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Experimental Transplantation

MICROENCAPSULATION OF NEONATAL PORCINE ISLETS: PROTECTION FROM HUMAN ANTIBODY/COMPLEMENT-MEDIATED CYTOLYSIS IN VITRO AND LONG-TERM REVERSAL OF DIABETES IN NUDE MICE1

Rayat, Gina R.; Rajotte, Ray V.; Ao, Ziliang; Korbutt, Gregory S.2

Author Information

Surgical-Medical Research Institute, Department of Surgery, University of Alberta, Edmonton, Alberta, Canada

2 Address correspondence to: Gregory S. Korbutt, PhD, Surgical-Medical Research Institute, 1074 Dentistry/Pharmacy Building, University of Alberta, Edmonton, Alberta T6G 2N8, Canada.

1 Supported by the Canadian Diabetes Association, Alberta Heritage Foundation for Medical Research, the Medical Research Council of Canada, MacLachlan Fund of the University of Alberta Hospitals, and the Edmonton Civic Employees Charitable Assistance Fund (GSK). GSK is a recipient of a scholarship from the Canadian Diabetes Association and the Alberta Heritage Foundation for Medical Research.

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Abstract

*Abbreviations: NPI, neonatal porcine islets, HBSS, Hank’s balanced salt solution.

Islet transplantation is an attractive alternative treatment for patients with type 1 diabetes (1–7). However, there are currently two major barriers to overcome before islet transplantation can be offered as a therapy for diabetic patients. One is the poor availability of human pancreatic tissue and the other is the need for permanent immunosuppression to prevent rejection. In respect to the former, pigs are an attractive source of islets because they breed rapidly, have large litters, and porcine insulin has been used to treat patients with type I diabetes for many years. Recently, we developed a simple and reliable method to efficiently isolate large numbers of islets from neonatal pigs (8). These islets are easily maintained in tissue culture, correct diabetes in nude mice and have growth potential both in vitro and in vivo (8, 9). Although these characteristics make neonatal porcine islets (NPI) a very attractive source of insulin-producing tissue for clinical transplantation, we have demonstrated that NPI are susceptible to lysis mediated by human antibody and complement in vitro (10, 11). One strategy to prevent this type of immune response is to place islets within an immunoisolation device such as a microcapsule. Several materials and methods have been developed for microencapsulation. Experiments using microencapsulated islets have been difficult to reproduce due in part to problems related to the encapsulation process (12), purity/biocompatibility of the alginate (13), capsule diameter (14), and the resulting islet viability. Nonetheless, we have shown that microencapsulation enhances canine islet survival in vitro and in vivo (15, 16) as well as partially protects NPI xenografts after implantation in nonobese diabetic (NOD) mice (17). However it remains unknown whether microencapsulated NPI can be protected from the cytolytic effects of human antibody and complement or if they can survive in vivo and correct diabetes long-term. The aim of this study was to examine the immunoprotective properties of microencapsulated NPI in an in vitro human antibody/complement cytotoxicity assay and to assess their ability of reversing hyperglycemia in diabetic nude mice.

MATERIALS AND METHODS

Animals.

The 1- to 3-day-old Landrace-Yorkshire neonatal pigs (1.5–2.0 kg body weight) of either sex were used as islet donors. Male, inbred, athymic nude Balb/c mice (age 6 to 8 weeks; The Jackson Laboratories, Bar Harbor, ME) were used as recipients of NPI. Mice were rendered diabetic by intravenous injection of 90 mg/kg body weight alloxan (Sigma Chemical Co., St. Louis, MO; freshly dissolved in 1 mmol/liter hydrochloric acid) 4 to 5 days before transplantation. All recipients in the study had blood glucose levels above 20 mmol/liter. Blood samples were obtained from the tail vein to monitor glucose levels (Medisense glucose meter; Medisense Canada, Mississauga, Ontario, Canada). Animals were maintained in virus-antigen-free rooms with free access to sterilized tap water and pelleted food.

Preparation and encapsulation of NPI.

The method used to isolate NPI has been previously described (8). Briefly, neonatal pigs were anesthetized with halothane and subjected to laparotomy and exsanguination. The pancreas was removed, placed in Hanks’ balance salt solution (HBSS), cut into small pieces, and digested with 2.5 mg/ml collagenase (Sigma). After filtration through a nylon screen (500 μm), the tissue was cultured for 5 to 7 days in HAM’s F10 medium (Gibco, Burlington, Ontario, Canada) containing 10 mmol/liter glucose, 50 μmol/liter isobutylmthylxanthine (ICN Biomedicals, Montreal, PQ), 0.5% bovine serum albumin (fraction V, radioimmunoassay grade; Sigma), 2 mmol/liter L-glutamine, 3 mmol/liter CaCl2, 10 mmol/liter nicotinamide (BDH Biochemical, Poole, England), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C (5% CO2, 95% air).

