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Microencapsulation of living cells — A long-term delivery system
137
2 (1985) 137-141 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Journal
of Controlled
Release,
MICROENCAPSULATION
OF LIVING
CELLS - A LONG-TERM
DELIVERY
SYSTEM*
A.M. Sun*+ and G.M. O’Shea Connaught University
Research of Toronto,
Institute,
1755 Steeles Avenue
Department
of Physiology,
West, Willowdale, Faculty
Ontario
of Medicine,
M2R
Toronto,
3T4
Ontario
(Canada) M5S
lA8
(Canada)
To provide a long-term delivery system for hormones and other biomolecules a new durable and biocompatible membrane has been developed for the microencapsulation of living cells, which can protect transplanted cells from immuno rejection. Evaluation of the surface finish of the microcapsules by scanning electron microscopy revealed essentially smooth interior and exterior capsule surfaces. The wall thickness was determined to be 4.00 + 0.28 urn. The water content of the wall was 93%. Single in traperitoneal transplantation of encapsulated islets reversed diabetes in streptozotocin-induced diabetic rats for up to 650 days indicating prolonged insulin release from the encapsulated cells. Intact capsules containing islet cells were recovered from animals almost 2 years after implantation. Implan tation of encapsulated hepatocytes into rats with galactosamine-induced liver failure increased the survival rate of the recipients. The biocompatible capsules, which survived in the animal body for nearly 2 years, have great clinical potential for a long-term drug and hormone delivery system.
INTRODUCTION
Immobilized enzymes and other macromolecules have been used in many long-term delivery systems with both industrial and medical applications. However these systems have been limited by such problems as: (i) limited amount of material entrapped in the delivery systems, (ii) uncontrolled release *Paper presented at the Second International Symposium on Recent Advances in Drug Delivery Systems, February 27, 28 and March 1, 1985, Salt Lake City, UT, U.S.A. **To whom correspondence should be addressed.
0168-3659/85/$03.30
of the substance, (iii) surgery to recover nondegradable matrix systems, and (iv) poor biocompatibility of the matrices. Theoretically, the microencapsulation of cells has certain advantages as a delivery system for hormones, and other enzymes macromolecules, Physiological release of secretory products from the immobilized cells would be prolonged by immunoisolation of the cells within biocompatible membranes, with few side effects anticipated. In this report we describe a long-term in vitro and in vivo study on islet cells and hepatocytes encapsulated in biocompatible alginate-polylysinealginate membranes.
0 1985 Elsevier Science Publishers B.V
138
MATERIALS
AND METHODS
Animals
Outbred male Wistar rats (Charles River Canada Inc.) weighing 200-300 g were used throughout the study. Animals were made diabetic by an i.v. injection of streptozotocin (65 mg/kg). Weekly blood samples for determination of fasting plasma glucose concentrations, were obtained by bleeding from the orbital sinus. Only rats with persisting fasting plasma glucose concentrations above 300 mg/dl were used as transplant recipients. Islet isolation and culture
Islets were isolated from rat pancreas by a standard collagenase digestion technique [1] and hand-picked with the aid of a dissecting microscope. The isolated islets were cultured prior to encapsulation, for 1 to 3 days at 37°C in CMRL-1969, supplemented with 7.5% bovine fetal serum and 300 mg/dl glucose. For long-term culture studies, groups of 100 encapsulated islets were maintained at 37°C in 7 ml medium and fluid changed at weekly intervals. Islet encapsulation
Islets were suspended in 1.3% (w/v) sodium alginate (Kelcogel@ LV, Kelco, NJ, U.S.A.) and 0.85% NaCl at a concentration of 1000 islets/ml. Spherical droplets of this suspension, formed by syringe pump extrusion, gelled upon collection in 1.1% CaCl,. Following washing steps with 25 ml volumes of 0.1% CHES (2-N-cyclohexylaminoethane sulfonic acid) and 0.85% NaCl, the gels were coated with polylysine by suspension in 30 ml polyL-lysine (0.05% w/v, M.W. 17,000-25,000, Sigma, St. Louis, MO, U.S.A.). The capsules were then washed with 25 ml volumes of 0.1% CHES, 1.1% CaC12 and 0.85% NaCl and suspended for 4 min in 0.12% sodium alginate which formed the outer layer of the membrane. Further washing with 0.85% NaCl
