- Email: [email protected]
Subcutaneous xenotransplantation of hybrid artificial pancreas encapsulating pancreatic B cell line (MIN6): Functional and histological study
Cell Transplantation, Vol. 6. No. 5, pp. 541-545.1997 0 1997ElsevierScienceInc. Printedin theUSA. All rightsreserved 0963.6897/97 $17.00+ .OO ELSEVIER
PI1 SO963-6897(97)00074-2
Original Contribution
SUBCUTANEOUS XENOTRANSPLANTATION OF HYBRID ARTIFICIAL PANCREAS ENCAPSULATING PANCREATIC B CELL LINE (MIN6): FUNCTIONAL AND HISTOLOGICAL STUDY YOSHIYUKI
KAWAKAMI,*’
MASAYUKI
KAZUTOMO INOUE,* HIROYUKI HAYASHI,*
IMAMLJRA,* HIROO IWATA,~
YOSHITO IKADA,t
W.J. WANG,*
HIROSHI SETOYAMA,*
MASUMI NOZAWA,$
Y.J. Gu,*
AND JUN-ICHI MIYAZAKI~
*First Departmentof Surgery, Faculty of Medicine, tResearch Center for Biomedical Engineering, Kyoto University, Kyoto 606, Japan; iDepartment of Surgery, Meikai University, Japan; $Department of Disease-Related Gene Regulation, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan
increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 f 10.0 vs. 37.4 f 10.7 vu/3 X lo6 cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation. 0 1997 Elsevier Science Inc.
IJ Abstract - The biohybrid artificial pancreas is designed to enclose pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harboring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times
0 Keywords - Biohybrid artificial pancreas; Pancreatic B cell line (MIN6); Subcutaneous xenotransplantation; Microbeads.
INTRODUCTION
The biohybrid artificial pancreas is designed to enclose the pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing the endocrine tissues to survive and control glucose metabolism for an extended period of time. We have previously demonstrated that three-layer microbeads composed of polybrene, carboxymethyl cellulose, and a mixture of agarose and polystyrene sulfonic acid (PSSa), used as the bioartificial pancreas seeded with rat islet grafts achieved long-term survival after xenotransplantation to diabetic mice without employing any immunosuppressants (10). The pancreatic B cell line MIN6 is established from pancreas B cell tumors that develop in transgenic mice harboring the human insulin promoter gene connected to the SV40
ACCEPTED512 1197.
Kyoto University, 54.Shogoin Kawaracho, Sakyo-ku, Kyoto, 606.01, Japan.
should be addressed to Yoshiyuki Kawakami, First Department of Surgery, Faculty of Medicine, ‘Correspondence
541
542
Cell Transplantation
0 Volume 6, Number 5, 1997
T-antigen hybrid gene. It has been demonstrated that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets (5,7). In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas.
MATERIALS
AND METHODS
Encapsulation of A4IN6 Cells MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 25 mM glucose, 15% fetal bovine serum) equilibrated with 5% CO, and 95% air at 37°C. Semiconfluent cells of passage numbers 25 in Petri dishes were removed by 0.1% trypsin-phosphate buffer solution (PBS). MIN6 cells were suspended in serumfree DMEM and mixed with 5% agarose and 5% PSSa dissolved in serum-free minimum Eagle’s essential medium (MEM) at 40°C in a 50 mL glass centrifuge tube. Droplets of agarose-PSSa mixture encapsulating MIN6 cells were shaped by addition of paraffin oil and by shaking manually. Gel formation of the microcapsules was then induced by gentle agitation in an ice bath for 5 min. Polybrene solution 1% in serum-free MEM was added to a glass centrifuge tube, which was then immersed and gently agitated in an ice bath for 30 min. After that, the tube was centrifuged at 800 rpm for 10 min and the supernatant of paraffin oil and polybrene solution was removed by suction. This procedure was repeated again. Finally, the microcapsules were washed three times with ice-cooled Hank’s balanced salt solution (HBSS), immersed in 1% carboxymethyl cellulose solution in serum-free MEM in an ice bath for 15 min, and again washed with HBSS. The microbeads were incubated for 24 h in the medium before the subcutaneous xenotransplantation.
