Problems associated with the use of dispersed pancreatic tissue for xenotransplantation: A possible solution

Abstract: In light of the disappointing clinical results with purified islets, we propose that a simple preparation of impure islets may provide a better graft for both future allogeneic and xenogeneic transplantation trials. We hypothesize that the simplest, least traumatic procedure to disperse the pancreas, which minimizes the disruption of normal cellular associations, will yield an islet graft of maximum viability. The peritoneal cavity is an attractive site for the transplantation of such preparations since it can accommodate nearly unlimited graft volumes, and is easily accessible. In the present study, we tested the ability of impure islet autografts transplanted in the peritoneal cavity to reverse diabetes in a preclinical model. Dogs (N=29) underwent total pancreatectomy and were divided into three groups: group I (N=7) received intraportal pure islets; group II (N=13) received intraperitoneal pure islets; group III (N=9) received intraperitoneal impure islets. There were no significant differences in islet mass transplanted in the groups: group I 11,686 ± 2,514 IE/kg, group II 10,274 ± 1,125 IE/kg, and group III 11,735 ± 1,497. At 15 months, all purified grafts in group I failed, and all but one purified graft in group II failed, while five impure grafts in group III continued to function ( P < 0.05, versus group I and II, X ²). Glucose disposal in dogs with functioning grafts did not differ between groups. In conclusion, free impure islets are able to engraft within the peritoneal cavity and reverse diabetes in a large animal. Impure islets grafts were superior to purified grafts in initial engraftment and long-term functional survival. Thus, the peritoneal cavity represents a site that can support the engraftment and long-term function of both impure islet allografts and possibly xenografts if sufficient immune suppression or tolerance is achieved.     

Functional assessment of microencapsulated porcine islets with agarose polystyrene sulfonic acid in vitro and in xenotransplantation

We evaluated the functional efficacy of microencapsulated porcine islet xenografts transplanted into nonobese diabetic (NOD) mice. Islets were isolated from the pancreata of CSK miniature swine by manual collagenase digestion and Ficoll purification. Purified porcine islets were immediately encapsulated into microbeads of agarose polystyrene sulfonic acid (Ag-PSSa). They remained morphologically intact by dithizone staining after 7 days in culture. Insulin secretion from encapsulated islets was determined in response to glucose challenge during perifusion. When encapsulated islets were exposed to 200 mg/dL glucose, within 5 minutes, insulin release became 5-fold greater than that at 80 mg/dL. However, a second phase insulin secretion appeared in response to 250 mg/dL glucose challenge. In xenotransplantation, microencapsulated porcine islets (1000 to 1800 MC islets) were transplanted into the peritoneal cavity of diabetic NOD mice (n = 4) without immunosuppression. The survival times after the onset of diabetes were observed after both MC islets transplanted NOD mice and nontransplanted NOD mice (n = 4). MC islets transplant recipients had significantly (P < .05) longer survival (47.5 +/- 18.6; mean +/- SD) than nontransplanted NOD mice (21.0 +/- 9.31), although random blood glucose levels were not normalized.     

国产胶原酶在小鼠胰岛分离中的效果研究#br#

目的 研究一种国产胶原酶在小鼠胰岛分离中的分离效果,探索该胶原酶在胰岛分离中应用的 可行性。方法 将国产胶原酶分别配成不同浓度的胶原酶溶液和含中性蛋白酶的胶原酶的溶液,经胰管逆行灌注胶原酶的方法进行小鼠胰岛分离,采用Ficoll液对胰岛进行纯化,计数所得的胰岛细胞团,培养 6 h后检测活性,对最佳分离结果的胰岛做同系糖尿病小鼠的肾被膜下移植,监测术后血糖和进行组织学 检查,以进口Sigma胶原酶V为对照。结果 国产胶原酶在小鼠胰腺消化时间、所得胰岛数量和活性效果 上跟进口胶原酶V有一定差距（P＜0.01）,但含中性蛋白酶的国产胶原酶组在小鼠胰岛分离数量和当量上 与进口胶原酶组无差异（P＞0.05）,分离的胰岛在体内移植后降血糖效果与进口胶原酶组无差异（P＞0.05）。 结论 国产胶原酶结合中性蛋白酶可用于小鼠胰岛的分离。     

Long-term survival and function of intraportal porcine and human islet xenografts in nondiabetic nude mice

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.     

