University of Groningen. Immunoprotection of pancreatic islets Groot, Martijn de

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1 University of Groningen Immunoprotection of pancreatic islets Groot, Martijn de IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 24 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Groot, M. D. (24). Immunoprotection of pancreatic islets: in vitro studies into causes of microencapsulated graft failure Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 1 maximum. Download date:

2 CHAPTER THREE IMPROVED IMMUNOPROTECTION BY MODIFIED CAPSULE PERMEABILITY MARTIJN DE GROOT 1, BART J. DE HAAN 2, PAULA P. M. KEIZER 1, ANNELIES L. DRAAISMA 1, HENRI G. D. LEUVENINK 1, THEO A. SCHUURS 1, REINOUT VAN SCHILFGAARDE 1, PAUL DE VOS 2 1 Department of Surgery, Surgical Research Laboratory 2 Department of Pathology and Laboratory Medicine, Section Medical Biology Submitted SUMMARY Immunoprotection of pancreatic islets by microencapsulation involves separation of the graft from the immune system. Macrophage derived cytokines, however, probably still enter microcapsules and disturb graft function. This study was conducted to assess IL-1β, TNF-α and insulin diffusion across the membrane of microcapsules with different grades of permeability, in search of an optimal balance between protection and function. Microcapsules with four different grades of permeability were made by 5, 1, 15 or 2 minutes incubation in.1% poly-l-lysine (PLL). Permeability of capsules was assessed by measuring insulin release from encapsulated islets and cytokine release (IL-1β, TNF-α) from encapsulated macrophages. The protective properties were determined by measuring the insulin output from encapsulated islets after culture with IL-1β and TNF-α. Insulin releases remained largely unaffected with all grades of permeability, except for the stimulated insulin release by 2 min PLL encapsulated islets. Cytokine release from 5 min PLL encapsulated macrophages was reduced to 18 % and 43 % for IL-1β and TNF-α, respectively. The output of both cytokines was even further inhibited towards 2.5 % by 1 min PLL capsules and less than 1 % for the 15 and 2 min PLL incubated capsules. Cytokine treatment during culture reduced the insulin releases from 5 and 1 min PLL encapsulated islets, but not the insulin releases from 15 and 2 min PLL encapsulated islets. Exposure of low-g alginate beads for 15 minutes to PLL provides improved protection against cytokines compared to 5 or 1 minutes exposure to PLL, while maintaining encapsulated islet function.

3 5 CHAPTER THREE INTRODUCTION Microencapsulation of islets of Langerhans is a potentially effective method to prevent islet graft rejection without immunosuppressive therapy. Although some successes have been made, the general limited graft survival of the encapsulated islets restricts the techniques clinical applicability. Graft failure is associated with hypoxia, and with pericapsular cellular overgrowth that consists of macrophages and fibroblasts (5,13). Macrophages produce several toxic factors including cytokines, nitric oxide and free radicals. The macrophage-derived IL- 1β (17.5 kd) and TNF-α (51 kd) have been shown to be toxic towards islets, and may be small enough to enter alginate-poly-l-lysine-alginate (APA) capsules (3,9). To prevent the passage of these cytokines may well improve islet graft survival. Permeability of APA capsules depends on the composition of the alginate, the molecular weight of poly-l-lysine (PLL), the concentration of PLL, and the exposure time of alginate beads to PLL during the encapsulation procedure (6,8,1,14). In this study, different grades of permeability were achieved by varying the exposure time of alginate beads to PLL. The diffusion of IL-1β, TNF-α and insulin across the different PLL membranes was assessed, in order to find a permeability grade for microcapsules that prevents cytokine entrance, while maintaining the insulin release of encapsulated islets. MATERIALS AND METHODS Design of the study Different grades of permeability were achieved by incubating alginate beads for either 5, 1, 15 or 2 minutes in poly-l-lysine during the encapsulation procedure. Functional properties were assessed by measuring the insulin secretion of encapsulated islets during two days of culture and during a glucose challenge test. Permeability for cytokines was studied by measuring cytokine release by encapsulated macrophages. The protective properties of the microcapsules were determined by measuring the insulin secretion of encapsulated islets after two days of culture in the presence of IL-1β (1 U/ml) and TNF-α (1 U/ml). Animals, islet isolation and culture Male outbred Wistar rats (Harlan CPB, Zeist, The Netherlands) weighing gram served as islet source. All animals received care in compliance with the guidelines of the local Animal Care and Use Committee following National Institutes of Health guidelines.