preceded treatment of the capsules with 0.05 M sodium citrate, pH 7.4 for 6 min, to liquify the gel inside the capsules. The excess citrate was removed by two NaCl washes and the encapsulated islets were cultured at 37°C. The capsules with a membrane of polylysine sandwiched between 2 layers of alginate were 0.7-0.8 mm in diameter and contained 1 to 2 islets per calsule. Characterization of capsular membranes
The surface finish and wall thickness were assessed by scanning electron microscopy (SEM), and an image-shearing optical test. Image-shearing optical tests were done by KMS Fusion, Inc., Ann Arbor, MI, U.S.A. Islet allografts Under light ether anaesthesia, diabetic rats received intraperitoneal transplants at 4.55.0 X lo3 encapsulated islets suspended in saline. The capsules were implanted in the peritoneal cavity using a wide-bore cannula attached to a 10 ml syringe. Histochemical study
Encapsulated islets were fixed in Bouin’s solution and embedded in paraffin wax. Sections 6 pm thick were stained with aldehyde thionin, hematoxylin and eosin. Assays
Plasma glucose concentration was determined using a Beckman Glucose Analyser II. Insulin was assayed by radioimmunoassay using polyethyleneglycol to separate the free and antibody-bound hormones [ 21. Hepatocyte en~apsuiation and altotransplantation
Outbred Wistar rats, weighing 250-300 g, were used for both donor and recipient animals. Hepatic necrosis was induced in recipient animals by intraperitoneal injection
139
of D-galactosamine hydrochloride (Sigma, 2.6 g/kg body weight). Hepatocytes were isolated from the donor animals using a collagenase digestion method [3]. The encapsulation procedure used was as described above for islet encapsulation except that the molecular weight of the poly-I.,-lysine used was 40,000.
RESULTS Physical-chemical
characterization
of capsular mem-
brane
The evaluation of the surface finish of the microcapsules by SEM revealed essentially exterior capsular interior and smooth
surfaces. Using an image-shearing technique, the wet wall thickness was calculated to be 4.00 f 0.28 pm. The water content, of the wall was determined to be 93% w/w. Long-term culture of encapsulated islets
Islets encapsulated in alginate-polylysinealginate membranes remained viable in culture for over three months. Histological studies showed that up to 93 days in culture the islets were still intact, stained uniformly and showed good p-granulation throughout all cross-sections (Fig. la). By 135 days the islets showed some evidence of deteriorating structure (Fig. lb) but were still well granulated. Allografts of encapsulated islets
a
_’ ,,.
,’ -..
b
Fig. 1. Photomicrographs 93(a) and 135(b) days
stain. Magnification
x
of encapsulated
in culture. 50.
islets after
Aldehyde
thionin
Single intraperitoneal transplants of 4.55.0 X lo3 encapsulated islets reversed diabetes in all 8 recipients within two days. Only one transplant failed within one month (Fig. 2H). The individual blood glucose profiles of the other 7 animals are shown in Figs. 2A-G. The mean fasting plasma glucose dropped from a pre-transplant value of 378 * 5.4 (mean + SEM) mg/dl to 85.4 + 5.4 mg/dl two days post-transplantation. Two of these animals were still normoglycemic when sacrificed at 365 (Fig. 2A) and 648 (Fig. 2C) days posttransplantation, indicating the continuous insulin secretion from the encapsulated cells to meet the physiological demand of the recipients. Capsules were recovered from animals at 156, 365 and 648 days posttransplantation. Histological staining revealed the presence of intact islets. The surfaces of most capsules were free of cell attachment and were physically intact with the enclosed islets clearly visible. Some capsules had fibroblast-like cells on the external surfaces. Transplant recipients showed a very rapid increase in body weight and no evidence of cataract development in their eyes was observed. Untreated diabetic controls showed no significant weight increase during the same
Fig. 3. Photomicrograph of encapsulated cytes. Magnification x 15.
rat hepato-
E
induced fulminant hepatic failure. The survival rate of the recipient rats was increased by 50% when approximately 1 X 10’ encapsulated rat hepatocytes were implanted intraperitoneally. When encapsulated hepatocytes were cultured, a significant amount of albumin was observed in the culture medium. H
0
50
100
1.50
200
250
300
350
400
DISCUSSION
0l)p
Fig. 2. Fasting plasma glucose profiles of streptozotocininduced diabetic rats after transplantation of 4.5-5.0 X 10’ encapsulated rat islets. Note different scales in Figs. 2a and 2b. Vertical bars denote time of second transplantation, where applicable (2B, 2D, 2E, 2F, 2G).
