Prolongation of islet allograft survival following in vitro culture (24 degrees C) and a single injection of ALS.

Isolated rat islets remain morphologically and functionally intact during a 7-day period of in vitro culture at 24 degrees C. In vitro culture of islets at 24 degrees C for 7 days prior to transplantation, in conjunction with a single injection of antiserum to lymphocytes into the diabetic recipient, results in islet allograft survival of 100 days when the islets are transplanted across a major histocompatibility barrier.

Prolongation of islet xenograft survival without continuous immunosuppression.

The survival of isolated rat islets transplanted into diabetic mice was prolonged markedly by maintaining the rat islets in vitro at 24 degrees C for 7 days before transplantation and administering to the recipients a single injection of antiserum to mouse and rat lymphocytes shortly before transplantation.

Induction of immune unresponsiveness to concordant islet xenografts by intrahepatic preimmunization and transient immunosuppression.

Streptozotocin-induced, diabetic mice (C57BL/6) were preimmunized by injecting 25 low temperature, cultured Wistar-Furth (WF) rat islets into the portal vein, and the recipients received one injection of mouse and rat antilymphocyte sera. 3 wk later, fresh WF islets were transplanted under the kidney capsule of the preimmunized recipients, and normoglycemia was maintained in all 13 recipients for 60 d. Removal of the grafts at 60 d returned the mice to a diabetic state. Transplants of fresh WF islets under the kidney capsule without pretreatment of the recipients had a mean survival time of 16.5 +/- 2.5 d. These findings demonstrate that immune unresponsiveness can be achieved across a concordant, islet xenograft barrier within 3 wk after intrahepatic preimmunization with a small number of donor rat islets and transient immunosuppression with antilymphocyte sera.

A method for the mass isolation of islets from the adult pig pancreas.

A method for mass isolation of islets from the pig pancreas is described. The procedure involves the use of an enzymatic and mechanical pancreatic digestion procedure followed by filtration and separation of the islets on Ficoll gradients. A remarkably high yield of purified islets has been obtained from the pig pancreas with this procedure. The islets are morphologically intact and respond to acute stimulation with glucose in vitro.

Use of reflected green light for specific identification of islets in vitro after collagenase isolation.

A simple technique is described that permits specific identification in vitro of isolated islets as contrasted with samll lymph nodes.

Long-term perfusion of isolated rats islets in vitro.

A perfusion system is described for long-term maintenance of isolated rat islets in vitro. This system permits the monitoring of the pattern, rate, and amount of insulin secretion following repeated, acute stimulations with glucose during the period of culture. Fibroblastic proliferation did not occur, thus making is possible to reclaim the islets for biochemical and morphologic studies at the conclusion of the experiments. Maintenance of the islets with a low concentration of glucose (1.0 mg./ml.) resulted in a marked decline in insulin secretion following acute stimulations with glucose (5.0 mg./ml.) during an eight-day interval. Stimulation with 10 mM theophylline and 5.0 mg./ml. glucose on day 9 resulted in enhanced insulin release. The decline in glucose sensitivity occurred even more rapidly when the islets were maintained in the presence of a lower concentration of glucose (0.5 mg./ml.). The pattern of insulin release was altered with an absence of a first phase of secretion. Adenylate cyclase activity of islets maintained with 0.5 mg./ml. glucose for four days was significantly decreased in comparison with islets from fed rats and islets maintained with 2.5 mg./ml. glucose. A means of maintaining the same biphasic pattern and amount of glucose-induced insulin release was achieved by using alternating levels of glucose (1.5 and 2.5 mg./ml) for maintenance of the islets during a 36-day interval.

Effect of culture on islet rejection.

In order to diminish or prevent rejection of transplanted allogeneic islets of Langerhans, in vitro culture was used. After digestion of the rat pancreas and Ficoll separation, islets were handpicked to be free of vascular and ductal tissue. Phenol red in the culture medium imparted a pink color to the islets when observed with a diffuse green light against a black background. Islets cultured at room temperature (24 degrees C) remained functionally and morphologically intact for 1--4 wk. Insulin secretion was 1--3 microU per islet per hour, increasing to 16 microU per islet per hour at 37 degrees C. Culture alone resulted in a modest prolongation of function across a major histocompatibility barrier, Wistar Furth to Lewis (mean survival time, 11.6 +/- 1.2 vs. 7.2 +/- 0.5 days). However, one injection of antilymphocytic serum (ALS) into 10 recipients at the time of transplantation prolonged survival to greater than 100 days in nine rats. In the combination ACI to Lewis, also a major barrier, the same regimen prolonged function to greater than 100 days in five out of five recipients. Injection of donor peritoneal exudate cells resulted in prompt rejection of islets. These results suggest that culture and ALS either damage or alter passenger leukocytes in the donor tissue, thereby preventing rejection of the islets.

