cytokines [9,50,84–86]. This cellular overgrowth, which represents the foreign body reaction against microcapsules, is not restricted only to alginate / PLL / alginate microcapsules [9]. Fibroblast growth is typically found near the membrane of alginate microspheres cross-linked with BaCl 2 and containing mouse islets

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[17]. Several months’ function of encapsulated allografts and xenografts of islets of Langerhans implanted in the peritoneal cavity was reported by Lacy et al. [20], Zekorn et al. [17], Chen et al. [87], Jain et al. [88] and Kessler et al. [68]. Although the membranes (polycarbonate, cellulose triacetate, AN69, agarose) were biocompatible, important fibrosis occurred, accompanied by the adhesion of fibroblasts and macrophages and consequently also by necrosis of some encapsulated islets. Four weeks after the implantation of microcapsules from HEMA–MMA copolymers it was possible to detect a layer of cellular overgrowth [38]. Transplants of human islets and human insulinoma tissue survive in non-obese diabetic mice NOD up to 6 months when encapsulated in hollow-fiber membranes. However, this type of fiber was also observed to elicit an inflammatory pericapsular response in the rat [89,90] and the porcine model [79]. Using a protamine– heparin multilayer membrane, Tatarkiewicz [56] reported, after a 4-month study, only traces of fibrotic tissue around capsules entrapped in the omentum and, rather surprisingly, no tissue reaction of phagocytic character. Five-percent agarose gel containing mouse fibroblasts Ltk-transfected with human insulin cDNA and implanted into streptozotocin (STZ)-induced diabetic mice revealed at 30 days pericapsular inflammation which was probably responsible for the malfunction of the encapsulated cells [91]. The cytotoxicity of the host macrophages, which are the most important cells responsible for the loss of functional activity of the encapsulated graft and transplantation failures [92,93], depends on their state of activation and on their ability to release cytotoxic soluble factors such as cytokines (IL-1b, TNF), growth factors and nitric oxide. Activated macrophage products progressively modify the local environment, initially leading to destruction of target cells or tissue and later, i.e. in chronic delayed type hypersensitivity (DTH) reaction, causing replacement by connective tissue. Fibrosis is the outcome of chronic DTH, when elimination of antigen and rapid resolution are unsuccessful. Wiegand et al. [94] demonstrated that 80% of the islets were destroyed during co-culturing in the presence of activated macrophages, while only 17% in the presence of resident macrophages. In agreement with this find-

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ing, the necrosis of pancreatic islets encapsulated in AN69 membrane [93] or alginate / PLL [94] was observed after co-culture in the presence of activated macrophages. Cell adhesion at the surface of implanted devices is, in addition to the selected polymeric material, greatly influenced by the device content. There is an inverse correlation of graft function and degree of tissue reaction of the host, and the involvement of xenoantigens (alloantigens) released from encapsulated cells is obvious. Implanted empty devices usually remain free from cellular adhesion, while those containing xenografts trigger the activation of macrophages in vitro, and a fibrotic reaction in vivo [93]. It has been reported that allografts can survive in microporous diffusion chambers, whereas xenografts are rejected under the same condition [95,96]. Encapsulation of xenogeneic pancreatic islets within a semipermeable acrylonitrile AN69 membrane leads, 6 days after transplantation, to 65% surface colonization with macrophages, while only 5% surface colonization was observed with empty control devices [93]. The fact that implanted empty devices made of AN69 membrane remained free from cellular adhesion indicated that the material by itself is not responsible for the activation of macrophages [93]. Similar discordance in the cellular reaction occurring at the surface of empty and islet containing capsules was also reported by Horcher et al. [85] using barium alginate and by Weber et al. [97] with alginate–poly-L-lysine microcapsules. These results suggest that the encapsulated graft somehow triggers the cellular reaction of the recipient. The destruction of the xenogeneic tissue inside the device is probably mediated by the release of antigenic products from encapsulated deteriorated and dying cells and induction of a local inflammatory reaction around the implanted material. Once outside the device, the xenoantigens are ingested and processed by host antigen-presenting cells such as macrophages adherent to the capsule surface. The processed antigens are then presented to CD4 1 T cells, which in turn stimulate an inflammatory reaction resulting in the death of the encapsulated cells. It is not known whether death of encapsulated xenogeneic cells results from specific immune killing or from starvation caused by the lack of essential nutrients. Among the potentially toxic factors secreted by CD4 1 T cells or macrophages are cyto-