period and developed eye cataracts within 2-3 months. Five animals each received a second transplant of 5 X lo3 encapsulated islets when they regressed to the diabetic state (Figs. 2B, 2D, 2E, 2F and 2G). Two of these transplants functioned for the life span of the recipients (Figs. 2B and 2D); only in one case did the second transplant function for a shorter period than the initial implant (Fig. 2E). Allografts of encapsulated hepatocytes
Encapsulated implanted into
hepatocytes rats with
(Fig. 3) were D-galactosamine-
This study demonstrates that pancreatic cells encapsulated in a biocompatible membrane can be used as a long-term hormone or enzyme delivery system. Intraperitoneal transof encapsulated pancreatic islets plant reversed diabetes for over a year. Intact capsules containing islets were recovered from the peritoneal cavity 12--21 months after transplantation. Histological staining and insulin secretion studies on encapsulated islets recovered from the recipients demonstrated that the capsules contained viable beta cells with insulin granules and retained the ability to secrete insulin in response to glucose challenge. These results clearly indicate that microencapsulated islet cells can provide a long-term insulin delivery system. We have previously reported that the alginate-polylysinealginate membrane has the properties of a hydrogel and is approximately 4 pm in thickness [4]. The biocom-
141
patibility of a number of hydrogels has been established and these materials have several biomedical applications. Recently Klom et al. [5] reported the use of a macroporous hydrogel membrane in a hybrid artificial this hydrogel membrane elicited pancreas; considerably less fibrous tissue reaction than the membranes used for other hybrid artificial pancreas studies. Portable devices providing continuous insulin infusion either subcutaneously, intraperitoneally or intravenously are now in use in a large number of centers. Whether the improved glycemic control achieved with these devices delays the progression of established lesions or prevents their development remains a controversy. Initial results indicate that 6 months of continuous subcutaneous insulin infusion (CSII) had a beneficial effect on retinal and kidney functions, but followup data revealed that after a further 6 months retinal morphology had deteriorated in the CSII group and there was no significant difference between these patients and a group receiving conventional treatment. Tamborlane et al. [6] found that despite an improvement in glycemic control, insulin pump treatment for two years did not reverse established diabetic microvascular lesions. Unexpected deaths occur in some patients on CSII and are believed to be attributable to hypoglycemia. It is interesting to note that in our study cataracts, a symptom of diabetic complications in experimental animals, did not develop in the recipient rats while every untreated diabetic control rat developed cataracts within 2-3 months. This observation would suggest that transplantation of encapsulated islets has the potential to prevent the development of the sequalae of diabetes. Although the results from experiments with microencapsulated hepatocytes are of a preliminary nature, they indicate a promising basis for the development of an adequate support system for the failing human and animal liver.
CdNCLUSlON
This study demonstrates that allografts can be successfully immunoisolated within biocompatible membranes and provide a longterm delivery system. In the future this technique could have clinical applications in the field of bioartifical organ transplantation.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the expert technical assistance of A. Wood, H. Van Rooy, R. Taylor and S. Chow. This research was supported in part by grants from the Medical Research Council of Canada and the Canadian Diabetes Association.
REFERENCES P.E. Lacy and M. Kostianovsky, Method for the isolation of intact islets of Langerhans from the rat pancreas, Diabetes, 16 (1967) 35-39. B. Desbuquois and G.D. Aurbach, Use of polyethyleneglycol to separate free and antibodybound peptide hormones in radioimmunoassay, J. Clin. Endocrinol., 33 (1971) 732-738. R.J. Bonney, J.E. Becker, P.R. Walker and V.R. Potter, Primary monolayer cultures of adult liver parenchymal cell suitable for study of the regulation of enzyme synthesis, In Vitro, 9 (1973) 399-413. A.M. Sun, G.M. O’Shea and M.F.A. Goosen, Injectable microencapsulated islet cells as a bioartificial pancreas, Appl. Biochem. Biotechnol., 10 (1984) 87-99. G.F. Klomp, H. Hashiguchi, P.C. Ursell, Y. Takeda, T. Taguchi and W.H. Dobelle, Macroporous hydrogel membranes for a hybrid artificial pancreas. II. Biocompatibility, J. Biomed. Mater. Res., 17 (1983) 865-871. W.V. Tamborlane, J.E. Puklin, M. Bergman, C. Verdonk, M.C. Rudolf, P. Felig, M. Gene1 and R. Sherwin, Long-term improvement of metabolic control with the insulin pump does not reverse diabetic microangiopathy, Diabetes Care, 5 (1982) 58-64.