Subcutaneous Xenotransplantation Male Lewis rats, from lo- to 12-wk-old, were used as transplant recipients made diabetic by a single intravenous injection of streptozotocin (55 mg/kg body weight, dissolved in citrate buffer). Diabetic rats were defined as those with two consecutive nonfasting blood glucose levels of more than 400 mg/dL. Microbeads encapsulating MIN6 cells (3 X 10’) were xenogenically implanted in the subcutaneous tissue of the back of a recipient rat through a skin incision. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation study and insulin secretion was observed. Retrieved microbeads were offered to the histological study.
Histological Examination For histological examination, microbeads containing MIN6 cells were fixed in 4% paraformaldehyde (PFA) for 24 h and dehydrated in 70% ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Immunocytochemical staining for insulin was performed using streptoavidin-biotin (SAB) method with HISTOFINE SAB-PO kit (Nichirei Co., Japan). Briefly, sections were placed on APS coated slides and fixed with 4% PFA for 1 h, deparaffined with 100% ethanol for 3 min, and repeated more two times, followed by the primary antibody (polyclonal guinea pig antiinsulin antibody). The slides were washed with PBS and incubated with biotin-labeled rabbit antimouse antibody for 10 min at the room temperature. The slides were washed with PBS and incubated with peroxidase-conjugated streptoavidin for 5 min at the room temperature. Then the slides were washed with PBS and treated with diaminobenzidin (DAB) solution for 15 min at the room temperature and washed with distilled water. Finally, the slides were stained with methylgreen and mounted.
Static Incubation Study Insulin secretory responses to glucose stimulation were observed in a static incubation study. The microbeads were pre-incubated for 1 h in Krebs-Ringer bicarbonate buffer (KRBB) containing 0.2% bovine serum albumin (BSA) and 3.3 mM glucose at 37”C, and then incubated in KRBB containing 3.3 mM glucose for 2 h. Next, the microbeads were incubated in KRBB containing 16.7 mM glucose for an additional 3 h. Buffer solutions were changed every hour and each time samples were taken for subsequent insulin assay with an enzyme immunoassay kit (Glazyme, Sanyo, Japan).
RESULTS
There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Grafted microbeads were easily retrieved without any complications.
Histological Examination Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells (Fig. 1). Histological study showed that these MIN6 cells in the microbeads appeared to be viable and cellular infiltration were not observed within or around the microbeads. Immunohistochemical analysis of the microbeads revealed that intense staining of insulin in the cytoplasm of encapsulated MIN6 cells was clearly ob-
Subcutaneous xenotransplantation of MIN6 0 Y.
KAWAKAMI
543
ET AL.
Fig. 1. Photomicrograph of microbeads neous xenotransplantation (X75).
retrieved
1 wk after subcuta-
Fig. 2. Histological examination of microbeads retrieved I wk after subcutaneous xenotransplantation (H-E staining, magnification X44).
Fig. 3. Immunohistochemical examination of microbeads retrieved 1 wk after subcutaneous xenotransplantation (Insulin staining, magnification X 110).
served. Insulin productivity is strongly suggested
Static Incubation
of MIN6 cells in the microbeads
to be preserved
(Figs. 2 and 3).
Study
Insulin secretory response before subcutaneous xenotransplantation; In the static incubation study, the insulin
secretion of microbeads encapsulated with MIN6 cells showed a significantly greater response after 16.7 mM glucose stimulation (34.2 + 2.8 pU/3 X lo6 cells/hr, II = 5, p < 0.01) than that observed in response to 3.3 mM glucose stimulation (14.8 I+_2.9 l.~U/3 X lo6 cells/hr, n = 5). Insulin secretory response 1 wk after subcutaneous
544
Cell Transplantation 0 Volume 6, Number 5, 1997
h
f
I
pco.01
-,
16.7
Glucose concentration(mM) Fig. 4. Glucose-stimulated insulin secretion from the microbeads retrieved 1 wk after subcutaneous xenotransplantation.
xenotransplantation; The insulin secretion of retrieved microbeads in response to 3.3 mM glucose was 37.4 2 10.7 p,U/3 X IO6 cells/hr (n = 5), whereas that of retrieved microbeads after stimulation with 16.7 mM glucose was 84.3 + 10.0 pU/3 X lo6 cells/h (n = 5), showing a significantly greater response (approximately 2.3-fold, p < 0.0 1) (Fig. 4).