4 IMPROVED IMMUNOPROTECTION Islets were isolated as previously described (16). Briefly, under halothane anesthesia, the abdomen was opened and the common bile duct was cannulated under non-sterile conditions. The pancreas was distended by infusion of 1 ml sterile Krebs-Ringer-Hepes solution containing 1 % Bovine Serum Albumin (BSA). Subsequently, the pancreas was excised and brought into a laminar flow cabinet. All further procedures were performed under sterile conditions. The pancreas was chopped into small pieces (1-2 mm) and digested at 37 C in two incubation steps of 1 minutes each, with 1. and.7 mg/ml collagenase P (Boehringer, Mannheim, Germany), respectively. Islets were seperated from exocrine tissue by centrifugation using a discontinuous Dextran gradient (15) and further purified by handpicking. Prior to encapsulation, islets were cultured overnight in CMRL 166 (containing 1% fetal calf serum (FCS, Gibco), 8.3 mm glucose, 1 mm Hepes and 1% Penicillin/Streptomycin (Gibco)) in portions of 1 islets per 25 cm 2 in non-treated petri-dishes (Greiner) at 37 C in humidified air containing 5% CO 2. Islet encapsulation Alginate (2.7 % w/v, Keltone LV; Kelco International, London, U.K., 6 % mannuronic acid) was mixed with cultured islets to a concentration of 1,5 islets/ml. Poly-L-lysine-alginate encapsulation was performed as described elsewhere (4), with the following modification: droplets were expressed in consecutive petri-dishes, instead of a funnel, containing 5 ml 1 mm CaCl 2 (1 mm HEPES, 2 mm KCl) solution. Islet encapsulation was performed in fractions of 7 minutes each; two minutes of droplet formation followed by a five minute incubation step in which the droplets were allowed to gelificate. After gelification the alginate-islets beads ( µm in diameter) were collected into 5 ml tubes, washed twice with KRH and kept in KRH until all droplets were generated. Then, all beads were pooled, divided into four portions and suspended for 1 minute in KRH containing 2.5 mmol/l CaCl 2. The poly-l-lysine (PLL) membrane was formed by incubating the four fractions of alginate beads in a.1% PLL solution for either 5, 1, 15 or 2 minutes (poly-l-lysine-hcl, molecular weight 22,; Sigma). Three successive washing steps of 1 minute each with Ca 2+ -free KRH containing 135 mmol/l NaCl removed the non-bound PLL. The outer alginate layer was subsequently applied by a 1-min incubation in ten-times diluted alginate solution. Three consecutive washings with Ca 2+ - free KRH removed the excessive alginate. 51

5 52 CHAPTER THREE Macrophage encapsulation Rat alveolar macrophages (NR 8383 cell line, ATCC, Maryland, USA) were cultured continuously in RPMI (1 x 1 6 cells/ml; RPMI containing 15 % FCS, 8.3 mm glucose, 1 % L-glutamine and 1 % Penicillin/Streptomycin) which was refreshed twice a week. Macrophage viability was assessed by tryphan blue exclusion staining and cell preparations were found to consist for more than 9% of viable cells. Macrophages were stimulated one hour prior to harvesting with LPS (.5 µg/ml cell suspension). Twelve million macrophages were spun down (2 g, 7 min) after which the supernatant was completely removed. The remaining pellet was mixed with.5 ml alginate and put in a 5 ml syringe. Droplets (~2 µm) were generated by gentle pressure (1 mm Hg) in 2 minutes and allowed to gelificate for 5 minutes in 1 mm CaCl 2. The CaCl 2 was removed and macrophage-containing beads were washed three times with RPMI. During the washing steps small beads (< 5 µm) and fragments were washed away. The pellet, comprised of macrophage containing Ca 2+ -alginate beads (~2 µm), was carefully resuspended in 2 ml of alginate and encapsulation was proceeded as described for islets. Culture Equal portions of free islets (~ 1 per 25 cm 2 ), encapsulated islets (~ 1 per cm 2 ) and encapsulated macrophages (concentration varied per experiment) were cultured. The culture period was 2 days and was performed in petri-dishes filled with 8 ml of CMRL 166 (containing 1% FCS, 8.3 mm glucose, 1 mm Hepes and 1% Penicillin/Streptomycin), at 37 C in humidified air containing 5% CO 2. Free and encapsulated islets were also cultured in the presence of 1 U/ml IL-1β and 1 U/ml TNF-α (both R&D, Minneapolis, USA). After two days, culture medium samples were taken. Islet culture samples were analysed for rat insulin using a radioimmunoassay (Linco, Ede, The Netherlands). Macrophage culture samples were analysed for IL-1β and TNF-α with the Quantikine M Immunoassay for rat IL-1β and TNF-α (R&D). Since the amount of encapsulated macrophages varied per experiment, cytokine releases were expressed relatively to the cytokine output of macrophages in capsules without PLL. Glucose challenge test Encapsulated islets were tested for their glucose-induced insulin response in three separate samples of 2 randomly picked encapsulated islets per experiment. Islets were pre-incubated for 45 minutes at 37 C in 2 ml Krebs- Ringer-Bicarbonate (KRB) containing.25 % BSA and 2.75 mm glucose, gassed with 95 % O 2 and 5 % CO 2. The in vitro insulin release was then assessed by

