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ORIGINAL ARTICLE

Glucose Metabolism In Vitro of Cultured and Transplanted Mouse Pancreatic Islets Microencapsulated by Means of a High-Voltage Electrostatic Field

Aileen King, BSC
Stellan Sandler, MD, PHD
Arne Andersson, MD, PHD
Claes Hellerström, MD, PHD
Bård Kulseng, MD
Gudmund Skjåk-Braek, MSC, PHD

The aim of this study was to assess the function of mouse pancreatic islets microencapsulated using a high-voltage electrostatic field. Islets were microencapsulated in alginate/poly-L-lysine/alginate (APA) capsules and maintained in tissue culture. Rates of glucose oxidation and insulin release were then assessed. Glucose metabolism was also measured in microencapsulated islets retrieved after transplantation to normal syngeneic mice. The high-voltage electrostatic system made possible the production of uniformly sized microcapsules, which were smaller than those produced by co-axial air-jet systems. Nonencapsulated islets were used as controls. Empty microcapsules or islet-containing microcapsules were transplanted intraperitoneally and retrieved after 2 weeks for assessment of foreign-body reactions and glucose oxidation rates. After 1 day and 2 weeks in tissue culture, both control islets and microencapsulated islets increased their rates of glucose oxidation and insulin release 7- to 10-fold in response to an increase in glucose concentration from 1.7 to 16.7 mmol/l. Both empty and islet-containing microcapsules, retrieved 2 weeks after transplantation, showed high rates of glucose oxidation at both low and high glucose concentrations, suggesting overgrowth with metabolically active fibroblasts. Morphological studies indicated a marked foreign-body reaction on the surface of all transplanted microcapsules. The islets in cultured microcapsules had a normal histological appearance, whereas the islets within transplanted microcapsules showed a range of morphological appearances, from intact islets to cell debris. In conclusion, this study shows that mouse pancreatic islets survive and remain functionally competent for at least 2 weeks in vitro after microencapsulation in APA capsules generated in an electrostatic field. However, a foreign-body reaction with cellular growth on the capsular surface was present after intraperitoneal syngeneic transplantation.

Diabetes Care 22 (Suppl. 2):B121–B126, 1999

Transplantation of islets of Langerhans to patients with type 1 diabetes has been carried out for a number of years, but the success has been limited (1). One of the main problems in pancreatic islet transplantation is immune rejection; another is the destruction of the graft because of recurrence of disease (2). Islet transplantation is therefore confined to patients already on immunosuppressive drug treatment, often as a result of kidney transplantation. However, this allows only a small population of type 1 diabetic patients to be treated and only at a stage of advanced complications.

A means of obviating immune suppression would be to surround the islets with an immune protective barrier that protects the graft from cellular or humoral assault. For this purpose, Lim and Sun (3) developed a technique for microencapsulation of islets in a alginate matrix covered with a layer of polylysine. Although this technique has been modified and applied in numerous studies, reproducibly successful results have so far not been reported. Remaining problems include foreign-body reactions, oversized capsules, slow production rate of capsules, and limited functional survival of microencapsulated islet grafts (4). In the present study, uniformly sized microcapsules formed in a high-voltage electrostatic field were smaller and produced faster than conventional microcapsules formed by co-axial air-jet systems. The aim of the study was to assess the viability of islets after microencapsulation using an electrostatic field. Viability of the microencapsulated and control islets was assessed by determinations of rates of glucose oxidation and insulin release 24 h and 2 weeks after microencapsulation. Glucose oxidation measurements were also carried out in transplanted microencapsulated islets and transplanted empty capsules, the latter being used to assess the amount of metabolically active cells on the outside of the capsule.

RESEARCH DESIGN AND METHODS

Animals, islet isolation, and culture
Inbred C57BL/6 mice, which were originally obtained from Jackson Laboratories (Bar Harbor, ME) and subsequently bred at the Biomedical Centre, Uppsala, Sweden, were used. Pancreatic islets were isolated by collagenase digestion (Collagenase A from Clostridium histolyticum; Boehringer Mannheim, Mannheim, Germany) and then hand-picked using a braking pipette according to a procedure described in detail elsewhere (5). Groups of ~150 islets were cultured free-floating in medium RPMI 1640 (11.1 mmol/l glucose) supplemented with 10% fetal calf serum (Sigma, St. Louis, MO), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Boehringer Mannheim). The culture medium was changed every 2nd day. Approximately half of the number of islets was kept in culture, whereas the other half was microencapsulated about 7 days after isolation. All animal experiments were approved by the local animal ethics committee.