DISCUSSION
Islet transplantation with a biohybrid artificial pancreas is a potential alternative to whole pancreas and islet transplantation (9). We have previously demonstrated that three-layer microbeads (agarose-PSSa) seeded with islets functioned effectively after xenotransplantation as well as after allotransplantation (6,lO). Recent study has shown that in subcutaneous allotransplantation, human islets encapsulated in hollow acrylic-copolymer fibers were protected from any immune reaction of the recipient for a period of 2 wk after transplantation (8). A variety of transplantation sites have been proposed, including the spleen, epiploic pouch, peritoneal cavity, testes, thymus, kidney capsule, brain, subcutaneous and intramuscular sites, and even the pancreas itself (1). The subcutaneous site would have the advantage of being accessible and implanted devices could be retrieved easily when needed. To solve the serious problem of a shortage of human donors, we have confirmed the
effectiveness of MIN6 cells enclosed in MRPB (meshreinforced polyvinyl alcohol hydrogel bag) devices each acting as a biohybrid artificial pancreas in xenotransplantation (4). In this study, we performed the histological and functional examination of three-layer microbeads employing MING cells after subcutaneous xenotransplantation. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation. In the histological study, we clearly found that MIN6 cells encapsulated in three-layer microbeads revealed an intense staining of insulin in the cytoplasm. This histological findings was supported by the functional study with the static incubation that demonstrated good secretory response of insulin from the microbeads. It has been pointed out that one of the crucial impediments to achieve the long-term survival of grafted bioartificial pancreas is immune cell-mediated inflammatory response induced by chronic leakage of soluble antigens (3). In the recent report, various biomedical polymers used in artificial membranes have been shown to activate macrophages involved in foreign body reaction and induce synthesis of interleukin-lp, a known P-cell toxin. Smaller molecules, such as cytokines, are considered to pass through the membrane, and their permeability is affected by not only molecular cutoff but other factors including molecular geometry, chemical, and physical properties and binding of proteins (2). Our histological findings showed no evidence of infiltration of inflammatory cells within or around the microbeads at least 1 wk after subcutaneous xenotransplantation, suggesting an excellent biocompatibility of our device. We have already confirmed that three-layer microbeads encapsulated with rat islets achieved long-term graft survival on intraperitoneal xenotransplantation to diabetic mice (10). The outmost layer of microbeads, carboxymethyl cellulose, may be also effective to prevent inflammatory response in the transplantation site of subcutaneous tissue. In this study, we demonstrate that three-layer microbeads encapsulating MIN6 cells show excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation. These results point to a possible future clinical application of this promising bioartificial pancreas to subcutaneous xenotransplantation. Finally, further studies must be done to evaluate the glucose metabolism in the diabetic recipient such as the change of serum glucose levels and insulin levels after subcutaneous xenotransplantation as well as the long term function of our subcutaneously xenotransplanted device.
Acknowledgments-This study was supported in part by a grant from the Ministry of Education, Japan (#B07457250, #A08557071).