6 IMPROVED IMMUNOPROTECTION three consecutive incubations of (i) 45 minutes in 2.75 mm glucose in KRB, (ii) 45 minutes in 16.5 mm glucose in KRB and.1 % IBMX (Iso-Butyl-Methyl Xanthine, Sigma) (iii) 45 minutes in 2.75 mm glucose in KRB. At the end of each incubation, incubation media were completely removed for insulin determination by a radioimmunoassay. The insulin releases were expressed as nanograms of insulin/ml/2 islets/45 minutes. Statistical analysis Results were statistically evaluated using the Student t-test with a onetailed distribution (* significance level <.5). Homoscedasticity was tested and affirmed using Bartlett s test. 53 RESULTS Capsule permeability and insulin output of encapsulated islets Functional properties of the different PLL capsules were tested by determining both the insulin accumulation during culture (FIGURE 1) and the insulin release in response to a glucose challenge (FIGURE 2). Increasing the PLL incubation time lead to some decrease in insulin release during culture, however, this was not significant (FIGURE 1, open bars). Insulin release was reduced for all permeability grades in the presence of cytokines, and this reduction was significant for 5 and 1 min PLL. 1 * * Insulin (ng/ml) 5pll 5pll+ 1pll 1pll+ 15pll 15pll+ 2pll 2pll+ FIGURE 1. Concentration of insulin (ng/ml ± sem) secreted by encapsulated islets during two days of culture with cytokines (closed bars) and without cytokines (open bars). Significant differences (P<.5, student t-test, n=6) are denoted with asterix (*).

7 54 CHAPTER THREE 35 Insulin (ng/ml) A 35 free islets basal stimulated recovery 5 min PLL a B basal stimulated recovery * free islets +cytokines 5 min PLL +cytokines * C 35 E 35 basal stimulated recovery 1 min PLL basal stimulated recovery 15 min PLL a D basal stimulated recovery 1 min PLL +cytokines F basal stimulated recovery * b 15 min PLL +cytokines G 35 basal stimulated recovery 2 min PLL H basal stimulated recovery b 2 min PLL +cytokines I a basal stimulated recovery J basal stimulated recovery b