After culture, NPI were washed with HBSS supplemented with 10 mmol/liter HEPES. Islets were then resuspended in 0.44 ml HBSS and 0.55 ml of 1.5% (w/v) highly purified alginate (Metabolex, Inc., Hayward, CA) dissolved in HBSS. The resulting islet/alginate mixture was vortexed to obtain a homogeneous solution, then transferred into a 1-ml syringe. Microcapsules (250–350 μm in diameter) were formed by passing the alginate/islet suspension through an electrostatic generator followed by collection in a 120 mmol/liter CaCl2 (10 mmol/liter HEPES, 0.01% Tween 20) solution for 10 min. The capsules were washed by gravity sedimentation in supplemented HAM’s F10 medium and cultured in the same medium at 37°C for 2 days. Controls included nonencapsulated islets which were also cultured in HAM’s F10 for an additional 2 days.

In vitro human antibody/complement-mediated cytotoxicity assay.

The method for the in vitro cytotoxicity assay has been previously described (10, 11). Briefly, encapsulated and nonencapsulated NPI were incubated for 24 hr in either heat-inactivated (complement-depleted) human AB serum or fresh human serum (containing complement) diluted 1:1 with HAM’s F10 medium (supplemented as above). After the incubation period, NPI were recovered from the petri dishes, washed in HAM’s F10 medium, and the preparations were assessed for recovery of cellular insulin and DNA contents as well as functional viability. Insulin content was measured after extraction in 2 mmol/liter acetic acid containing 0.25% bovine serum albumin (8). Samples were sonicated in acetic acid, centrifuged (800Ă—g, 15 min), then supernatants were collected and stored at −20°C until assayed for insulin content by ELISA (Boehringer Mannheim, Laval, PQ). For DNA content, aliquots were washed in citrate buffer (150 mmol/liter NaCl, 15 mmol/liter citrate, 3 mmol/liter EDTA, pH 7.4) and stored as cell pellets at −20°C. Before the assay, cell pellets were placed in 450 μl of lysis buffer (10 mmol/liter Tris, 1 mmol/liter EDTA, 0.5% Triton X-100, 4°C, pH 7.5), sonicated, supplemented with 25 μl of Proteinase K solution (8 mg/ml), vortexed, and incubated at 65 and 70°C for 45 and 10 min, respectively. Lysates were supplemented with 25 μl of RNAse A solution (10 mg/ml), vortexed and incubated for 1 hr at 37°C. Aliquots of 25 and 50 μl were assayed in duplicate by diluting them in 1 ml of DNA buffer (10 mmol/liter Tris, 1 mmol/liter EDTA, pH 7.5) and measuring fluorescence at a wavelength of 490 nm (excitation) and 515 nm (emission) after addition of 1 ml Pico Green reagent (1/200 dilution with DNA buffer). Samples were run in parallel with and diluted in proportion to a seven-point (0–400 ng/ml) standard curve that was generated using calf thymus DNA.

For assessment of in vitro functional viability, the secretory response to glucose of nonencapsulated and encapsulated NPI was determined using a static incubation assay (8, 10). The cultured fractions were recovered from the petri dishes, washed, and aliquots of 50–100 NPI were incubated for 120 min in 1.5 ml of RPMI medium supplemented with 2 mmol/liter L-glutamine, 0.5% BSA and either 2.8 mmol/liter glucose, 20 mmol/liter glucose, or 20 mmol/liter glucose plus 10 mmol/liter theophylline. Tissue and medium were then separated by gravity sedimentation and assayed for their respective insulin contents. The insulin content of the medium was expressed as a percentage of the total content (i.e., tissue plus medium). Stimulation indices were calculated by dividing the amount of insulin release at 20 mmol/liter glucose (±theophylline) by that released at 2.8 mmol/liter glucose.

Transplantation of encapsulated NPI and metabolic follow-up.

