Ivan Vacek

Normalization of diabetes in spontaneously diabetic cynomologus monkeys by xenografts of microencapsulated porcine islets without immunosuppression.

Porcine pancreatic islets were microencapsulated in alginate-polylysine-alginate capsules and transplanted intraperitoneally into nine spontaneously diabetic monkeys. After one, two, or three transplants of 3-7 x 10(4) islets per recipient, seven of the monkeys became insulin independent for periods ranging from 120 to 804 d with fasting blood glucose levels in the normoglycemic range. Glucose clearance rates in the transplant recipients were significantly higher than before the graft administration and the insulin secretion during glucose tolerance tests was significantly higher compared with pretransplant tests. Porcine C-peptide was detected in all transplant recipients throughout their period of normoglycemia while none was found before the graft administration. Hemoglobin A1C levels dropped significantly within 2 mo after transplantation. While ketones were detected in the urine of all recipients before the graft administration, all experimental animals became ketone free 2 wk after transplantation. Capsules recovered from two recipients 3 mo after the restoration of normoglycemia were found physically intact with enclosed islets clearly visible. The capsules were free of cellular overgrowth. Examination of internal organs of two of the animals involved in our transplantation studies for the duration of 2 yr revealed no untoward effect of the extended presence of the microcapsules.

The Use, in Diabetic Rats and Monkeys, of Artificial Capillary Units Containing Cultured Islets of Langerhans (Artificial Endocrine Pancreas)

A unit was constructed that consisted of a core of hollow fibers through which low-molecular-weight substances, such as glucose and insulin, could pass freely but were impermeable to high-molecular-weight proteins, such as antibodies. Islets of Langerhans from normal rats were planted in the space surrounding the fibers, and either blood or nutrient medium was circulated through the fibers themselves. In experiments with animals, the units were attached to the vascular system of diabetic rats and monkeys. Blood glucose concentrations in the rats were reduced to nondiabetic levels within one hour and were maintained for the duration of the experiments. In monkeys the blood glucose level declined from 210 mg./100 ml. to 90 mg./100 ml. in four hours and insulin in the serum rose to 93 μU./ml. in one-half hour. Also, we have found that islets from monkeys cultivated in the artificial endocrine pancreas (AEP) continue to release insulin into circulating tissue culture medium for over eight months.

Prolonged Reversal of Diabetic State in NOD Mice by Xenografts of Microencapsulated Rat Islets

Transplantation of the islets of Langerhans could be the most promising approach to the clinical treatment of insulin-dependent (type I) diabetes mellitus. In this study, we report on a modified encapsulation technique that produces small alginate-polylysine capsules (0.25–0.35 mm diam). In an in vitro study, both encapsulated and unencapsulated islets showed comparable responses to glucose challenge in terms of insulin secretion. With the new capsules, 16 spontaneously diabetic NOD mice received transplants of 800 encapsulated rat islets/animal. Nonfasting blood glucose concentration decreased from 24.4 ± 1.4 to 4.0 ± 1.3 mM. At 4 and 5 mo posttransplantation, the capsules were removed from 2 recipients. Both animals regressed to a hyperglycemic state after capsule removal. However, after another islet transplantation, normoglycemia was again restored in these 2 animals. In control mice, which received unencapsulated islets, the xenografts remained functional for <10 days. A high mortality rate was observed among these animals within 2 mo of the recurrence of the hyperglycemic state. Our results clearly indicate that encapsulation of pancreatic islets in the improved capsules can effectively prolong xenograft survival without immunosuppression in an animal model that mimics human type I diabetes mellitus.

Generation of Alginate-Poly-L-Lysine-Alginate (APA) Biomicrocapsules: the Relationship Between the Membrane Strength and the Reaction Conditions

Alginate-poly-l-lysine-alginate (APA) microcapsules have proven effective in protecting enclosed live cells from immune rejection following transplantation into experimental animals, thereby eliminating the need for immunosuppressive therapy. However, in order for the capsules to remain intact for extended periods in vivo, the thickness of the membrane material must be optimized. In this study, the membrane thickness was examined as an indicator of membrane strength and measured under different reaction conditions. The thickness was found to increase 1) from 4.6 microns to 6.6 microns with an increase in the concentration of sodium alginate from 1.25 (w/v) to 2.0% (w.v); 2) from 4.2 microns to 6.2 microns with an increase in the concentration of the calcium solution from 20 mM to 100mM; 3) from 3.9 microns to 10.3 microns with an increase in the concentration of poly-l-lysine (PLL) from 0.02% (w/v) to 0.08% (w/v); and 4) from 2.3 microns to 7.4 microns with an increase in the reaction time with the PLL from two to seven minutes. On the other hand, membrane thickness decreased 1) from 9.8 microns to 8.6 microns with an increase of the pH in the PLL solution from 5.8 to 9.2; 2) from 13.2m to 5.8 microns with an increase in the molecular weight of PLL from 14,000 to 57,000; 3) from 8.4 microns to 6.0 microns with an increase in the treatment time with 0.9 (w/v) NaCl solution from zero to fifteen minutes and; 4) from 7.5 microns to 6.1 microns with an increase in the treatment time of the second sodium alginate coating from zero to ten minutes. Membrane thickness was inversely proportional to capsule volume expansion during membrane synthesis. By replacing calcium chloride by calcium lactate and eliminating the use of CHES in the construction of capsule membranes, we improved the strength and biocompatibility of our capsules, as evidenced by marked improvements in the survival rates of diabetic mice treated with islet transplants enclosed in the new capsules. These results indicate that it is possible to obtain optimal membrane thickness for a given purpose by creating specific reaction conditions under which membranes are synthesized.