8 IMPROVED IMMUNOPROTECTION FIGURE 2. (previous page) The insulin secretion (ng/ml/2 islets/45 min ± sem) of free and encapsulated islets during a glucose challenge test after two days of culture with and without cytokines. Significant differences (P<.5, student t-test, n=6) between stimulated insulin secretions are denoted with asterix (*).The stimulated insulin secretion of 2 min PLL encapsulated islets (2I) was significantly lower compared to free islets and 1 min PLL encapsulated islets (a). The stimulated insulin secretion of 2 min PLL encapsulated islets after cytokine culture (2J) was significantly lower compared to 1 and 15 min PLL encapsulated islets after cytokine culture (b). With the glucose challenge test, no significant differences were found between the stimulated insulin releases of free islets (26.64 ± 4.23 ng/ml), 5 min PLL (22.4 ± 3.86 ng/ml) and 1 min PLL (23.47 ± 3.31 ng/ml) encapsulated islets (FIGURE 2A, C and E). With 15 min PLL (14.42 ± 2.45 ng/ ml, Figure 2G) the decrease with approximately one-third as compared to 5 and 1 min PLL was substantial, but it was statistically not significant. The only significant decrease in the stimulated insulin release was found with 2 min PLL encapsulated islets (FIGURE 2I; ± 3.25 ng/ml) when compared to free islets and to 1 minutes PLL encapsulated islets. Capsule permeability and cytokine output of encapsulated macrophages To assess the diffusion of IL-1β and TNF-α from microcapsules with different grades of permeability, macrophages were encapsulated in capsules with, 5, 1, 15 and 2 min PLL coating, and cultured for two days. Macrophage encapsulation was performed in two steps. First, single cell-containing alginate beads were generated (~2 µm), after which these beads were encapsulated just like islets. Figure 3 shows that this two-step encapsulation method resulted in complete encapsulation of all single cells. 55 A ~ 75 µm B FIGURE 3. Macrophages-containing alginate bead encapsulated in APA microcapsules by a two-step encapsulation procedure (original magnification A: 4x and B: 1 x).

9 56 CHAPTER THREE The permeability of capsules for cytokines is illustrated by the release of IL-1β and TNF-α by encapsulated macrophages during culture. The amount of cytokines (ranging from 2 ng/ml) was related to the cytokine output of macrophages in capsules without a PLL layer ( min PLL). As shown in Figure 4, the amount of cytokines found in the medium of 5 min PLL encapsulated macrophages was reduced towards 18 % and 43 % for IL-1β and TNF-α, respectively. Cytokine release from 1 min PLL encapsulated macrophages was reduced to approximately 2.5 % for both cytokines. Increased PLL incubation times (15 and 2 minutes) resulted in an IL-1β and TNF-α output of less than 1 % when compared to the ouput from macrophages in microcapsules without PLL. Microcapsules and their ability to protect against cytokine mediated dysfunction In order to test the microcapsules with different grades of permeability for their ability to protect against IL-1β and TNF-α, encapsulated islets were cultured in the presence of IL-1β (1 U/ml) and TNF-α (1 U/ml). As a result of the cytokine treatment, free, 5 and 1 min PLL encapsulated islets showed significantly lower insulin releases, both during culture (Figure 1, closed bars) and during the glucose challenge test (Figure 2B, D and F). The insulin release from 15 min PLL (1.42 ± 2.63 ng/ml) and 2 min PLL (4.68 ± 1.81 ng/ml) encapsulated islets after cytokine culture, however, did not differ significantly from controls, neither during culture (Figure 1) nor during the glucose challenge test (Figure 2H and J). 75 percentage Il-1ß A a a, b a, b, c a, b, c 5 PLL 1 PLL 15 PLL 2 PLL FIGURE 4. Percentages (± sd) of IL-1β (A) and TNF-α (B) released by encapsulated macrophages during two days of culture. Cytokine release of macrophages encapsulated in microcapsules without PLL was set on 1%. Significant differences (P<.5, student t- test, n=4) are denoted with a (P<.5 vs PLL), b (P<.5 vs 5 PLL) and c (P<.5 vs 1 PLL). 75 percentage TNF-α a a, b a, b, c a, b, c 5 PLL 1 PLL 15 PLL 2 PLL