The morphology of encapsulated NPI immediately before transplantation was examined by dithizone staining (0.02% final concentration), immunohistochemical staining for insulin (8, 11), and electron microscopy (8). Before immunohistochemistry, samples were fixed in Bouin’s solution for 2 hr, washed three times with 70% ethanol, and embedded in paraffin. Next, 5-μm sections were stained with guinea pig antiporcine insulin antibody (1:1000 dilution; Dako Laboratories, Mississauga, Ontario, Canada) for 30 min followed by biotinylated goat anti-guinea pig IgG secondary antibody (1:200 dilution; Vector Laboratories, Burlingame, CA). For electron microscopic analysis, NPI were fixed in 2.5% (v/v) glutaraldehyde (Millonig’s buffer, pH 7.2), postfixed in 1.5% (w/v) OsO4, washed in distilled water, then dehydrated successively in 50, 70, 80, 90, and 100% ethanol before embedding in araldite. Sections were stained with lead citrate and uranyl acetate then subsequently examined in a Hitachi H 7000 (Hitachi Ltd., Tokyo, Japan) transmission electron microscope.

Nonencapsulated or encapsulated islets were transplanted under the kidney capsule or i.p. into alloxan-induced diabetic nude mice, respectively. Recipients were monitored for blood glucose levels once a week between 8:00 and 11:00 a.m. When the blood glucose level was ≤8.4 mmol/liter, the graft was deemed a success. At 40 weeks posttransplantation, an oral glucose tolerance test and then an i.p. glucose tolerance test 48 hr later were performed on NPI recipients with normalized basal glycemia and in normal controls. After a 2-hr fast, D-glucose (3 mg/g body weight) was administered as a 50% solution into nonanesthetized mice. Blood samples were obtained from the tail vein at 0, 15, 30, 60, and 120 min.

Five to 7 days after the GTTs, capsules were recovered by an I.P. lavage for dithizone and immunohistochemical staining or EM analysis (as described above). To confirm the efficacy of the encapsulated NPI at correcting diabetes, the pancreas of each recipient was assayed for insulin content as previously described (8). Similarly, animals with renal subcapsular grafts were nephrectomized and subsequently monitored to confirm a return to hyperglycemia.

Statistical analysis.

Data are expressed as means±SEM of n independent observations. Statistical significance of differences was determined using one-way analysis of variance (P <0.05 was considered significant).

RESULTS

Cytotoxicity of human serum and complement.

Incubation of nonencapsulated NPI for 24 hR in fresh human serum containing complement resulted in a 53% loss of cellular insulin mass (P <0.001) and a 51% reduction in recoverable DNA content (P <0.001) when compared with nonencapsulated NPI cultured in complement depleted heat-inactivated human serum (Table 1). In contrast, exposure of encapsulated islets to fresh human serum had no cytotoxic effect on the islets (91.0±8.0% insulin and 94.0±8.0% DNA recovered). Similar results were observed when encapsulated islets were exposed to heat-inactivated human serum (96.0±9.0% insulin and 89.0±3.0% DNA recovered). The secretory activity of NPI was tested by comparing the percentage of cellular insulin released at low glucose (2.8 mM), high glucose (20 mM), and high glucose plus 10 mM theophylline. Nonencapsulated NPI previously treated with complement deficient heat-inactivated human serum exhibited a mean stimulation index of 4.9±0.7 when comparing release at high glucose versus at low glucose. When exposed to 20 mM glucose in combination with 10 mM theophylline, the stimulation index increased to more than 22-fold. However, insulin secretion of nonencapsulated islets exposed to human serum containing complement was markedly altered (Table 2). Incubation at low glucose significantly (P <0.05) increased the secretory rate of nonencapsulated NPI pretreated with fresh human serum; whereas insulin secretion was significantly reduced when exposed to high glucose plus theophylline. Thus the calculated stimulation indices after incubation with either 20 mM glucose or 20 mM glucose plus 10 mM theophylline were significantly (P <0.001 and <0.05, respectively) lower compared to nonencapsulated NPI previously cultured with heat-inactivated human serum. In contrast, no significant differences were observed in the insulin secretory activity between controls (nonencapsulated NPI exposed to heat-inactivated serum) and microencapsulated NPI pretreated with either fresh or heat-inactivated human serum. Thus, the calculated stimulation indices of these groups were comparable.

T1-11
Table 1:
Effect of human serum and complement on nonencapsulated and encapsulated NPI
T2-11
Table 2:
Effect of human serum and complement on the insulin secretory capacity of NPI.

Transplantation of NPI into diabetic nude mice.