Microencapsulation of islets
Islets were suspended in sterile filtered 1.8% (wt/vol) alginate (Drammen, Norway), which contained mannitol as osmolythe, and pushed through a needle into an 0.8% (wt/vol) CaCl₂ solution, also containing mannitol. An electric field of 7 kV was generated between the solution and the needle, thus pulling the droplets down to form beads. The size of the microcapsules formed (525 ± 10 µm; n = 4; 25 microcapsules were measured in each preparation) was controlled by the voltage applied. Microcapsules produced in our laboratory using a co-axial air-jet system are 850 ± 25 µm in diameter. The microcapsules were washed three times in saline after retrieval from the gelling solution. A poly-L-lysine hydrochloride coating with a molecular weight of 19.8 kD (Sigma) was then added to the beads by gently shaking them in 0.1% poly-L-lysine hydrochloride (wt/vol) in saline for 10 min. After washing the beads in saline, a further coating of alginate was applied by suspending them in 0.1% alginate in saline (wt/vol) and shaking gently for 10 min. All solutions contained 1 mmol/l HEPES (Sigma). The microcapsules were finally washed three times in saline. Microcapsules containing islets were picked and cultured in RPMI 1640 and fetal calf serum, as described above. Empty microcapsules were produced separately using the same technique, but fetal calf serum was not added to the medium when they were maintained in culture. Using the high-voltage electrostatic field technique to form the capsules, ~1,500 capsules were produced per minute.

Experimental groups
Islets from each isolation (four mice) were assigned randomly to the following groups.

Recently microencapsulated islets. Islets were kept in culture for 1 week, microencapsulated, resuspended in culture medium (RPMI 1640 and 10% fetal calf serum), and examined the next day.

Cultured microencapsulated islets. Islets were microencapsulated 1 week after isolation and were then cultured for a further 2 weeks.

Control islets. Nonmicroencapsulated control islets were cultured in parallel to the above groups, i.e., for 1 or 3 weeks.

Transplanted microencapsulated islets. Islets were maintained in culture for 1 week before being microencapsulated. Approximately 200 microcapsules were then transplanted intraperitoneally the day after microencapsulation.

Empty microcapsules. Approximately 200 empty microcapsules were transplanted intraperitoneally for 2 weeks, and control empty microcapsules were kept maintained in vitro for 2 weeks.

Transplantation and microcapsule recovery
Male C57BL/6 mice were anesthetized with a combination of xylazine hydrochloride (16 mg/kg; Bayer Leverkusen AG, Leverkusen, Germany) and ketamine (150 mg/kg; Parke-Davis, Barcelona, Spain) administered subcutaneously. An incision was made in the skin and the peritoneal membrane. Approximately 200 microencapsulated islets were then delivered into the peritoneal cavity by means of a pipette. After a period of 2 weeks, capsules were retrieved by washing the abdominal organs with Hanks' solution.The retrieval rate of microcapsules varied between mice and was estimated to be between 50 and 80%.

Glucose oxidation rate
Groups of 10 microcapsules or control islets in triplicate were incubated in glass vials containing a Krebs-Ringer bicarbonate buffer (6) supplemented with 10 mmol/l HEPES (Sigma), D-[U-¹⁴C]glucose (Amersham International, Amersham, U.K.) and nonradioactive glucose to give a final concentration of 1.67 or 16.7 mmol/l. The vials were inserted into glass scintillation vials, gassed with 95% O₂ and 5% CO₂, and sealed tightly. The flasks were incubated for 90 min at 37°C until the glucose oxidation was terminated by injection of 100 µl of 0.05 mmol/l antimycin A (Sigma) into the glass vials. ¹⁴CO₂ formed by cell metabolism was released from the incubation medium by injection of 100 µl of 0.4 mol/l Na₂HPO₄ (pH 6.0) and trapped in 250 µl Hyamine 10-X (Packard Instruments, Downers Growe, IL) during a further 120-min incubation. Five milliliters of Ultima Gold (Packard Instruments) were finally added, and the radioactivity was measured by liquid scintialltion counting.