Subcutaneous
xenotransplantation
of MIN6 0 Y. KAWAKAMI
REFERENCES R.G.; Hering, B.J.; Stroedter, D.; Zekom, T.; Federlin, K.F. Experimental islet transplantation in small animals. In: Ricordi, C., eds. Pancreatic islet cell transplantation Austin, TX: R.G. Landes: 250; 1992. Cole, D.R.; Waterfall, M.; McIntyre, M.; Baird, J.D. Microencapsulated islet grafts in the BB/E rat: A possible role for cytokines in graft failure. Diabetologia 35:231237; 1992. Colton, C.K.; Avgoustiniatos, E.S. Bioengineering in development of the hybrid artificial pancreas. J. Biochem. Eng. 113:152-170; 1991. Hayashi, H.; moue, K.; Aung, T.; Tun, T.; Echigo, Y.; GU, Y.J.; Shinohara, S.; Kaji, H.; Kato, M.; Imamura, M.; Maetani, S.; Morikawa, N.; Iwata, H.; Ikada, Y.; Miyazaki, J.-I. Xenotransplantation of a novel B-cell line (MIN6) in mesh-reinforced polyvinyl alcohol hydrogel bag. Transplant. Proc. 27:3358-3361; 1995. Ishihara, H.; Asano, T.; Tsukuda, K.; Katagiri, H.; Inukai, K.; Anai, M.; Kikuchi, M.; Yazaki, Y.; Miyazaki, J.-I.; Oka, Y. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 36:1139-1145; 1993. Iwata, H.; Takagi, T.; Kobayashi, K.; Oka, T.; Tsuji, T.;
1. Bretzel,
2.
3.
4.
5.
6.
7.
8.
9.
10.
ET AL.
545
Ito, F. Strategy for developing microbeads applicable to islet xenotransplantation into a spontaneous diabetic NOD mouse. J. Biomed. Mater. Res. 28:1201-1207; 1994. Miyazaki, J.-I.; Araki, K.; Yamato, E.; Ikegami, H.; Asano, T.; Shibasaki, Y.; Oka, Y.; Yamamura, K.-I. Establishment of a pancreatic cell line that retains glucose-inducible insulin secretion: Special reference to expression of glucose transporter isoforms. Endocrinology 127:126-132; 1990. Scharp, D.W.; Swanson, C.J.; Olack, B.J.; Latta, P.P.; Hegre, O.D.; Doherty, E.J.; Gentile, F.T.; Flavin, K.S.; Ansara, M.F.; Lacy, P.E. Protection of encapsulated human islets implanted without immunosuppression in patients with type I or type II diabetes and in nondiabetic control subjects. Diabetes 43:1167-l 170; 1994. Soon-Shiong, P.; Heintz, R.E.; Merideth, N.; Yao, Q.X.; Yao, Z.; Zheng, T.; Murphy, M.; Moloney, M.K.; Schmehl, M.; Harris, M.; Mendez, R.; Mendez, R.; Sandford, P.A. Insulin independence in a type 1 diabetic patient after encapsulated islet transplantation. Lancet 343:950951; 1994. Tein, T.; Inoue, K.; Hayashi, H.; Tun, A.; Gu, Y.J.; Doi, R.; Kaji, H.; Echigo, Y.; Wang, W.J.; Setoyama, H.; Imamura, M.; Maetani, S.; Morikawa, N.; Iwata, H.; Ikada, Y. A newly developed three-layer agarose microcapsule for a promising biohybrid artificial pancreas; Rat to mouse xenotransplantation. Cell Transplant. 5:59-63; 1996.
PI1 SO963-6897(97)00074-2
Original Contribution
SUBCUTANEOUS XENOTRANSPLANTATION OF HYBRID ARTIFICIAL PANCREAS ENCAPSULATING PANCREATIC B CELL LINE (MIN6): FUNCTIONAL AND HISTOLOGICAL STUDY YOSHIYUKI
KAWAKAMI,*’
MASAYUKI
KAZUTOMO INOUE,* HIROYUKI HAYASHI,*
IMAMLJRA,* HIROO IWATA,~
YOSHITO IKADA,t
W.J. WANG,*
HIROSHI SETOYAMA,*
MASUMI NOZAWA,$
Y.J. Gu,*
AND JUN-ICHI MIYAZAKI~
*First Departmentof Surgery, Faculty of Medicine, tResearch Center for Biomedical Engineering, Kyoto University, Kyoto 606, Japan; iDepartment of Surgery, Meikai University, Japan; $Department of Disease-Related Gene Regulation, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan
increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 f 10.0 vs. 37.4 f 10.7 vu/3 X lo6 cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation. 0 1997 Elsevier Science Inc.