Figure 1 A illustrates the blood glucose values during posttransplant follow-up period in the following groups of recipients: (1) nonencapsulated NPI transplanted under the kidney capsule, (2) nonencapsulated NPI implanted i.p., and (3) encapsulated NPI placed i.p. After alloxan administration, all recipients exhibited blood glucose values above 20 mmol/liter. All animals transplanted i.p. with encapsulated NPI exhibited blood glucose values ≤8.4 mmol/liter within 8 weeks posttransplantation (Table 3, Fig. 1A). This metabolic state was maintained over the 40 weeks follow-up period. Similar results were achieved when nonencapsulated NPI were implanted under the kidney capsule of diabetic nude mice (Table 3, Fig. 1A). However, when nonencapsulated NPI were transplanted i.p., all recipients failed to achieve euglycemia and survived for only 21.0±4.0 days posttransplant.

F1-11
Figure 1:
A, Blood glucose values in alloxan-induced diabetic nude mice transplanted with NPI under the kidney capsule (nonencapsulated; •; n=10) or i.p. (encapsulated; Ă™; n=16 or nonencapsulated; â–ª; n=6). Blood glucose values during oral (B; oral glucose tolerance test); and i.p. (C; i.p. glucosre tolerance test) administration of glucose to nude mice transplanted with non-encapsulated (♦; kidney; n=10) or encapsulated (•; i.p.; n=12) NPI in comparison to age-matched normal control (â–ª; n=6) mice at 40 weeks posttransplantation. Values are means±SEM. Statistical significance of differences between groups was calculated by one-way analysis of variance. *P <0.05 versus normal controls.
T3-11
Table 3:
Metabolic follow-up of diabetic nude mice transplanted with 2000 NPI

Glucose tolerance tests were performed on normoglycemic recipients and controls at 40 weeks posttransplantation. Compared to age-matched normal control mice, animals implanted with encapsulated NPI (i.p.) or nonencapsulated NPI (kidney capsule) showed significantly lower (P <0.05) glucose values at 15 and 30 min in both OGTT and IPGTT (Fig. 1, B and C). There was no significant difference in the glucose values at all time points between the two transplant groups. In all groups, the glucose values at 120 min postglucose administration were not significantly different from the values at 0 min.

Examination of recovered encapsulated NPI at 42 weeks posttransplantation using dithizone revealed intact NPI with more intense dithizone staining compared to NPI before transplantation (Fig. 2 , A and B). Similarly, immunohistochemical (Fig. 2, C and D) and electron microscopic (Fig. 2, E and F) analyses, showed fully differentiated islets containing significantly more numerous insulin-producing cells and well-granulated endocrine cells compared to islets before transplantation that contain less endocrine cells and more nonendocrine cells. Throughout the experimental period, the capsules remained intact with no signs of fibrosis observed on the capsules’ exterior.

F2-11
Figure 2:
Morphology of encapsulated NPI before (A, C, and E) and after (B, D, and F) implantation to alloxan-induced diabetic nude mice. NPI were treated with dithizone (A and B), immunostained for the presence of insulin-positive cells (C and D), and processed for transmission electron microscopy (E and F).

In all recipients of nonencapsulated NPI (kidney capsule), removal of the islet graft-bearing kidney was followed by a rapid return to the diabetic state. The pancreatic insulin content of recipients of encapsulated NPI was <2% (<0.7 μg) of the insulin content of normal nude mice (38 μg; data not shown). These results demonstrate that the normoglycemia observed in these recipients was attributable to insulin production from grafts and not from residual pancreatic cells.