2 (1985) 137-141 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Journal
of Controlled
Release,
MICROENCAPSULATION
OF LIVING
CELLS - A LONG-TERM
DELIVERY
SYSTEM*
A.M. Sun*+ and G.M. O’Shea Connaught University
Research of Toronto,
Institute,
1755 Steeles Avenue
Department
of Physiology,
West, Willowdale, Faculty
Ontario
of Medicine,
M2R
Toronto,
3T4
Ontario
(Canada) M5S
lA8
(Canada)
To provide a long-term delivery system for hormones and other biomolecules a new durable and biocompatible membrane has been developed for the microencapsulation of living cells, which can protect transplanted cells from immuno rejection. Evaluation of the surface finish of the microcapsules by scanning electron microscopy revealed essentially smooth interior and exterior capsule surfaces. The wall thickness was determined to be 4.00 + 0.28 urn. The water content of the wall was 93%. Single in traperitoneal transplantation of encapsulated islets reversed diabetes in streptozotocin-induced diabetic rats for up to 650 days indicating prolonged insulin release from the encapsulated cells. Intact capsules containing islet cells were recovered from animals almost 2 years after implantation. Implan tation of encapsulated hepatocytes into rats with galactosamine-induced liver failure increased the survival rate of the recipients. The biocompatible capsules, which survived in the animal body for nearly 2 years, have great clinical potential for a long-term drug and hormone delivery system.
INTRODUCTION
Immobilized enzymes and other macromolecules have been used in many long-term delivery systems with both industrial and medical applications. However these systems have been limited by such problems as: (i) limited amount of material entrapped in the delivery systems, (ii) uncontrolled release *Paper presented at the Second International Symposium on Recent Advances in Drug Delivery Systems, February 27, 28 and March 1, 1985, Salt Lake City, UT, U.S.A. **To whom correspondence should be addressed.
0168-3659/85/$03.30
of the substance, (iii) surgery to recover nondegradable matrix systems, and (iv) poor biocompatibility of the matrices. Theoretically, the microencapsulation of cells has certain advantages as a delivery system for hormones, and other enzymes macromolecules, Physiological release of secretory products from the immobilized cells would be prolonged by immunoisolation of the cells within biocompatible membranes, with few side effects anticipated. In this report we describe a long-term in vitro and in vivo study on islet cells and hepatocytes encapsulated in biocompatible alginate-polylysinealginate membranes.
0 1985 Elsevier Science Publishers B.V
138
MATERIALS
AND METHODS
Animals
Outbred male Wistar rats (Charles River Canada Inc.) weighing 200-300 g were used throughout the study. Animals were made diabetic by an i.v. injection of streptozotocin (65 mg/kg). Weekly blood samples for determination of fasting plasma glucose concentrations, were obtained by bleeding from the orbital sinus. Only rats with persisting fasting plasma glucose concentrations above 300 mg/dl were used as transplant recipients. Islet isolation and culture
Islets were isolated from rat pancreas by a standard collagenase digestion technique [1] and hand-picked with the aid of a dissecting microscope. The isolated islets were cultured prior to encapsulation, for 1 to 3 days at 37°C in CMRL-1969, supplemented with 7.5% bovine fetal serum and 300 mg/dl glucose. For long-term culture studies, groups of 100 encapsulated islets were maintained at 37°C in 7 ml medium and fluid changed at weekly intervals. Islet encapsulation
Islets were suspended in 1.3% (w/v) sodium alginate (Kelcogel@ LV, Kelco, NJ, U.S.A.) and 0.85% NaCl at a concentration of 1000 islets/ml. Spherical droplets of this suspension, formed by syringe pump extrusion, gelled upon collection in 1.1% CaCl,. Following washing steps with 25 ml volumes of 0.1% CHES (2-N-cyclohexylaminoethane sulfonic acid) and 0.85% NaCl, the gels were coated with polylysine by suspension in 30 ml polyL-lysine (0.05% w/v, M.W. 17,000-25,000, Sigma, St. Louis, MO, U.S.A.). The capsules were then washed with 25 ml volumes of 0.1% CHES, 1.1% CaC12 and 0.85% NaCl and suspended for 4 min in 0.12% sodium alginate which formed the outer layer of the membrane. Further washing with 0.85% NaCl