kines, superoxide, and nitric oxide [93,95,96]. The gradually increasing level of anti-islet antibodies detected in recipients of xenogeneic porcine islet-like cell clusters (ICC) as well as in recipients of ICC microencapsulated in alginate–poly-L-lysine–alginate capsules documents that islet antigens are released, penetrate through the capsule membrane, activate the recipient’s immune system, and trigger complement activation and subsequently a local inflammation [64]. The humoral response raised against the encapsulated ICC can be prevented by treating the animals with a low dose of CsA from the day of transplantation; this may indicate that immunosuppression should be considered in the transplantation of encapsulated xenografts [64]. Weber et al. [97] found that xenogeneic transplantation of rat islets into NOD mice does not induce adhesion of inflammatory cells to the surface of poly-L-lysine alginate microcapsules if rat islets are irradiated with UV-B. Based on this observation it was concluded that UV-B irradiation somehow decreases immunogenicity of the antigen(s) released from encapsulated islets to which the NOD cellular reaction is directed. The possibility of preventing the diffusion of antigens from encapsulated cells and consequently of avoiding the activation of surface adherent macrophages should considerably improve the functional survival of xenografts [93]. Iwata et al. [67] reduced the amount of soluble antigens released from xenogeneic cells encapsulated in 5% agarose microbeads by culturing them in vitro at 378C. This treatment restored the islets damaged during isolation and microencapsulation and the amount of released islet antigens was decreased. However, such in vitro culture effectively prolonged xenograft survival only in recipients with a low level of anti-xenograft antibodies. Xenograft survival in recipients with a high level of antibodies was limited [24]. An effective prevention of the release of highly immunogenic soluble antigens, lack of the recipient’s immune response and consequently protection of an encapsulated cell from destruction were also reported for microbeads with a highly dense membrane composed of agarose and poly(styrene sulphonic acid) (PSSa) mixed gel [24]. There is an evident effect of the autoimmune status on the biocompatibility of the devices used for cell encapsulation. Numerous data [4–6,61,67] indicate that encapsulated xenogeneic islets, regardless

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of the capsule model, readily reverse streptozotocininduced diabetes in mice, and that the islet function is maintained for many months. Eventual failure of microcapsules transplanted into animals with chemically induced diabetes did not result from immune attack, as capsules retrieved from those animals are free-floating and they do not show any signs of fibrosis. The failure most likely results from islet death within the capsule due to nutrient deficiency [6]. However, with a few exceptions [3,98], longterm normoglycemia has not been demonstrated in autoimmune NOD mice receiving islets encapsulated in classical barrier systems [67,83,99–103]. In contrast to chemically induced diabetes, the failure of encapsulated islets transplanted into NOD mice resulted from an immune or autoimmune attack. Capsules retrieved from those animals stick together and demonstrate marked fibrosis around the capsule surface [6]. Histological sections of semipermeable hollow fibres composed of a hydrogel of a polyacrylonitrile–sodium methallylsulphonate copolymer (AN69) with encapsulated xenogeneic islets retrieved from the peritoneal cavities of recipient NOD mice showed surrounding inflammation with adherent cells and fibroblastic reaction [21]. Regardless of the fibroproductive reaction, the transplantation of AN 69 encapsulated islets reversed the diabetic state in 37% of autoimmune NOD mice. On the other hand, using multicomponent microcapsules with the correct choice of pore size and membrane thickness, Wang et al. [60] demonstrated normoglycemia in NOD mice for up to 241 days, suggesting an effective protection of the graft against autoimmune attack by the NOD mice immune system. Siebers et al. [104] reported that immunization with donor islets did not influence the survival of an encapsulated graft in normal rats. In contrast, only short-term function of encapsulated graft was observed after application into autoimmune animals (BB rats). The authors suggest an influence of macrophages, which are known to be involved in the very early stages of islet destruction in BB rats and which are of utmost importance in the formation of the pericapsular infiltrate. Additional factors that affect cell adhesion are surface roughness and charge. It was shown that membranes with smooth external surfaces lasted for more than 10 weeks in streptozotocin-induced diabetic rats with minimal fibrotic reaction, whereas rough surfaces elicited significant fibrotic response in