IJ Abstract - The biohybrid artificial pancreas is designed to enclose pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harboring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times
0 Keywords - Biohybrid artificial pancreas; Pancreatic B cell line (MIN6); Subcutaneous xenotransplantation; Microbeads.
INTRODUCTION
The biohybrid artificial pancreas is designed to enclose the pancreatic endocrine tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing the endocrine tissues to survive and control glucose metabolism for an extended period of time. We have previously demonstrated that three-layer microbeads composed of polybrene, carboxymethyl cellulose, and a mixture of agarose and polystyrene sulfonic acid (PSSa), used as the bioartificial pancreas seeded with rat islet grafts achieved long-term survival after xenotransplantation to diabetic mice without employing any immunosuppressants (10). The pancreatic B cell line MIN6 is established from pancreas B cell tumors that develop in transgenic mice harboring the human insulin promoter gene connected to the SV40
ACCEPTED512 1197.
Kyoto University, 54.Shogoin Kawaracho, Sakyo-ku, Kyoto, 606.01, Japan.
should be addressed to Yoshiyuki Kawakami, First Department of Surgery, Faculty of Medicine, ‘Correspondence
541
542
Cell Transplantation
0 Volume 6, Number 5, 1997
T-antigen hybrid gene. It has been demonstrated that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets (5,7). In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas.
MATERIALS
AND METHODS
Encapsulation of A4IN6 Cells MIN6 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 25 mM glucose, 15% fetal bovine serum) equilibrated with 5% CO, and 95% air at 37°C. Semiconfluent cells of passage numbers 25 in Petri dishes were removed by 0.1% trypsin-phosphate buffer solution (PBS). MIN6 cells were suspended in serumfree DMEM and mixed with 5% agarose and 5% PSSa dissolved in serum-free minimum Eagle’s essential medium (MEM) at 40°C in a 50 mL glass centrifuge tube. Droplets of agarose-PSSa mixture encapsulating MIN6 cells were shaped by addition of paraffin oil and by shaking manually. Gel formation of the microcapsules was then induced by gentle agitation in an ice bath for 5 min. Polybrene solution 1% in serum-free MEM was added to a glass centrifuge tube, which was then immersed and gently agitated in an ice bath for 30 min. After that, the tube was centrifuged at 800 rpm for 10 min and the supernatant of paraffin oil and polybrene solution was removed by suction. This procedure was repeated again. Finally, the microcapsules were washed three times with ice-cooled Hank’s balanced salt solution (HBSS), immersed in 1% carboxymethyl cellulose solution in serum-free MEM in an ice bath for 15 min, and again washed with HBSS. The microbeads were incubated for 24 h in the medium before the subcutaneous xenotransplantation.
Subcutaneous Xenotransplantation Male Lewis rats, from lo- to 12-wk-old, were used as transplant recipients made diabetic by a single intravenous injection of streptozotocin (55 mg/kg body weight, dissolved in citrate buffer). Diabetic rats were defined as those with two consecutive nonfasting blood glucose levels of more than 400 mg/dL. Microbeads encapsulating MIN6 cells (3 X 10’) were xenogenically implanted in the subcutaneous tissue of the back of a recipient rat through a skin incision. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation study and insulin secretion was observed. Retrieved microbeads were offered to the histological study.
Histological Examination For histological examination, microbeads containing MIN6 cells were fixed in 4% paraformaldehyde (PFA) for 24 h and dehydrated in 70% ethanol, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Immunocytochemical staining for insulin was performed using streptoavidin-biotin (SAB) method with HISTOFINE SAB-PO kit (Nichirei Co., Japan). Briefly, sections were placed on APS coated slides and fixed with 4% PFA for 1 h, deparaffined with 100% ethanol for 3 min, and repeated more two times, followed by the primary antibody (polyclonal guinea pig antiinsulin antibody). The slides were washed with PBS and incubated with biotin-labeled rabbit antimouse antibody for 10 min at the room temperature. The slides were washed with PBS and incubated with peroxidase-conjugated streptoavidin for 5 min at the room temperature. Then the slides were washed with PBS and treated with diaminobenzidin (DAB) solution for 15 min at the room temperature and washed with distilled water. Finally, the slides were stained with methylgreen and mounted.