preceded treatment of the capsules with 0.05 M sodium citrate, pH 7.4 for 6 min, to liquify the gel inside the capsules. The excess citrate was removed by two NaCl washes and the encapsulated islets were cultured at 37°C. The capsules with a membrane of polylysine sandwiched between 2 layers of alginate were 0.7-0.8 mm in diameter and contained 1 to 2 islets per calsule. Characterization of capsular membranes
The surface finish and wall thickness were assessed by scanning electron microscopy (SEM), and an image-shearing optical test. Image-shearing optical tests were done by KMS Fusion, Inc., Ann Arbor, MI, U.S.A. Islet allografts Under light ether anaesthesia, diabetic rats received intraperitoneal transplants at 4.55.0 X lo3 encapsulated islets suspended in saline. The capsules were implanted in the peritoneal cavity using a wide-bore cannula attached to a 10 ml syringe. Histochemical study
Encapsulated islets were fixed in Bouin’s solution and embedded in paraffin wax. Sections 6 pm thick were stained with aldehyde thionin, hematoxylin and eosin. Assays
Plasma glucose concentration was determined using a Beckman Glucose Analyser II. Insulin was assayed by radioimmunoassay using polyethyleneglycol to separate the free and antibody-bound hormones [ 21. Hepatocyte en~apsuiation and altotransplantation
Outbred Wistar rats, weighing 250-300 g, were used for both donor and recipient animals. Hepatic necrosis was induced in recipient animals by intraperitoneal injection
139
of D-galactosamine hydrochloride (Sigma, 2.6 g/kg body weight). Hepatocytes were isolated from the donor animals using a collagenase digestion method [3]. The encapsulation procedure used was as described above for islet encapsulation except that the molecular weight of the poly-I.,-lysine used was 40,000.
RESULTS Physical-chemical
characterization
of capsular mem-
brane
The evaluation of the surface finish of the microcapsules by SEM revealed essentially exterior capsular interior and smooth
surfaces. Using an image-shearing technique, the wet wall thickness was calculated to be 4.00 f 0.28 pm. The water content, of the wall was determined to be 93% w/w. Long-term culture of encapsulated islets
Islets encapsulated in alginate-polylysinealginate membranes remained viable in culture for over three months. Histological studies showed that up to 93 days in culture the islets were still intact, stained uniformly and showed good p-granulation throughout all cross-sections (Fig. la). By 135 days the islets showed some evidence of deteriorating structure (Fig. lb) but were still well granulated. Allografts of encapsulated islets
a
_’ ,,.
,’ -..
b
Fig. 1. Photomicrographs 93(a) and 135(b) days
stain. Magnification
x
of encapsulated
in culture. 50.
islets after
Aldehyde
thionin
Single intraperitoneal transplants of 4.55.0 X lo3 encapsulated islets reversed diabetes in all 8 recipients within two days. Only one transplant failed within one month (Fig. 2H). The individual blood glucose profiles of the other 7 animals are shown in Figs. 2A-G. The mean fasting plasma glucose dropped from a pre-transplant value of 378 * 5.4 (mean + SEM) mg/dl to 85.4 + 5.4 mg/dl two days post-transplantation. Two of these animals were still normoglycemic when sacrificed at 365 (Fig. 2A) and 648 (Fig. 2C) days posttransplantation, indicating the continuous insulin secretion from the encapsulated cells to meet the physiological demand of the recipients. Capsules were recovered from animals at 156, 365 and 648 days posttransplantation. Histological staining revealed the presence of intact islets. The surfaces of most capsules were free of cell attachment and were physically intact with the enclosed islets clearly visible. Some capsules had fibroblast-like cells on the external surfaces. Transplant recipients showed a very rapid increase in body weight and no evidence of cataract development in their eyes was observed. Untreated diabetic controls showed no significant weight increase during the same
Fig. 3. Photomicrograph of encapsulated cytes. Magnification x 15.
rat hepato-
E
induced fulminant hepatic failure. The survival rate of the recipient rats was increased by 50% when approximately 1 X 10’ encapsulated rat hepatocytes were implanted intraperitoneally. When encapsulated hepatocytes were cultured, a significant amount of albumin was observed in the culture medium. H
0
50
100
1.50
200
250
300
350
400
DISCUSSION
0l)p
Fig. 2. Fasting plasma glucose profiles of streptozotocininduced diabetic rats after transplantation of 4.5-5.0 X 10’ encapsulated rat islets. Note different scales in Figs. 2a and 2b. Vertical bars denote time of second transplantation, where applicable (2B, 2D, 2E, 2F, 2G).