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less than 1 week. Thus, if surface wrinkles are minimized the stimulation of fibrosis overgrowth is reduced [80]. Using atomic force microscopy for analysis, Xu et al. [105] reported that multicomponent capsules could be prepared with different morphologies and surface roughness depending on the composition of the cation solution; namely, on the mole ratio of anti-gelling and gelling cations [Na 1 ] / [Ca 21 ]. Cells interact directly with charged surfaces through cell surface proteoglycans. The way to decrease unwanted cell adsorption is to shield the charged surface by the use of a non-ionic polymer. Sawhney and Hubbell [9] documented in a mouse intraperitoneal implant model reduced adsorption of albumin, fibrinogen and complement components and consequently lowered fibroblast adhesion by shielding the surface charge of alginate / PLL capsules using a non-ionic water-soluble poly(ethylene glycol). On alginate / PLL microcapsules, 59% of cells from a total adherent cell density were loosely attached, but 41% appeared well spread. In contrast, only 3.5% of attached cells and no signs of spreading were observed with alginate / PLL-g-MPEG microspheres. The surface quality of artificial membranes can be modified to fit the purpose. The permeability and adhesivity of hemocompatible acrylonitrile membrane AN69 was modified by adsorption of a hydrophilic copolymer or by a protein coating. Protein coating mainly decreased the diffusion of glucose and insulin, whereas glucose diffusion was preserved and that of insulin doubled after adsorption of a copolymer onto the membrane. In addition, copolymer coating increased considerably the adhesion of macrophages from peritoneal liquid [68]. The fibrotic surface cell reaction was avoided if pentalayered microcapsules containing alginate / PLL-g-MPEG were implanted instead of classical alginate / PLL capsules [9].

3. Immunoisolation and immunoprotection The effectiveness of immunoisolation and consequently satisfactory immunoprotection of encapsulated living cells in all immunoisolation devices depends on membrane permeability. This is closely linked with the pore size of the capsule membrane.

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To provide proper immunoisolation, the bulk of the surface area must have a pore size smaller than all potentially immunotoxic substances. The best known among them are alloantibodies, xenoantibodies, autoantibodies, components of the complement system and cytokines [60,106]. However, the precise knowledge of the involvement of other potentially cytotoxic substances such as oxygen radicals, nitric oxide and some other substances which might be involved in the death of encapsulated cells or in insufficient production of biosubstances is still missing. Not only the pore size, but also non-homogeneous pore distribution, is a problem associated with the long-lasting ability of encapsulated cells to supply the host with a sufficient quantity of the selected protein [10,60]. If there is a considerable heterogeneity in permeability among capsules, the implanted cells are protected against the cytotoxic effect of immunoglobulins, components of the complement system and against selected cytokines to a varying degree depending on the individual capsule. Uludag et al. [10] reported significant hydroxymethyl methacrylate–methyl methacrylate (HEMA–MMA) capsule-to-capsule variation in the secretion of a 1 antitrypsin (52 kDa) and fibrinogen (340 kDa), as well as in their relative secretion ratio. Despite the fact that the average capsule molecular weight cutoff was determined as 100 kDa [58], almost 40% were still permeable to fibrinogen. Recently, a new multicomponent system of an entrapper model was proposed offering the possibility of a correct choice of pore size and membrane thickness [60].