Static Incubation Study Insulin secretory responses to glucose stimulation were observed in a static incubation study. The microbeads were pre-incubated for 1 h in Krebs-Ringer bicarbonate buffer (KRBB) containing 0.2% bovine serum albumin (BSA) and 3.3 mM glucose at 37”C, and then incubated in KRBB containing 3.3 mM glucose for 2 h. Next, the microbeads were incubated in KRBB containing 16.7 mM glucose for an additional 3 h. Buffer solutions were changed every hour and each time samples were taken for subsequent insulin assay with an enzyme immunoassay kit (Glazyme, Sanyo, Japan).
RESULTS
There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Grafted microbeads were easily retrieved without any complications.
Histological Examination Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells (Fig. 1). Histological study showed that these MIN6 cells in the microbeads appeared to be viable and cellular infiltration were not observed within or around the microbeads. Immunohistochemical analysis of the microbeads revealed that intense staining of insulin in the cytoplasm of encapsulated MIN6 cells was clearly ob-
Subcutaneous xenotransplantation of MIN6 0 Y.
KAWAKAMI
543
ET AL.
Fig. 1. Photomicrograph of microbeads neous xenotransplantation (X75).
retrieved
1 wk after subcuta-
Fig. 2. Histological examination of microbeads retrieved I wk after subcutaneous xenotransplantation (H-E staining, magnification X44).
Fig. 3. Immunohistochemical examination of microbeads retrieved 1 wk after subcutaneous xenotransplantation (Insulin staining, magnification X 110).
served. Insulin productivity is strongly suggested
Static Incubation
of MIN6 cells in the microbeads
to be preserved
(Figs. 2 and 3).
Study
Insulin secretory response before subcutaneous xenotransplantation; In the static incubation study, the insulin
secretion of microbeads encapsulated with MIN6 cells showed a significantly greater response after 16.7 mM glucose stimulation (34.2 + 2.8 pU/3 X lo6 cells/hr, II = 5, p < 0.01) than that observed in response to 3.3 mM glucose stimulation (14.8 I+_2.9 l.~U/3 X lo6 cells/hr, n = 5). Insulin secretory response 1 wk after subcutaneous
544
Cell Transplantation 0 Volume 6, Number 5, 1997
h
f
I
pco.01
-,
16.7
Glucose concentration(mM) Fig. 4. Glucose-stimulated insulin secretion from the microbeads retrieved 1 wk after subcutaneous xenotransplantation.
xenotransplantation; The insulin secretion of retrieved microbeads in response to 3.3 mM glucose was 37.4 2 10.7 p,U/3 X IO6 cells/hr (n = 5), whereas that of retrieved microbeads after stimulation with 16.7 mM glucose was 84.3 + 10.0 pU/3 X lo6 cells/h (n = 5), showing a significantly greater response (approximately 2.3-fold, p < 0.0 1) (Fig. 4).