DISCUSSION

Neonatal porcine islets constitute an attractive source of insulin-producing tissue for clinical transplantation; however, several immunological obstacles must first be overcome. In particular, we have shown that human IgG and IgM natural xenoreactive antibodies bind to NPI cells (10, 11), NPI cells express the Galα (1, 3)Gal epitope (11), and NPI cells are susceptible to human antibody/complement-mediated cytolysis (10, 11). Furthermore, Galα (1, 3)Gal has also been detected on fetal porcine islet cell clusters (18, 19) whereas, other studies suggest that this epitope is not expressed on fully differentiated adult islet endocrine cells, but rather on intraislet ductal and endothelial cells (18–21). In addition, it has also been shown that natural xenoreactive antibodies bind to fetal (20) and adult (22, 23) porcine islet cells and exposure to human sera containing complement in vitro results in rapid destruction of these tissues (22, 23). Taken together, one potential approach to prevent humoral-mediated destruction of porcine islet grafts is to place them in an immunoisolation device to block access of human antibody and/or complement. In our study we examined whether a highly purified alginate microcapsule can protect NPI against the cytolytic effects of human serum and complement in vitro. Our data demonstrate that microencapsulated NPI are protected from the deleterious effects of fresh human serum containing complement because cellular insulin and DNA recoveries as well as insulin secretory activity were not adversely altered. In contrast, 24-hr exposure of nonencapsulated NPI to human serum and complement resulted in more than 50% loss of cellular insulin/DNA contents and an abnormal glucose responsiveness during an in vitro static incubation. The mechanism responsible for the protective effect of our alginate capsule is not known. We have however, determined that human IgM, but not IgG, is unable to diffuse across the alginate capsule used in our study (data not shown). It is therefore possible that the alginate capsule blocks the passage of human IgM and more importantly large molecular weight products of complement activation and thereby prevents destruction of the NPI. This concept is supported by the observation that human IgM as opposed to IgG is the predominant type of human antiporcine xenoreactive antibodies (24–30) and that alginate-poly-L-lysine microencapsulated sensitized sheep erythrocytes were protected against activated complement fragments (31). Furthermore, we previously reported (17) that the same alginate microcapsule improved survival and function of NPI as well as NOD mouse islets transplanted into diabetic NOD mice. Survival of microencapsulated NPI and syngeneic NOD mouse islet grafts was markedly improved when the NOD mice were given immunosuppressive therapy (17).

Diabetic nude mice were transplanted with encapsulated and nonencapsulated NPI to examine their efficacy at correcting diabetes in these animals. At week 8 posttransplantation, 100% of the mice receiving 2000 encapsulated (i.p.) or nonencapsulated (kidney capsule) NPI exhibited blood glucose levels ≤ 8.4 mmol/liter. These data are comparable to our previous findings where 2000 nonencapsulated NPI gradually achieved normoglycemia within 8 weeks after implantation under the kidney capsule of diabetic nude mice (8). Moreover, our study demonstrates that encapsulated NPI are equally effective at correcting diabetes in nude mice when transplanted in the peritoneum. Furthermore, 2000 nonencapsulated NPI were not able to reverse diabetes after i.p. implantation. This was an expected result because it has been previously reported that i.p. islet grafts are not very effective at correcting diabetes (32, 33). It is possible that our alginate microcapsule provides a support matrix for the NPI, thereby allowing them to survive and engraft in the peritoneal cavity.

However, unexpected results were observed during the glucose tolerance tests, because mice transplanted with encapsulated grafts not only exhibited comparable glucose tolerance to recipients of renal subcapsular NPI, but also had lower blood glucose levels at 15 and 30 min after glucose challenge when compared with normal controls. To our knowledge, this is the first report demonstrating that microencapsulated islet grafts placed i.p. can not only achieve long-term euglycemia but also glucose tolerance comparable to normal control animals. Our interpretation of these data is that the alginate capsule is highly biocompatible thereby permitting optimal islet engraftment/survival, avoiding fibrotic reactions around the encapsulated islets, resulting in sufficient islet cell mass to achieve normoglycemia and glucose tolerance. Furthermore, the phenomenon that both transplant groups had lower blood glucose levels compared to normal controls likely results from different insulin secretory properties of porcine islets compared with mouse islets, as previously shown in other studies when porcine islets were implanted into diabetic nude mice (8).

Although NPI placed in either site were unable to achieve euglycemia immediately posttransplant, they eventually developed the ability to establish and maintain euglycemia during the 40-week follow-up period. At the time of transplantation, NPI were not fully differentiated/matured, thus it is likely that after transplantation they exhibited growth and/or differentiation of new β cells until a critical mass was achieved to establish and maintain euglycemia. Our morphological data indirectly support this concept, because at the time of transplantation NPI were composed of relatively few insulin-positive cells, whereas several weeks after implantation the recovered grafts were largely composed of β cells. These observations indicate that microencapsulated immature NPI grafts continue to grow and differentiate when transplanted into the peritoneum of diabetic nude mice.

We conclude that microencapsulation protects NPI from the cytolytic effects of human antibody and complement. Because we have shown that NPI are susceptible to human natural antibody/complement lysis in vitro (10, 11), microencapsulation may be a strategy to prevent this form of humoral-mediated destruction. Furthermore, microencapsulation does not effect NPI survival and function after transplantation into the peritoneum of diabetic nude mice. Moreover, microencapsulated NPI grafts appear to mature and differentiate posttransplantation and they are not only capable of maintaining long-term euglycemia in diabetic nude mice, but can also achieve glucose tolerance. These results provide further information supporting the feasibility of using neonatal porcine islet transplantation as a treatment for diabetes in humans.

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