3.1. Penetration of antibodies and components of the complement system The physical barrier provided by the capsule may be sufficient to prevent contact between the transplanted tissue and host immunocytes [107], but the complement-mediated lysis of encapsulated graft, which is responsible for graft failure, suggests that the microcapsule membrane is permeable to components of the complement system and immunoglobulins. IgG is the major and most abundant immunoglobulin in normal serum and it is an important immunoglobulin in the defense system against foreign antigens. It has a molecular weight of

approximately 150 kDa compared with 950 kDa and 300–400 kDa for IgM and IgA, respectively, the two other most important Ig classes. Immunoglobulin penetration into the capsule depends on the exclusion limit of the capsule membrane [2,6]. If the selected membrane blocks IgG penetration, it is unlikely, but not excluded, that larger substances or even immune cells would come into contact with the encapsulated graft. If antibodies, by chance, penetrate inside the capsule and bind to encapsulated cells, the activation of the complement cascade starts. Such membranes which block the passage of antibodies preserve pancreatic beta cells from complement-dependent cytoxicity [108]. The complement system comprises a group of more than 30 serum and cell surface proteins with MW in the range of 25 to 750 kDa. One of them, the C5 convertase, generated by either the alternative or the classical pathway, initiates the activation of the terminal components of the complement system, culminating in the formation of the cytocidal membrane attack complex (MAC). MAC mediates cytolysis by polymerizing on cell surfaces to form pores or by disrupting the integrity of the phospholipid bilayer in the membranes of encapsulated cells. According to the molecular weight, the penetration of the largest component of complement (C1q; MW 410 kDa) would be the factor limiting the complement-mediated lysis of encapsulated grafts. It seems, however, that the molecular weight is only one aspect determining the diffusion of molecules through the microbead membrane. Using 5% agarose microbeads, Iwata et al. [24] observed that the rate-determining step of encapsulated cell destruction is not diffusion of C1q, but the penetration of IgG. Similarly, Kulseng et al. [109] described the penetration of transferrin (MW 81 kDa) into microcapsules impermeable to tumor necrosis factor (TNF) (MW 51 kDa). It has been shown that the molecular weight of PLL in alginate / PLL capsules affects the permeability of the membrane to hemoglobin and to albumin [110], but not to IgG [111]. Molecular shape, tridimensional structure, charge and concentration outside and inside the device are other factors influencing the diffusion rate of molecules from the extracapsular space into the microcapsular interior [67,109]. Despite extensive studies of the permeability of

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alginate / PLL / alginate capsules [110–113], the results from different laboratories are rather contradictory. This contradiction might partly arise from minor modifications of the encapsulation procedure and it may also be related to the technical limitation of measuring technologies as well as selection of test solutes [2]. It was reported that only alginate beads with an outer surface not coated with PLL are permeable to IgG [111]. On the other hand, the results of inverse column size exclusion chromatography (SEC) suggest that the standard alginate / PLL / alginate capsules are always permeable to IgG immunoglobulin [2]. Halle´ et al. [111] observed that alginate–poly-Llysine membranes are not permeable to IgG when PLL is used ranging from 21 to 390 kDa. Heald et al. [114] compared the ability of alginate microcapsules with a high content of mannuronic acid (MALG) and with a high content of guluronic acid (GALG) to prevent complement-mediated lysis of encapsulated porcine islets of Langerhans. Either poly-L-lysine (PLL) or poly-L-ornithine (PLO) were used for the stabilization of microcapsules and the microcapsules were exposed overnight to xenogeneic human or autologous pig serum. Cell metabolism was determined by MTT assay. No significant differences in the ability to prevent complement-mediated lysis were found among all four types of microcapsules tested (MALG–PLL, MALG–PLO, GALG–PLL, GALG–PLO). However, the encapsulation process was imperfect. In two of the six experiments a significant reduction in metabolism occurred after cultivation of porcine islets encapsulated within MALG–PLL, MALG–PLO and GALG–PLL membranes with human serum [114]. The encapsulated cell survival is inversely proportional to the diffusion coefficient of IgG in the immunoprotecting material. Five percent agarose microbeads restrict the diffusion of IgG, but some antibodies can still penetrate as the microencapsulation of hamster islets in 5% agarose prolonged xenograft survival only in recipients with a low level of pre-formed anti-hamster antibodies. If a high level of anti-hamster antibodies was induced in recipients by a prior injection of free hamster islets, the encapsulated graft was destroyed by complementmediated lysis [67]. If the network of an agarose hydrogel membrane was rendered denser by increas-