period and developed eye cataracts within 2-3 months. Five animals each received a second transplant of 5 X lo3 encapsulated islets when they regressed to the diabetic state (Figs. 2B, 2D, 2E, 2F and 2G). Two of these transplants functioned for the life span of the recipients (Figs. 2B and 2D); only in one case did the second transplant function for a shorter period than the initial implant (Fig. 2E). Allografts of encapsulated hepatocytes
Encapsulated implanted into
hepatocytes rats with
(Fig. 3) were D-galactosamine-
This study demonstrates that pancreatic cells encapsulated in a biocompatible membrane can be used as a long-term hormone or enzyme delivery system. Intraperitoneal transof encapsulated pancreatic islets plant reversed diabetes for over a year. Intact capsules containing islets were recovered from the peritoneal cavity 12--21 months after transplantation. Histological staining and insulin secretion studies on encapsulated islets recovered from the recipients demonstrated that the capsules contained viable beta cells with insulin granules and retained the ability to secrete insulin in response to glucose challenge. These results clearly indicate that microencapsulated islet cells can provide a long-term insulin delivery system. We have previously reported that the alginate-polylysinealginate membrane has the properties of a hydrogel and is approximately 4 pm in thickness [4]. The biocom-
141
patibility of a number of hydrogels has been established and these materials have several biomedical applications. Recently Klom et al. [5] reported the use of a macroporous hydrogel membrane in a hybrid artificial this hydrogel membrane elicited pancreas; considerably less fibrous tissue reaction than the membranes used for other hybrid artificial pancreas studies. Portable devices providing continuous insulin infusion either subcutaneously, intraperitoneally or intravenously are now in use in a large number of centers. Whether the improved glycemic control achieved with these devices delays the progression of established lesions or prevents their development remains a controversy. Initial results indicate that 6 months of continuous subcutaneous insulin infusion (CSII) had a beneficial effect on retinal and kidney functions, but followup data revealed that after a further 6 months retinal morphology had deteriorated in the CSII group and there was no significant difference between these patients and a group receiving conventional treatment. Tamborlane et al. [6] found that despite an improvement in glycemic control, insulin pump treatment for two years did not reverse established diabetic microvascular lesions. Unexpected deaths occur in some patients on CSII and are believed to be attributable to hypoglycemia. It is interesting to note that in our study cataracts, a symptom of diabetic complications in experimental animals, did not develop in the recipient rats while every untreated diabetic control rat developed cataracts within 2-3 months. This observation would suggest that transplantation of encapsulated islets has the potential to prevent the development of the sequalae of diabetes. Although the results from experiments with microencapsulated hepatocytes are of a preliminary nature, they indicate a promising basis for the development of an adequate support system for the failing human and animal liver.
CdNCLUSlON
This study demonstrates that allografts can be successfully immunoisolated within biocompatible membranes and provide a longterm delivery system. In the future this technique could have clinical applications in the field of bioartifical organ transplantation.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the expert technical assistance of A. Wood, H. Van Rooy, R. Taylor and S. Chow. This research was supported in part by grants from the Medical Research Council of Canada and the Canadian Diabetes Association.
REFERENCES P.E. Lacy and M. Kostianovsky, Method for the isolation of intact islets of Langerhans from the rat pancreas, Diabetes, 16 (1967) 35-39. B. Desbuquois and G.D. Aurbach, Use of polyethyleneglycol to separate free and antibodybound peptide hormones in radioimmunoassay, J. Clin. Endocrinol., 33 (1971) 732-738. R.J. Bonney, J.E. Becker, P.R. Walker and V.R. Potter, Primary monolayer cultures of adult liver parenchymal cell suitable for study of the regulation of enzyme synthesis, In Vitro, 9 (1973) 399-413. A.M. Sun, G.M. O’Shea and M.F.A. Goosen, Injectable microencapsulated islet cells as a bioartificial pancreas, Appl. Biochem. Biotechnol., 10 (1984) 87-99. G.F. Klomp, H. Hashiguchi, P.C. Ursell, Y. Takeda, T. Taguchi and W.H. Dobelle, Macroporous hydrogel membranes for a hybrid artificial pancreas. II. Biocompatibility, J. Biomed. Mater. Res., 17 (1983) 865-871. W.V. Tamborlane, J.E. Puklin, M. Bergman, C. Verdonk, M.C. Rudolf, P. Felig, M. Gene1 and R. Sherwin, Long-term improvement of metabolic control with the insulin pump does not reverse diabetic microangiopathy, Diabetes Care, 5 (1982) 58-64.