DISCUSSION
Islet transplantation with a biohybrid artificial pancreas is a potential alternative to whole pancreas and islet transplantation (9). We have previously demonstrated that three-layer microbeads (agarose-PSSa) seeded with islets functioned effectively after xenotransplantation as well as after allotransplantation (6,lO). Recent study has shown that in subcutaneous allotransplantation, human islets encapsulated in hollow acrylic-copolymer fibers were protected from any immune reaction of the recipient for a period of 2 wk after transplantation (8). A variety of transplantation sites have been proposed, including the spleen, epiploic pouch, peritoneal cavity, testes, thymus, kidney capsule, brain, subcutaneous and intramuscular sites, and even the pancreas itself (1). The subcutaneous site would have the advantage of being accessible and implanted devices could be retrieved easily when needed. To solve the serious problem of a shortage of human donors, we have confirmed the
effectiveness of MIN6 cells enclosed in MRPB (meshreinforced polyvinyl alcohol hydrogel bag) devices each acting as a biohybrid artificial pancreas in xenotransplantation (4). In this study, we performed the histological and functional examination of three-layer microbeads employing MING cells after subcutaneous xenotransplantation. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation. In the histological study, we clearly found that MIN6 cells encapsulated in three-layer microbeads revealed an intense staining of insulin in the cytoplasm. This histological findings was supported by the functional study with the static incubation that demonstrated good secretory response of insulin from the microbeads. It has been pointed out that one of the crucial impediments to achieve the long-term survival of grafted bioartificial pancreas is immune cell-mediated inflammatory response induced by chronic leakage of soluble antigens (3). In the recent report, various biomedical polymers used in artificial membranes have been shown to activate macrophages involved in foreign body reaction and induce synthesis of interleukin-lp, a known P-cell toxin. Smaller molecules, such as cytokines, are considered to pass through the membrane, and their permeability is affected by not only molecular cutoff but other factors including molecular geometry, chemical, and physical properties and binding of proteins (2). Our histological findings showed no evidence of infiltration of inflammatory cells within or around the microbeads at least 1 wk after subcutaneous xenotransplantation, suggesting an excellent biocompatibility of our device. We have already confirmed that three-layer microbeads encapsulated with rat islets achieved long-term graft survival on intraperitoneal xenotransplantation to diabetic mice (10). The outmost layer of microbeads, carboxymethyl cellulose, may be also effective to prevent inflammatory response in the transplantation site of subcutaneous tissue. In this study, we demonstrate that three-layer microbeads encapsulating MIN6 cells show excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation. These results point to a possible future clinical application of this promising bioartificial pancreas to subcutaneous xenotransplantation. Finally, further studies must be done to evaluate the glucose metabolism in the diabetic recipient such as the change of serum glucose levels and insulin levels after subcutaneous xenotransplantation as well as the long term function of our subcutaneously xenotransplanted device.
Acknowledgments-This study was supported in part by a grant from the Ministry of Education, Japan (#B07457250, #A08557071).
Subcutaneous
xenotransplantation
of MIN6 0 Y. KAWAKAMI
REFERENCES R.G.; Hering, B.J.; Stroedter, D.; Zekom, T.; Federlin, K.F. Experimental islet transplantation in small animals. In: Ricordi, C., eds. Pancreatic islet cell transplantation Austin, TX: R.G. Landes: 250; 1992. Cole, D.R.; Waterfall, M.; McIntyre, M.; Baird, J.D. Microencapsulated islet grafts in the BB/E rat: A possible role for cytokines in graft failure. Diabetologia 35:231237; 1992. Colton, C.K.; Avgoustiniatos, E.S. Bioengineering in development of the hybrid artificial pancreas. J. Biochem. Eng. 113:152-170; 1991. Hayashi, H.; moue, K.; Aung, T.; Tun, T.; Echigo, Y.; GU, Y.J.; Shinohara, S.; Kaji, H.; Kato, M.; Imamura, M.; Maetani, S.; Morikawa, N.; Iwata, H.; Ikada, Y.; Miyazaki, J.-I. Xenotransplantation of a novel B-cell line (MIN6) in mesh-reinforced polyvinyl alcohol hydrogel bag. Transplant. Proc. 27:3358-3361; 1995. Ishihara, H.; Asano, T.; Tsukuda, K.; Katagiri, H.; Inukai, K.; Anai, M.; Kikuchi, M.; Yazaki, Y.; Miyazaki, J.-I.; Oka, Y. Pancreatic beta cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets. Diabetologia 36:1139-1145; 1993. Iwata, H.; Takagi, T.; Kobayashi, K.; Oka, T.; Tsuji, T.;
1. Bretzel,
2.
3.
4.
5.
6.
7.
8.
9.
10.
ET AL.
545
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