Insulin release
Groups of 10 control islets or microencapsulated islets were incubated at 37°C for a total of 2 h in triplicate in glass vials containing 0.25 ml Krebs-Ringer bicarbonate buffer supplemented with 10 mmol/l HEPES and 2 mg/ml bovine serum albumin (fraction V; Miles, Slough, U.K.). This medium contained either 1.7 or 16.7 mmol/l glucose, with the former being used for the 1st hour of incubation and the latter for the 2nd hour of incubation. The medium was removed after each incubation, and the insulin concentrations were measured by radioimmunoassay (7).

DNA measurement
To establish whether there were differences in the size of control islets after 1 and 3 weeks of culture, DNA measurements were carried out. A total of 50 islets were placed in culture (RPMI 1640 and 10% fetal calf serum) for either 1 or 3 weeks (medium changed every 2nd day), after which the islets were counted. The remaining islets were counted, collected, and placed in 200 µl redistilled water. They were sonicated, and DNA was measured fluorphotometrically (8,9). Each observation represented a different isolation. The amount of DNA per islet was then calculated.

Histology
Microcapsules were fixed in 10% formalin overnight and then stored in 70% ethanol until further treatment. The microcapsules were dehydrated by placing them in increasing concentrations of ethanol (70, 80, 90, 95, and 99.9%) for 15 min. They were then cleared in xylene for 2 X 10 min and placed in melted paraffin (56°C) for 90 min, after which they were embedded in paraffin. The microcapsules were sectioned at 5 µm and stained with hematoxylin and eosin.

Statistical analysis
A mean was calculated from each duplicate or triplicate group (DNA and glucose oxidation, respectively) of islets and then considered as one separate observation. Each observation represented different isolations. The data were normally distributed, and when multiple comparisons were made between groups, analysis of variance (ANOVA), including Fisher's protected least statistical difference test, was performed using Statview (Abacus Concepts, Berkeley, CA).

RESULTS

Glucose oxidation rate in recently microencapsulated islets
When glucose oxidation measurements were carried out the day after microencapsulation, both control islets and microencapsulated islets responded well to a glucose challenge, with an ~8- to 10-fold stimulation at the high glucose concentration (Fig. 1). No values greater than the blanks were obtained when glucose oxidation was measured in empty microcapsules produced the previous day, thus showing that the microcapsules per se did not affect the values obtained (data not shown).

Figure 1—Glucose oxidation rates of cultured nonmicroencapsulated islets and microencapsulated islets 1 day after microencapsulation. The islets were maintained in medium RPMI 1640 + 10% fetal calf serum. Oxidation rates were measured after incubation with D-[U-14C]glucose. Values are means ± SEM for eight experiments. ***P< 0.001 vs. corresponding islets incubated at 1.7 mmol/l glucose, using ANOVA. Note that the ordinal axis in this figure differs from that in Figs. 2 and 3. Glucose: , 1.7 mmol/l; , 16.7 mmol/l.

Glucose oxidation rate in cultured microencapsulated islets
Islets cultured for 2 weeks after microencapsulation showed glucose oxidation rates similar to rates found 24 h after microencapsulation (Fig. 2). The response to glucose was still significant. However, the control islets had a greater response to glucose compared with microencapsulated islets at this time point. A comparison between the values obtained the day after microencapsulation and those obtained after 2 weeks in culture showed that the differences between microencapsulated and control islets after 2 weeks of culture depend on an increased glucose oxidation rate of the control islets rather than a deterioration of the function of the microencapsulated islets (cf. Fig. 1 and Fig. 2).

Figure 2—Glucose oxidation rates of cultured nonmicroencapsulated islets and microencapsulated islets 2 weeks after microencapsulation. Values are means ± SEM for seven or eight experiments. ***P< 0.001 vs. corresponding islets incubated at 1.7 mmol/l glucose. §P< 0.01 vs. nonmicroencapsulated islets incubated at 16.7 mmol/l glucose, using ANOVA. Glucose: , 1.7 mmol/l; , 16.7 mmol/l.