An improved method for the isolation of islets from the beef pancreas.

An improved method for the isolation of islets from the beef and pig pancreas is described. The procedure involves the use of strips of Velcro that retain the partially-digested collagen during the isolation of islets by the collagenase technique. The spiny portion of the Velcro is ideally suited to retain the collagen and yet permit the separation of islets from the pancreatic parenchyma. A remarkably high yield of islets has been obtained from the beef and pig pancreas using this procedure.

Induction of tolerance to islet xenografts in a concordant rat-to-mouse model.

Induction of tolerance to concordant rat islet xenografts (150 Wistar-Furth [WF] islets) in streptozocin-induced (STZ) diabetic mice (C57BL/6) was determined at three different sites for islet implantation (thymus, kidney capsule, and liver). Islets transplanted into the thymus or kidney capsule were either fresh or cultured at 24 degrees C for 7 days, and the mice received a single injection of either anti-mouse lymphocyte serum (MALS) alone or anti-rat lymphocyte serum (RALS) and MALS. Islets transplanted into the liver via the portal vein were cultured at 24 degrees C for 7 days, and the mice received a single injection of MALS and RALS. To document the induction of tolerance, recipients with islet xenografts surviving > 100 days were made diabetic again by STZ (thymus and liver) or nephrectomy (kidney capsule) and received a second transplant of 150 fresh WF islets in the kidney capsule. Kidney capsule placement of fresh or cultured islets with MALS alone or MALS and RALS did not induce tolerance in a significant number of recipients. The intrathymic transplantation of fresh or cultured islets with MALS alone resulted in prolonged WF islet xenograft survival (mean survival time of 39.7 +/- 7.9 days) but did not result in tolerance, whereas the administration of MALS and RALS with the intrathymic placement of fresh or cultured islets induced tolerance in approximately 50% of the mice. Intrahepatic transplantation of cultured islets with MALS and RALS resulted in tolerance to donor islets in 90% of the recipients. Donor specificity was evaluated by a third major histocompatibility complex-disparate fresh Lewis islet xenograft.(ABSTRACT TRUNCATED AT 250 WORDS)

Automated method for isolation of human pancreatic islets.

We describe an automated method for the isolation of human pancreatic islets. The procedure meets the following requirements: 1) minimal traumatic action on the islets, 2) continuous digestion in which the islets that are progressively liberated can be saved from further enzymatic action, 3) minimal human intervention in the digestion process, and 4) high yield and purity of the isolated islets. After purification on Ficoll gradients, an average of 164,600 islets/pancreas was obtained (2279 islets/g), with an average purity of 78.5% islets. The average volume and average insulin content of the final islet preparation were 348 mm3 and 93.4 U, respectively. The islets were morphologically intact with a normal degree of beta-granulation and responded to glucose stimulation with a fivefold increase of insulin secretion over basal levels. The procedure is now being used for the initiation of the second phase of clinical trials on human islet transplants.

Transplantation of insulin-producing tissue.

A series of new findings have shown that it is possible to prevent rejection of islet allografts and islet xenografts in animals without the continued use of immunosuppressive agents. The survival of allografts of rat islets has been prolonged for more than 100 days by in vitro culture of the islets at 24 degrees C for seven days prior to transplantation in conjunction with a single injection of rat antilymphocyte serum at the time of transplantation, Xenograft survival of rat islets transplanted into diabetic mice has been prolonged for more than 100 days by the use of culture of rat islets at low temperature, with a single injection of antiserums to mouse and rat lymphocytes at the time of transplantation. Rejection of established islet allografts in rats was induced by the injection of donor peritoneal exudate cells and donor T lymphocytes, whereas the injection of B lymphocytes did not induce rejection. The pretreatment regimens used for prolonging islet allograft and xenograft survival apparently destroy or alter passenger leukocytes in the grafts, and it would appear that these cells are needed for the induction of immune recognition by the recipient. Islet cells in mice express products of the H-2K and H-2D loci, but the cells do not express the I region (Ia) antigens. These new developments in the prevention of immune rejection of the islets raise the question as to whether these approaches may be applicable to the transplantation of islets into human subjects with diabetes.

Effect of transplantation site and alpha L3T4 treatment on survival of rat, hamster, and rabbit islet xenografts in mice.