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ing the concentration of agarose to 7.5%, islet xenotransplantation was successful in normal animals with a high level of pre-formed antibodies, but failed in NOD mice with an acute autoimmune disease [67]. This suggests that, in addition to autoantibodies, some other factors such as cytokines released by activated immunocytes are responsible for the destruction of encapsulated xenografts in autoimmune organisms. Effective protection of encapsulated xenogeneic islets of Langerhans against immune attack from autoimmune NOD mice was reported using microbeads composed of agarose and poly(styrene sulphonic acid) (PSSa) mixed gel. PSSa microbeads have a heterogeneous membrane composed of a thin polyanion complex layer and a thick agarose / PSSa mixed gel layer which prevents the release of antigens from the interior of the capsule and penetration of cytotoxic substances into the capsule [67]. It was found that these microcapsules are unable to prevent the permeation of IgG for more than a few days, but protect the encapsulated cells from cytolytic attack by the complement. It is assumed that the cytolytic complement activity is lost during permeation through the microcapsule, probably because of the strong charge interaction of PSSa in the membrane with components of the complement system [115]. Another successful transplantation in poly(styrene sulphonic acid) (PSSa) microbeads involved dopamine-secreting PC12 cells grafted into a xenogeneic brain [116]. A Ba 21 cross-linked alginate matrix effectively immunoisolates encapsulated rat and porcine islets as complement-mediated lysis was not observed after incubation with fresh xenogeneic human plasma [53]. Also, calcium alginate beads, which were subsequently coated with a membrane made by a transacylation reaction between propylene glycol alginate and human serum albumin, represent an effective barrier and prevent diffusion of immunoglobulin and / or components of the complement system. No lysis was observed after a 6-h incubation of encapsulated HLA-A2 cells cultured in media with anti-HLA-A2 IgG and complement [26]. IgG was easily able to enter the multicomponent capsules with an exclusion limit of 230 kDa and even 110 kDa, while the entry of IgG was markedly reduced for capsules with a 40 kDa exclusion limit [6,61]. A negatively charged AN69 membrane with a molecu-

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lar mass cut-off at 50 kDa and a pore size ranging from 1.9 to 5 nm was permeable to insulin (5.8 kDa) and glucose (0.18 kDa), while preventing the passage of immune cells and the diffusion of antibodies [93]. Surface roughness, which affects cell adhesion, does not necessarily influence the penetration of immunoglobulins inside the microcapsules. Halle´ et al. [111] observed that alginate–poly-L-lysine microcapsules with PLL of 390 kDa always have an irregular shape, with striations and / or craters, but are impermeable to IgG. Although morphological irregularities do not change the permeability of microcapsules to IgG, they could have other possible consequences for capsule durability, biocompatibility and immunoprotection [113].

3.2. Penetration of cytokines The effector phases of both innate and specific immunity are in large part mediated by cellular protein hormones (growth factors) called cytokines. In innate immunity, the effector cytokines are mostly produced by mononuclear phagocytes and are therefore often called monokines. Monokines elicit an inflammatory reaction that helps to eradicate potentially dangerous foreign cells or materials. In addition to activated macrophages and monocytes, sources of pro-inflammatory cytokines include fibroblasts, endothelial cells, dendritic cells, T cells and NK cells. Cytokines which might eventually be involved in the inflammatory reaction against transplanted encapsulated cells are interferon-a (IFNa, MW 18 kDa), interferon-b (IFNb, MW 18 kDa), interleukin-15 (IL-15, MW 13 kDa), interleukin-12 (IL-12, MW 35–40 kDa), tumor necrosis factor-a (TNF-a, MW 17–51 kDa), interleukin-1 (IL-1, MW 17 kDa), interleukin-6 (IL-6, MW 26 kDa), interleukin-10 (IL-10, MW 20 kDa) and some chemokines with MW 8–10 kDa. The principal mediators of acute inflammation are IL-1, TNF-a and IL-6. Interleukin-1 was first defined as a co-stimulator of T cell activation derived from activated mononuclear phagocytes. However, it is now clear that the principal function of IL-1, similar to that of TNF, is to mediate the host inflammatory response in innate immunity. It does not produce tissue injury by itself, but potentiates tissue injury caused by TNF. Upon