10 IMPROVED IMMUNOPROTECTION 57 DISCUSSION The macrophage derived cytokines IL-1β and TNF-α may contribute to the limited survival of the encapsulated islet graft (3,9). Capsule permeability can be reduced to prevent cytokine diffusion across the membrane, but this could also affect insulin release from encapsulated islets. In this study capsules with varying grades of permeability were analysed for their ability to provide protection against the deleterious effects of cytokines while maintaining encapsulated islet function. The only significant reduction of the stimulated insulin release was observed with 2 min PLL encapsulated islets. The insulin release from encapsulated islets during culture was not significantly affected by the reduction in capsule permeability (i.e. from 5 towards 2 min PLL), although a decreasing trend was observed. Apparently, the insulin release in response to a constant level of glucose (8.3 mm) is not affected by 2 min PLL capsules, whereas the potential insulin release of islets in response to a high level of glucose (16.5 mm) is. Thus, the lowest permeability not associated with impaired insulin output from encapsulated islets is achieved by incubating alginate beads for 15 minutes in.1 % PLL. This is the first permeability study that combines encapsulated islets with encapsulated macrophages to determine the diffusion of insulin and cytokines, respectively. Molecules with different sizes such as antibodies, proteins, Bovine Serum Albumin, Dextran and haemoglobin have been used to study capsule permeability in general (2,6,7,12). These molecules can be employed for the initial evaluation of the membrane permeability, and as an indication whether other molecules, like cytokines, can pass the capsule. However, actual diffusion of molecules across the capsule membrane depends on more than the molecule size alone. Factors involved are charge, hydrophobicity, spatial structure and possibly even the ability of the molecule to interact with the capsule membrane through Van der Waal or hydrogen bonds. The preference for actual molecules rather than surrogate molecules was the rationale for using both encapsulated islets and encapsulated macrophages to assess membrane permeability for insulin and cytokines, respectively. Encapsulation of macrophages is, however, technically different from encapsulation of islets, because macrophages are single cells that may cause irregularities on the capsule surface (11,17). To solve this problem, we have used a two-step encapsulation protocol in which small singlecell-containing beads were made that were subsequently encapsulated just like islets. Complete encapsulation of single cells was achieved with this method, as evidenced morphologically by the absence of protruding cells (FIGURE 3) and functionally by the almost complete blockade of cytokine diffusion, as discussed

11 58 CHAPTER THREE below. By use of encapsulated macrophages, we found that 5 min PLL capsules were more permeable to TNF-α (43 % output) than to IL-1β (18 % output). This was not expected since the 51 kd TNF-α trimer is larger than, and the 17 kd monomers of TNF-α are comparable to, the 17.5 kd of the IL-1β molecule. The diffusional difference may be explained by the difference in electrochemical properties, which is basidic for IL-1β and acidic for TNF-α (personal communication A. Carter, R&D Systems Europe Ltd, UK). Our data show that both the Il-1β and the TNF-α output are equally decreased towards 2.5 % by 1 min PLL capsules, and to 1 % by 15 and 2 min PLL capsules. This is not in accordance with Kulseng et al., who, by use of dynal beads, found capsules exposed for 1 minutes (or more) to.1 % PLL to be permeable to IL-1β (17.5 kd), but not to TNF-α (51 kd) (1). Based on insulin releases and glucose oxidation of islets after culture with cytokines, King et al. found their capsules to be permeable not only to IL-1β ( U/ml), but also to TNF-α (1 U/ ml) (8). The controversial results may be attributed to differences in methodology, i.e. a cytotoxicity study, the use encapsulated dynabeads and the use of encapsulated macrophages, but also to the use of different types of alginate (8,1). King et al. used alginate with a higher guluronic acid content (7 %) than Kulseng s (63 %) and our group (4 %). The lower concentration of guluronic acid is associated with a denser alginate structure and therefor a higher binding capacity to PLL (8,1). This explains the low permeability leading to the low diffusion rate of cytokines that is achieved here. Taken together, these results show that the decrease in permeability is associated with a decrease of cytokine diffusion across the capsule membrane. The best protective properties are obtained with 15 min PLL and 2 min PLL, where cytokine diffusion is most effectively inhibited. It is noted that although decreasing capsule permeability can decrease the cytokine diffusion towards 2.5 % and even 1 %, full protection is not achieved. A further decrease in permeability restricts the insulin release, which illustrates the dilemma between the preservation of the functional performance of encapsulated tissue on the one hand, and protection against cytokine mediated dysfunction on the other. Since even small amounts of cytokines produced by macrophages can be deleterious to islet function (1), other means of protection may prove necessary to increase graft survival in the macrophage rich peritoneal cavity. The combined data on insulin and cytokine releases by encapsulated islets and encapsulated macrophages, respectively, suggest that 15 min PLL encapsulated islets will provide the best protection against cytokines, without significantly affecting insulin output. This is corroborated by our findings that

12 IMPROVED IMMUNOPROTECTION the presence of IL-1β and TNF-α during culture is associated with significantly reduced insulin releases from 5 and 1 min PLL encapsulated islets, but not from 15 and 2 min PLL encapsulated islets. With 2 min PLL encapsulated islets the insulin output was restriced even in the absence of cytokines. Thus 15 minutes is the optimal duration of PLL-incubation to provide protection against cytokines while simultaneously maintaining adequate encapsulated islets function. This study provides support for the use of poly-amino acid treatment of alginate microcapsules, which may well become clinical applicable in the future. 59