To test whether the increased glucose oxidation rate in the control islets was related to a difference in islet size, we measured the DNA content of cultured islets after 1 and 3 weeks. However, a decline in size was observed (21.6 ± 3.0 at 1 week vs. 15.6 ± 3.6 ng/islet at 3 weeks, P< 0.05, using paired Student's t test). Empty microcapsules that were maintained in vitro in parallel showed no glucose oxidation after 2 weeks of culture (data not shown).

Glucose oxidation rate in transplanted microcapsules
Empty microcapsules retrieved 2 weeks after transplantation to normal mice oxidized glucose, indicating the presence of metabolically active cells. The oxidation rate was slightly increased at a higher glucose concentration, but this difference did not attain statistical significance (Fig. 3). Microcapsules containing syngeneic islets had a glucose oxidation rate at basal glucose concentrations that was about three times higher than the empty capsules, but there was no further increase at the higher glucose concentration.

Figure 3—Glucose oxidation rates of retrieved empty microcapsules and microencapsulated islets after transplantation. Microcapsules remained intraperitoneally for 2 weeks after transplantation to C57BL/6 mice. Values are means ± SEM for six to nine experiments. ***P< 0.001 vs. empty capsules incubated at 1.7 mmol/l glucose. **P< 0.01 vs. empty capsules incubated at 16.7 mmol/l glucose, using ANOVA. Glucose: , 1.7 mmol/l; , 16.7 mmol/l.

Insulin release in recently microencapsulated and cultured islets
Insulin release in response to 1.7 mmol/l glucose was similar in control and microencapsulated islets, irrespective of the culture period (Fig. 4). Insulin release in all groups increased seven- to ninefold in response to 16.7 mmol/l glucose. However, it was found that control islets had a lower insulin release in response to 16.7 mmol/l glucose after 1 week's culture compared with after 3 weeks' culture.

Figure 4—Insulin release of cultured nonmicroencapsulated islets and microencapsulated islets 1 day and 2 weeks after microencapsulation (1 week and 3 weeks, respectively, after islet isolation). The islets were exposed to 1.7 mmol/l glucose for 1 h and then to 16.7 mmol/l glucose for a 2nd hour. Values are means ± SEM for four experiments. ***P< 0.001 vs. corresponding islets incubated at 1.7 mmol/l glucose. §P< 0.01 vs. nonmicroencapsulated islets cultured 1 week, using ANOVA. Glucose: , 1.7 mmol/l; , 16.7 mmol/l.

Light microscopy
Both at the macroscopic and light microscopic level, it was seen that both transplanted empty microcapsules and transplanted microcapsules with islets were surrounded by fibrosis. The fibrotic reaction seen on the outside of the microcapsules tended to vary between animals, and it was evidently more severe in islet-containing microcapsules. The islets within transplanted microcapsules showed a range of morphological appearances, from intact islets (Fig. 5A) to cell debris. Islet-containing microcapsules that were maintained in culture only did not show any pericapsular fibrosis, and the encapsulated islets appeared to be structurally viable (Fig. 5B). However, occasionally, islets with necrotic areas were seen.

CONCLUSIONS— The present study shows that normal mouse pancreatic islets that had been microencapsulated using a high-voltage electrostatic field survived well in vitro and could respond metabolically to increased glucose concentrations both the day after and 2 weeks after microencapsulation. Insulin release in response to glucose was also well preserved in micro-encapsulated islets both 1 day and 2 weeks after microencapsulation. Our data thus show that the electrostatic field procedure does not detrimentally affect the islets. Previous studies have shown the feasibility of using a high-voltage electric field for the microencapsulation of islets (10), but there are no detailed reports on the metabolic function of such islets.