stimulation, monocytes / macrophages secrete IL-1, which activates a variety of cells involved in inflammation, such as lymphocytes, granulocytes and fibroblasts. IL-6 is a cytokine with pleiotropic activities that is synthesized by many different cell types including mononuclear phagocytes, vascular endothelial cells, fibroblasts, keratinocytes, some activated T cells, glial cells and other cells in response to IL-1 and, to a lesser extent, TNF. It causes hepatocytes to synthesize several plasma proteins, such as fibrinogen, that contribute to the acute phase response. IL-6 serves as a growth factor for activated B cells, T cells and as a co-factor with other cytokines for the growth of early bone marrow hematopoietic stem cells. Except for tumor cells that produce IL-6 constitutively, normal cells do not produce IL-6 unless appropriately stimulated. The production of IL-6 is positively or negatively regulated by a variety of stimuli including exposure to some polymeric materials. Overproduction of IL-6 due to chronic stimulation by a foreign material may be responsible for pathogenesis and / or several symptoms of a variety of diseases, including autoimmune diseases, malignancies, and viral diseases [117]. IL-6 is known to be induced by IL-1b and TNF in mouse islets of Langerhans and could be involved in the destruction of beta-cells [109]. TNF is a multipotent modulator of immune system activation. The major cellular source of TNF is activated mononuclear phagocytes, antigen-stimulated T cells, activated NK cells and activated mast cells. TNF acts locally as a paracrine and autocrine regulator of leukocytes and endothelial cells, it exerts direct anti-tumor effects and has many other biological functions [118,119]. The effects of macrophage-derived cytokines and growth factors occur in two phases. Acutely, TNF, IL-1 and macrophage-derived chemokines augment inflammatory reactions initiated by T cells. Chronically, these same cytokines also stimulate fibroblast proliferation and collagen production. The consequence of these slow actions of cytokines and growth factors released from chronically activated macrophages is the formation of fibrous tissue around the transplanted device, a process called fibrosis. Fibrosis is the outcome of a chronic delayed type of hypersensitivity reaction (DTH) when elimination of antigen and its rapid resolution are unsuccessful. Ideally, the capsules must be constructed in a way

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that isolates the allografts or xenografts not only from the components of the complement system and from immunoglobulins, but also from cytokines such as TNF, IL-1 and IL-6 [109]. However, this might be a difficult task to perform. The encapsulation should simultaneously protect the cells against different cytotoxic substances, including cytokines, but should allow penetration of molecules of similar or even larger molecular weight important for cell survival or synthesis of desired encapsulated cell bioproducts [61,109]. Due to the rather low molecular weight, cytotoxic cytokines and nitric oxide might readily pass through the semipermeable membrane and damage the encapsulated cells. Some lymphokines (interferon-g and IL-2), monokines (IL-1b, tumor necrosis factor) and soluble factors such as nitric oxide involved in macrophage cytotoxicity cause both degeneration of transplanted cells and inhibition of bioproduct release from encapsulated graft [109,120–126]. When cells are encapsulated and transplanted in a potentially immunogenic material such as alginate / PLL, pro-inflammatory cytokines might appear in considerable concentrations near the capsules as a result of an immunological response against the polymeric material [63]. If the microcapsule membrane is permeable to the immunomodulatory polypeptide products of lymphocytes, macrophages / monocytes, and natural killer cells which recruit to the surroundings of the microcapsule, a number of cytokines (interferong and interleukin-2) and monokines (interleukin-1 and tumor necrosis factor) induce the degeneration of encapsulated graft [80]. The permeability of an alginate / PLL microcapsule membrane to immunomodulatory cytokines released by lymphocytes, macrophages / monocytes, and natural killer cells was reported by Soon-Shiong et al. [107]. Wiegand et al. [94] observed necrosis of alginate / PLL encapsulated islets after 24 h of coculture in the presence of activated macrophages, and Cole et al. [99] reported inhibition of insulin release and progressive damage to alginate / PLL encapsulated islets cultured in the presence of IL-1b, tumor necrosis factor-a and interferon-g. Increased exposure to poly-L-lysine has been shown to reduce the permeability of the alginate capsule membrane [127]. Kulseng et al. [109] reported that alginate polycation capsules may be able to give some