Rat, hamster, and rabbit islets were transplanted into diabetic C57BL/B6 mice. The effect on islet xenograft survival of low-temperature culture of the donor islets, alpha L3T4 treatment of the recipients for seven days, and transplantation of the grafts either in the renal capsule or in the liver via the portal vein was determined. Renal capsule transplants of control rat, hamster, and rabbit islets cultured at 37 degrees C for one day produced normoglycemia in the recipients, with the mean survival time (MST) of the grafts ranging from 14 to 19 days. Low temperature culture alone did not produce a significant increase in the survival time. Treatment of the recipients with alpha L3T4 produced a marked prolongation of xenograft survival for all three species receiving renal subcapsular transplants of control or low temperature cultured islets. The range of MST* was from 34 to 46 days. The intrahepatic site produced an even further prolongation of the survival of concordant rat islet xenografts treated in this manner, with 60% of the recipients still normoglycemic at 100 days after transplantation. This enhancing effect of the intrahepatic site on survival did not occur with the discordant xenografts of hamster and rabbit islets.

A new site for islet transplantation--a peritoneal-omental pouch.

A new site for islet transplantation is described. A peritoneal-omental pouch was constructed in diabetic rats by encasing the omentum in a pouch formed from a strip of parietal peritoneum obtained from the recipient. Isografts of rat islets placed in the pouch maintained normoglycemia in the recipients and removal of the pouch resulted in a rapid return to a diabetic state. This site may be applicable to the transplantation of islets in human diabetes.

Prolongation of islet xenograft survival (rat to mouse) by in vitro culture at 37 C.

Meticulously selected rat islets were maintained in vitro for 7 days in a minimal volume of tissue culture medium at 37 C in air and 5% CO2. The cultured islets were transplanted into diabetic mice, either into the liver via the portal vein, or beneath the renal capsule. The survival of the cultured islets, following intrahepatic or renal subcapsular transplantation, was significantly prolonged compared with that of fresh islets. The renal subcapsular site apparently provides some additional immunologic advantage, because the survival time of the cultured islets in this site was approximately twice as long as in the intrahepatic transplants.

Automated large-scale isolation, in vitro function and xenotransplantation of porcine islets of Langerhans.

To evaluate the potential of utilizing porcine islet tissue as an alternative to human islet tissue for transplantation, we developed a method for the isolation of large amounts of highly purified porcine islets, and assessed the in vitro and in vivo function of the isolated islets after 1, 4, and 7 days of culture. The pancreatic duct of the splenic lobe was cannulated and distended by injection of Hanks' balanced salt solution containing 1.5 mg/ml collagenase. The pancreas was then processed by a modification of the automated digestion-filtration method developed in this laboratory, and with purification accomplished by Euro-Ficoll gradients (dialyzed Ficoll in Eurocollins solution), consisting of two layers of 1.108 and 1.091 g/cm3 density, topped with a layer of HBSS. The postpurification yield was 5203 +/- 645 (mean +/- SEM) islets per gram of pancreas with a number of islet equivalents (IE) per gram pancreas (islet equivalence: 150-microns-sized islets) of 3551 +/- 305, and a volume of 6.27 +/- 1.7 mm3 islet tissue per gram of pancreas. The islet purity exceeded 90%. Overnight-cultured, perifused porcine islets released 53.1 +/- 8.2 pM insulin/200 IE at 3.3 mM glucose, and 114.9 +/- 25.4 pM insulin/200 IE at 16.7 mM glucose (P less than 0.001 vs. basal output). When theophylline was added, insulin secretion increased to 264.2 +/- 63.2 pM/200 IE (P less than 0.001 vs. basal secretion and P less than 0.005 vs. secretion at 16.7 mM glucose). After 4 days of culture, the islets still responded to secretagogues. The functional integrity of the isolated islets was confirmed by reversal of diabetes in aL3T4 antibody-treated C57B/B6 diabetic mice: normoglycemia was promptly restored by transplanting 1000 overnight- or 7-day-cultured (24 degrees C) islets under the kidney capsule. These results suggest that continued improvements of porcine islet isolation and culture could permit the use of porcine islets in immunoalteration and immunoisolation studies that may lead to eventual human transplantation.

Prolongation of islet allograft survival.

Pretreatment of donor rats with irradiation and silica followed by in vitro culture of the islets for 1 to 2 days prolonged survival of allografts across a minor histocompatibility barrier if "hand-picked," clean islets were used for transplantation. Pretreatment of donor rats with irradiation and silica in conjunction with a single injection of antilymphocyte serum (ALS) into the recipient produced a prolongation of survival of hand-picked islets transplanted across a major histocompatibility barrier.

Characterization of lymphocytes from rejected and nonrejected islet xenografts.