The higher glucose oxidation rate and insulin release of the control islets 2 weeks after the first measurements does not seem to reflect a fusion of free-floating islets during prolonged culture, as the measurements of islet DNA content indicated a decline in size with time. Thus, the increased glucose oxidation and insulin release rates seen in control islets after a further 2 weeks of culture appear to be due to an enhanced functional activity of the -cells. The reason why the microencapsulated islets demonstrate neither enhanced glucose oxidation nor insulin release after 2 weeks' culture has yet to be elucidated. The observation that transplanted capsules showed adherence of cells to the surface indicates that the material forming the capsules was not sufficiently inert to prevent a foreign-body reaction. This has been observed before (11–15), but other studies report an almost complete lack of overgrowth (16,17). Empty microcapsules removed from the mice showed a significant rate of glucose oxidation, which indicates that cells found on the outside of the microcapsules are metabolically active. It is therefore difficult to evaluate presently how much the islet cells within the microcapsules were contributing to the total glucose oxidation rate measured, in relation to that exerted by the fibroblasts. However, there was no increased response to an increased glucose concentration of islet-containing transplanted microcapsules, and the basal oxidation rate of these capsules was high. These observations show that the rate of glucose oxidation reflected the metabolism of fibroblasts rather than that of islets. It is probable that the islets did not contribute to the glucose oxidation measurements because of decreased viability of the islet cells and/or diffusion difficulties of glucose into the capsule (see below). It is likely that the pericapsular cells are also metabolically active in vivo and thus reduce nutrient diffusion into the capsule—as recently suggested by Weir and Bonner-Weir (18)—which leads to decreased cell viability. This could also cause difficulties from an experimental viewpoint, as it is likely that the fibroblasts covering the microcapsules would impair diffusion of glucose to the inside during the glucose oxidation experiments. This would most likely hamper the assessment of the glucose metabolism of any viable islet cells inside. In an attempt to overcome this potential problem, we tried to remove fibrosis, using trypsin, after the retrieval of transplanted capsules. However, although in some microcapsules the fibrotic layer was easily removed, other microcapsules needed so much trypsin that the effect on the islets would be detrimental.

There was an increased amount of fibrosis on the capsules containing syngeneic islets compared with empty capsules. This could be seen histologically, although we did not carry out a systematic investigation. However, the glucose oxidation rates showed this to be the case, as the rate of glucose oxidation of transplanted encapsulated islets in 1.7 mmol/l glucose was significantly increased compared with that of transplanted empty capsules. Such an increase cannot be explained by islet glucose oxidation in the capsules while taking into account the low basal oxidation rate of microencapsulated islets (Figs. 1 and 2). The reason for this difference in fibrosis on islet-containing capsules compared with empty capsules is unknown.

In conclusion, this study shows that mouse pancreatic islets remain functionally competent after microencapsulation in alginate/poly-L-lysine/alginate capsules produced by means of a high-voltage electrostatic field technique. However, as has been the case with other preparations of microcapsules, a fibrotic overgrowth of the microcapsules in vivo after transplantation remains a problem.

Acknowledgments— This study was supported by grants from the Swedish Medical Research Council (12P–10739; 12X–109; 12X–8273 12X–9237), BIOMED 2 Medical Health Research of the European Community (BMH4-CT95-1561), the Swedish Diabetes Association, the Nordic Insulin Fund, the Family Ernfors Fund, and the Juvenile Diabetes Foundation International, Norwegian Research Council, Norwegian Diabetes Association.

We thank Margareta Engkvist and Astrid Nordin for excellent assistance.

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From the Department of Medical Cell Biology (A.K., S.S., A.A., C.H.), Uppsala University, Uppsala, Sweden; the Institute for Cancer Research (B.K.), University of Trondheim, Trondheim, Norway; and the Department of Biotechnology (G. S.-B.), Norwegian Institute of Biotechnology, University of Trondheim, Trondheim, Norway.

Address correspondence and reprint requests to Aileen King, Department of Medical Cell Biology, Biomedicum, P.O. Box 571, S-751 23 Uppsala, Sweden. E-mail: aileen.king@medcellbiol.uu.se.

Received for publication 27 May 1998 and accepted in revised form 9 September 1998.

Abbreviations: ANOVA, analysis of variance.

This article is based on a presentation at a satellite symposium of the 16th International Diabetes Federation Congress. The symposium and the publication of this article were made possible by educational grants from Hoechst Marion Roussel AG.

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