Xenografts of rat islets into diabetic mice : an evaluation of new smaller capsules

Healthy rat islets were encapsulated in alginate-polylysine-alginate capsules measuring 0.25-0.35 mm in diameter using a modified encapsulation technique. The encapsulated islets were transplanted intraperitoneally in nonimmunosuppressed streptozotocin-induced diabetic BALB/c mice. The diabetic condition of the experimental animals was reversed within two days following the transplantation and the animals remained normoglycemic for up to 308 days, with a mean xenograft survival of 219.8 +/- 46.2 days. Four and six months posttransplant the capsules were removed from two recipients. This resulted in regression to a hyperglycemic state. After a second transplant of encapsulated islets, the animals returned to normoglycemia. In control mice that received free unencapsulated islets, the xenografts remained functional for no more than 12 days. Our study clearly demonstrates that the encapsulation of islets in the new smaller capsules can effectively prolong xenograft survival without immunosuppression.

Normalization of diabetes by xenotransplantation of cryopreserved microencapsulated pancreatic islets : application of a new strategy in islet banking

To develop a requisite islet bank for the clinical implementation of an injectable bioartificial endocrine pancreas, microencapsulated islets were cryopreserved and assessed both in vitro by static glucose challenge and in a transplantation study. The insulin response of cryopreserved encapsulated rat islets was comparable with fresh islets. Transplantation of 800-900 banked rat islets resulted in the normalization of the metabolic blood glucose perturbation, body weight, and general health characteristics in 8 out of 8 diabetic mice for the study duration of 90 days. Whereas free islets are easily fragmented and lost during the freezing process, the capsule protects the fragile islets from freezing damage, increasing the retrieval rate from 79.5 +/- 9.8% to 97.2 +/- 1.3.

Cryopreservation of microencapsulated porcine pancreatic islets : In vitro and in vivo studies

BACKGROUND If the transplantation of immunoisolated porcine islets into human diabetics is to become reality, the development of a long-term storage method represents an important prerequisite. However, information on cryogenic storage of porcine islets is scanty and fragmentary. METHODS Porcine pancreatic islets microencapsulated in alginate-polylysine-alginate membranes were cryopreserved and assessed both in vitro by static glucose challenge and in vivo in a transplantation study. Two separate methods of islet cryopreservation were compared: method A, using the Bio Cool III freezing machine, and method B, which uses the Nalgene isopropyl alcohol insulated cooler. RESULTS Method A was found to have better preserved the ability of the microencapsulated cryopreserved islets to respond to high-glucose static challenge (7 out of 10 lots) compared with method B (1 out of 10 lots). Upon exposure to high glucose, the islet batches that did retain the ability to respond to glucose were shown to have secreted an average of 1220+/-73 pM/24 hr/islet of insulin as compared with 1528+/-118 pM/24 hr/islet for fresh islets. The presence of isobutyl methylxanthine further potentiated insulin secretion to 1805+/-81 pM/24 hr/islet and to 2410+/-104 pM/24 hr/islet for cryopreserved and free islets, respectively. Intraperitoneal transplantation of 2000 cryopreserved microencapsulated porcine islets into streptozotocin-diabetic mice resulted in the reversal of hyperglycemia in 6 out of 10 recipients for the duration of the 90-day study. CONCLUSIONS The effective protection of the delicate porcine endocrine tissue during the cryopreservation process and the subsequent long-term storage were demonstrated with considerable success in this study.

Microencapsulation and Transplantation of Genetically Engineered Cells: A New Approach to Somatic Gene Therapy

In order to develop a model for gene therapy which avoids dependence on an autologous source of target cells and immunosuppressive therapy, mouse Ltk fibroblasts transfected with a human growth hormone (hGH) fusion gene were encapsulated in a semipermeable alginate-poly-L-lysine-alginate (APA) membrane. The encapsulated cells were cultured in vitro or transplanted intraperitoneally into mice to monitor cell viability, cell growth, and hGH secretion. The effect of Zn2+ ions on vector expression was also monitored in vitro and in vivo. Results indicate that: (1) the capsule environment is compatible with cell viability and cell growth; (2) the capsule limits cell growth; (3) the capsule membrane is permeable to the exit of hGH; (4) gene product expression may be stimulated by external means; (5) the novel gene product is delivered in vivo; and (6) encapsulated cells recovered from transplant recipients continue to secrete hGH in vitro. The results suggest therapeutic potential of this approach to somatic gene therapy.

In vitro and in vivo evaluation of microencapsulated porcine islets

To provide a plentiful supply of pancreatic islets for future clinical transplants into diabetic patients, the authors have developed a simple and consistent method of isolation of porcine islets. Both in vitro and in vivo studies demonstrated that the islets were viable and functional. Xenotransplants of 1.5 x 10(3) - 2.5 x 10(3) of microencapsulated porcine islets into diabetic mice resulted in restoration of normoglycemia in 13 of 18 experimental animals for up to 10 months. A xenograft of 50 x 10(3) microencapsulated porcine islets into a spontaneously diabetic monkey normalized hyperglycemia for more than 150 days. This experiment indicated that the transplantation of encapsulated porcine islets has great potential as a clinical treatment in diabetes mellitus.

Effects of collagenase concentration on the purity and viability of isolated porcine pancreatic islets for use in xenotransplantation studies

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.