A procedure is described for obtaining lymphocytes from xenografts of rat islets transplanted beneath the renal capsule of diabetic mice. In acute rejection of transplants of fresh rat islets, the lymphoid reaction was composed of 90% T lymphocytes with a predominance of Ly-2 cells. The Ly-2 cells were presumably cytotoxic T lymphocytes. On the other hand, if the islets are pretreated to avoid rejection, by culture in 95% O2 and administration of antilymphocyte serum to the recipients, the lymphocytes that are attracted by the graft are quite different. First, the percentage of T lymphocytes decreased, although they continue to be the most common cell. Second, however, the Ly phenotype was altered. Early after transplantation the Ly-2 population was decreased relative to Ly-1 cells. By day 70, the proportion of Ly-2 cells had returned to that of infiltrates actively rejecting the grafts, even though no rejection was evident. It is possible that the Ly-2+ cells present in nonrejected, established islet xenografts may be suppressor lymphocytes.

Prolongation of islet xenograft survival by in vitro culture of rat megaislets in 95% O2.

Individual rat islets could be aggregated into single megaislets in vitro and the megaislets remained morphologically and functionally intact after a 7-day period of culture in the presence of 95% O2 and 5% CO2. Cultured rat megaislets transplanted beneath the renal capsule of diabetic mice produced normoglycemia in the recipients and the survival of the xenografts was markedly prolonged by the 7-day exposure of a high oxygen tension. A single injection of antilymphocyte sera to mouse and rat lymphocytes into the recipients receiving cultured megaislets did not produce a further increase in the percentage of survival of the grafts at 70 days after transplantation. Lymphoid aggregates were present around xenografts of cultured negaislets at 60 and 90 days after transplantation. This lymphoid reaction did not interfere with the function of the xenografts since the recipients were normoglycemic and removal of the grafts resulted in a rapid return to a diabetic state. Intraportal and intrasplenic transplants of cultured rat megaislets did not survive as long as the xenografts of megaislets transplanted beneath the renal capsule. The renal subcapsule site apparently provided some immunological advantage for delaying acute rejection since transplants of individual, fresh rat islets survived for twice as long under the renal capsule as compared wtih intraportal transplants of fresh rat islets.

Cryogenic storage of isolated, purified porcine pancreatic islets.

Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25 degrees C, supercooled to -7.5 degrees C, nucleated, slowly cooled at 0.25 degrees C/min to -40 degrees C, and stored at -130 degrees C. Then, after variable periods of storage, the islets were rapidly thawed at 37 degrees C. Postthaw actual islet and islet equivalent (150-microns sized islets) recovery were 75 +/- 7% and 66 +/- 4%, respectively. The frozen-thawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43 +/- 10 and 67 +/- 18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P = 0.2). Peak insulin release at 16.7 mmol/L glucose was 85 +/- 28 pmol/L from noncryopreserved islets and 157 +/- 48 pmol/L from the frozen-thawed islets (P = 0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221 +/- 83 (control islets) and 479 +/- 140 pmol/L (cryopreserved islets, P = 0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412 +/- 306 vs. 3756 +/- 764 pmol/L, respectively, P = 0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161 +/- 371 vs. 7505 +/- 2075 pmol/L, respectively, P = 0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500-2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39 +/- 3 days) was similar to that of noncryopreserved islet xenografts (43 +/- 6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozen-thawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets.

Prolonged survival of discordant porcine islet xenografts.

Purified porcine islets were prepared by collagenase digestion and density gradient purification, and transplanted under the kidney capsule of C57B/B6 mice with streptozotocin-induced diabetes which were receiving varying temporary immunosuppressive therapies. Islets that had been cultured for 1 day at 37 degree C were rejected after : 9+/-0.1 (mean+/-SE) days in control mice: 14+/-3 days in mice receiving mouse antilymphocyte serum (MLS) plus porcine antilymphocyte serum (PLS) on day of transplant (day 0); 43+/-6 days in mice treated for 1 week with anti-CD4 antibody (aCD4); 36+/-4 days in mice given aCD4 for 1 week plus PLS on days 0 and 7; 47+/-3 days in mice treated with aCD4 for 1 week plus MLS and PLS on day 21. Porcine islet survival in these latter three groups was significantly (P<0.01) and similarly longer than in the control and MLS plus PLS groups. Then, we transplanted islets that had been either cultured at 24 degrees C for 7 days or cryopreserved into 7-day aCD4-treated mice, to evaluate whether low temperature culture or the freezing-thawing procedure could affect survival. Neither 7-day, low temperature culture (mean survival time: 37+/-2 days) nor cryopreservation (mean survival time: 39+/-2 days) prolonged islets function further. Thus, the present study demonstrates that prolonged survival can be achieved with discordant porcine islet xenografts, and shows the greater efficacy of aCD4 treatment, which was not improved by additional immunosuppressive therapies we tested, nor by culture or cryopreservation of the islets.