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protection against toxic concentrations of cytokines even though the cytokines are able to pass through the capsule membrane. They studied the alginate / PLL capsule membrane permeability and selectivity to IgG (150 kDa), transferrin (81 kDa), interleukin-1 (IL-1, 17 kDa), interleukin-6 (IL-6, 26 kDa), tumor necrosis factor (TNF, 51 kDa), interleukin-1b (IL1b, 17.5 kDa), and insulin (5.8 kDa) by measuring the binding of 125 I-labeled proteins to encapsulated antibody-coated magnetic monodispersed polymer particles (Dynabeads). Alginate capsules with a high guluronic (high G) or high mannuronic (high M) acid content were exposed for 10 min to poly-Llysine (PLL) or to poly-D-lysine (PDL) of concentrations varying from 0.05 to 0.2%. IL-1 and IL-6 were found to penetrate all of the different capsule types. The observation that the high-G capsules could be made impermeable to TNF and still allow transferrin, with a larger molecular mass, to pass through supports the conclusion that not only the molecular mass, but also the molecular shape and its tridimensional structure is involved in the permeability of microbead membranes. TNF in its active form is a trimeric molecule consisting of three identical subunits of 17 kDa molecular mass, while transferrin is formed by one single polypeptide chain [109]. One of the reasons why it is difficult to compare and interpret data from different laboratories is the impurity of the polymer used for encapsulation. Alginate is a linear polysaccharide composed of mannuronic and guluronic acids. Their quantity and proportion, which are responsible for the biochemical properties of the alginate, its biocompatibility and mitogenicity, vary extensively from batch to batch. The use of purified material is necessary in order to reveal the parameters that control the biocompatibility of the implanted material. Commercially available alginate can be purified using a patented electrophoretic method, resulting in an amitogenic alginate suitable for clinical use [54,128]. Amitogenic alginate induces lower release of TNF-a and some other cytokines and subsequently lower fibroblast activation in vitro [54] and in vivo, and provides better long-term in vivo function than mitogenic alginate [31]. IL-1b entry into a multicomponent capsule formed by polyelectrolyte complexation of sodium alginate

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and cellulose sulphate (CS) with poly(methylene-coguanidine) hydrochloride in the presence of Ca 21 and Na 1 was significantly delayed, but not completely prevented, when the capsule exclusion limit was 3.2 kDa [61]. IL-1b was involved in the destruction of xenogeneic cells encapsulated within the acrylonitrile membrane AN69, since functional alteration together with a decrease in glucose-stimulated insulin release were seen during co-culture in the presence of macrophages and were similar in free and encapsulated islets [93]. Hydroxyethyl methacrylate–methyl methacrylate copolymer (HEMA– MMA) microcapsules are also permeable to IL-6, as encapsulated cells respond to IL-6 in the medium by an elevation of haptoglobin secretion [58]. The cytotoxicity of IL-1b for graft encapsulated in alginate poly-L-lysine microcapsules was also reported by Cole et al. [99]. In contrast to this observation, Tai et al. [129] and Zekorn et al. [130] reported that alginate / PLL microcapsules and capillary hollow fibers with a molecular mass cut-off of 50 kDa protected allogeneic and xenogeneic islets against the cytotoxic effect of IL-1b. As both alginate poly-L-lysine and AN69 membranes are negatively charged the explanation offered by Tai et al. [129] that the net negative charge of alginate– PLL repels IL-1b is probably not correct. Results concerning the intracapsular effects of cytokines are sometimes conflicting, reflecting the complexity of the permeability and biocompatibility problems. Moreover, antibodies against IL-1b did not protect encapsulated islets against the loss of insulin secretion, suggesting the involvement of other factors [93].

Acknowledgements This research was supported by the Ministry of Industry and Trade (grant No. PZ-Z2 / 24 / 99) and by the Ministry of Health (grant No. 5050-3). The ´ Jelınkova, ´ ´ PhD, author is grateful to Drs. Marketa ˇ´ Ivan Rıha, MD, PhD, and Pavel Rossmann, MD, PhD, for reviewing the manuscript.

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