13 6 CHAPTER THREE REFERENCES 1. Bendtzen K., Mandrup-Poulsen T., Nerup J., Nielsen J.H., Dinarello C.A., and Svenson M. Cytotoxicity of human pi 7 interleukin-1 for pancreatic islets of Langerhans. Science 232 (4757): , Brissova M., Lacik I., Powers A.C., Anilkumar A.V., and Wang T. Control and measurement of permeability for design of microcapsule cell delivery system. J. Biomed. Mater. Res. 39 (1): 61-7, Colton C.K., and Avgoustiniatos E.S. Bioengineering in development of the hybrid artificial pancreas. J. Biomech. Eng. 113 (2): , de Vos P., De Haan B.J., Wolters G.H., Strubbe J.H., and van Schilfgaarde R. Improved biocompatibility but limited graft survival after purification of alginate for microencapsulation of pancreatic islets. Diabetologia 4 (3): , de Vos P., van Straaten J.F., Nieuwenhuizen A.G., de Groot M., Ploeg R.J., De Haan B.J., and van Schilfgaarde R. Why do microencapsulated islet grafts fail in the absence of fibrotic overgrowth? Diabetes 48 (7): , Goosen M.F., O Shea G., Gharapetian H.M., chou S., and Sun A.M. optimization of microencapsulation parameters: semipermeable microcapsules as a bioartificial pancreas. Biotechnology and Bioengineering 27: , Halle J.P., Bourassa S., Leblond F.A., Chevalier S., Beaudry M., Chapdelaine A., Cousineau S., Saintonge J., and Yale J.F. Protection of islets of Langerhans from antibodies by microencapsulation with alginate-poly-llysine membranes. Transplantation 55 (2): , King A., Andersson A., and Sandler S. Cytokine-induced functional suppression of microencapsulated rat pancreatic islets in vitro. Transplantation 7 (2): , Kulseng B., Skjak-Braek G., Ryan L., Andersson A., King A., Faxvaag A., and Espevik T. Transplantation of alginate microcapsules: generation of antibodies against alginates and encapsulated porcine islet-like cell clusters. Transplantation 67 (7): , Kulseng B., Thu B., Espevik T., and Skjak-Braek G. Alginate polylysine microcapsules as immune barrier: permeability of cytokines and immunoglobulins over the capsule membrane. Cell Transplant. 6 (4): , Peirone M., Ross C.J., Hortelano G., Brash J.L., and Chang P.L. Encapsulation of various recombinant mammalian cell types in different alginate microcapsules. J. Biomed. Mater. Res. 42 (4): , 1998.

14 IMPROVED IMMUNOPROTECTION 12. Powers A.C., Brissova M., Lacik I., Anilkumar A.V., Shahrokhi K., and Wang T.G. Permeability assessment of capsules for islet transplantation. Ann. N. Y. Acad. Sci. 831: , Siebers U., Horcher A., Brandhorst H., Brandhorst D., Hering B., Federlin K., Bretzel R.G., and Zekorn T. Analysis of the cellular reaction towards microencapsulated xenogeneic islets after intraperitoneal transplantation. J. Mol. Med. 77 (1): , Thu B., Bruheim P., Espevik T., Smidsrod O., Soon-Shiong P., and Skjak- Braek G. Alginate polycation microcapsules. I. Interaction between alginate and polycation. Biomaterials 17 (1): , van Suylichem P.T., Wolters G.H., and van Schilfgaarde R. The efficacy of density gradients for islet purification: a comparison of seven density gradients. Transpl. Int. 3 (3): , Wolters G.H., van Suylichem P.T., van Deijnen J.H., and van Schilfgaarde R. Factors influencing the isolation process of islets of Langerhans. Horm. Metab. Res. Suppl. 25: 2-26, Zhou D., Sun A.M., Li X., Mamujee S.N., Vacek I., Georgiou J., and Wheeler M.B. In vitro and in vivo evaluation of insulin-producing beta TC6-F7 cells in microcapsules. Am. J. Physiol. 274 (5 Pt 1): C1356-C1362,

15 62 CHAPTER THREE