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.

Insulin inhibits its own secretion from isolated, perifused human pancreatic islets.

It is still a controversial question whether insulin suppresses its own secretion. We prepared pure human islets from three pancreases by collagenase digestion and density gradient purification. Aliquots of 200 islet equivalents (IE, 150-microns sized-islets) were sequentially perifused at 37 degrees C with 3.3 mmol/l glucose (3.3G, 40 min), 16.7 mmol/l glucose (16.7G, 30 min) and again 3.3G (30 min) after 24 h, 37 degrees C culture in CMRL 1066 medium with or without the addition of either 200 or 400 microU/ml human insulin in the incubation medium (6 replicates each). Insulin secretion was assessed by C-peptide (Cp) measurement in the perifusate. Without added insulin (C) and with 200 (Ins200) or 400 (Ins400) microU/ml added insulin, basal Cp release was 0.12 +/- 0.03, 0.14 +/- 0.02 and 0.14 +/- 0.04 ng/ml, respectively. At 16.7G, the first-phase secretion peak (expressed as Cp value) was significantly lower with Ins200 (0.47 +/- 0.13 ng/ml, P < 0.02) and Ins400 (0.68 +/- 0.15 ng/ml, P < 0.05) than C (0.83 +/- 0.15 ng/ml). The second-phase secretion peak was also significantly (P < 0.05) reduced with added insulin (Ins200: 0.47 +/- 0.08 ng/ml; Ins400: 0.45 +/- 0.07 ng/ml) than in its absence (C: 0.65 +/- 0.09 ng/ml). Accordingly, total Cp secretion was lower with Ins200 (10.6 +/- 2.3 ng/ml, P = 0.03) and Ins400 (11.8 +/- 2.3 ng/ml) than with C (16.0 +/- 2.2 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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 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.

Activation of intraislet lymphoid cells causes destruction of islet cells.

In vitro culture of rat islets at 24 degrees C for 7 days in tissue culture medium CMRL 1066 almost completely eliminated lymphoid cells from the islets. Immunostaining of the islets with monoclonal antibody OX4 for demonstration of class II major histocompatibility complex (MHC)-expressing cells revealed a decrease from 13.1 +/- 0.6 positive cells per islet on day 0 to 0.7 +/- 0.1 cells per islet on day 7. A comparable decrease was found using OX1 for demonstration of all leukocytes. In contrast, culture of rat islets at 24 degrees C for 7 days with tissue culture Roswell Park Memorial Institute (RPMI) 1640 medium was not as effective in eliminating lymphoid cells as in medium CMRL 1066 (3.0 +/- 0.2 class II MHC positive cells per islet at 7 days). Effective elimination of intraislet lymphoid cells apparently is due to the combined effect of low temperature culture and the tissue culture medium CMRL-1066. The second goal of the study was to determine whether the destructive effect of interferon gamma (IFN-gamma) on rat islets in culture was due to intraislet lymphoid cells. In vitro culture of rat islets with IFN-gamma (1000 units/ml) at 37 degrees C caused almost complete destruction of the islets at 7 days. If intraislet lymphoid cells were eliminated from the islets by in vitro culture at 24 degrees C followed by exposure to IFN-gamma (1000 units/ml) for 7 days at 37 degrees C, then IFN-gamma did not cause destruction of the islets and transplants of the treated islets produced normoglycemia in diabetic recipient mice. These findings indicate that intraislet lymphoid cells are responsible for destruction of islet cells when these cells (presumably macrophages) are activated by IFN-gamma. Intraislet lymphoid cells may play a significant role in destroying islet cells in autoimmune 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.

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.

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.

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.

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.

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.