Multiple donor allotransplantation. A new approach to pancreatic islet transplantation

Currently it is not feasible to isolate sufficient numbers of islets from a single pancreas for clinical transplantation. We examined whether small numbers of islets obtained from multiple donors could be used for transplantation. Islets were isolated from four inbred strains of mice (DBA/2, DBA/1, C3H, and A.SW) by a stationary collagenase digestion and Ficoll separation and transplanted into the renal subcapsular space of streptozotocin-induced diabetic B6AF1 mice. At least 200 handpicked islets were required to produce normoglycemia in syngeneic and allogeneic diabetic recipient mice. None of the mice given 50 islets became normoglycemic within 2 weeks postgrafting. When various numbers of purified islets from a single donor were transplanted, the survival was significantly better for 200-islet allografts than for 800-islet and 400-islet allografts. When a 200-islet composite graft was prepared from four donors (50 fresh handpicked islets from each donor), the survival of the composite graft as measured by sustained normoglycemia in nonimmunosuppressed recipients was dramatic, with 17 of 18 recipients maintaining normoglycemia indefinitely (greater than 200 days). Similarly, when islets isolated from four donors and cultured for various periods were mixed and transplanted (200 islets/recipient) all recipient mice (n = 8) enjoyed indefinite graft survival. Use of higher numbers of purified islets or crude islets in a composite multiple-donor islet allograft was less effective in achieving indefinite graft survival. Thus, transplantation of a composite graft made up with subtherapeutic numbers of islets from multiple histoincompatible donors to provide adequate therapeutic numbers is a practical solution to the lack of islet availability. In addition, composite islet grafts appear to possess immunological advantages, with significantly prolonged survival over that produced by single-donor islet grafts.     

Intrathymic islet transplantation in the canine: I. Histological and functional evidence of autologous intrathymic islet engraftment and survival in pancreatectomized recipients

BACKGROUND: Although an attractive alternative to daily insulin therapy, allogeneic pancreatic islet transplantation has yielded suboptimal results in clinical trials, in contrast to islet allotransplantation in animal models, which have demonstrated consistent success. The successful transplantation of isolated islets to the thymus, with a single concomitant dose of antilymphocyte serum, has been demonstrated in rodents, and more significantly, such intrathymic islet allografts have been shown to induce recipient tolerance toward subsequent extrathymic donor strain islet allografts. Intrathymic islet autotransplantation has been pursued, as a prelude to studies of allogeneic IT islet transplantation and tolerance induction, in canine, porcine, and non-human primate models, to assess the large animal thymus as a site capable of supporting a viable islet graft. However, little functional or histological evidence has established definitive survival of islets transplanted within the thymus of a phylogenetically advanced species, which may be requisite to tolerance induction. This study describes the successful intrathymic autotransplantation of isolated islets using a canine model. METHODS: Purpose-bred juvenile dogs, aged 4-6 months, underwent partial (n=4), or total pancreatectomy (n=11), and transplantation of autologous islets. The pancreas (or pancreatic limb) was distended with collagenase solution, and digested using a modification of the semiautomated system of Ricordi. Islets were purified by discontinuous gradient centrifugation, using Euroficoll (ficoll in Euro-Collin's kidney preservation solution). Partially pancreatectomized canines underwent IT transplantation of purified autologous islets (8000+/-4000 IEs), and were killed 8 weeks posttransplant. Totally pancreatectomized canines underwent transplantation of autologous islets to the liver (via portal vein embolization, n=5, IPO group) or the thymus (via direct IT injection, n=6, IT group), and were serially evaluated for a period of 8 weeks posttransplant to assess fasting blood glucose (FBG), serum insulin (SI) levels, and i.v. glucose tolerance (IVGTTs). K values (defined as the %-decrease/minute of the log(e) of blood glucose values) were calculated from IVGTT results. RESULTS: After autotransplantation in this cohort of animals, five of five IPO, and three of six IT islet recipients, remained normoglycemic (mean FBG< or =250 mg%) immediately posttransplant, and all recipients exhibited significantly elevated SI levels compared to apancreatic controls (n=10, followed 72 hr postpancreatectomy). Normal k values (=-1.1) were observed in two of five IPO, and in one of six IT recipients, 8 weeks after transplantation, and thymic tissue insulin content was increased compared to non-islet-bearing thymi (93.7+/-48.6 ng/g tissue vs. 0.7+/-0.4 ng/g tissue). At 8 weeks posttransplantation thymi from both partially and totally pancreatectomized animals were resected and processed for histological examination. Microscopic analysis of islet-bearing thymi revealed positive staining for islet-specific hormones (insulin and glucagon) within all IT recipients., Identification of islets within thymi of hyperglycemic IT recipients was problematic as islet beta cells were highly degranulated as a result of the recipients glycemic state. CONCLUSIONS: These results indicate that autologous islets, transplanted to the canine thymus, engraft, function, and survive for up to 8 weeks after islet autotransplantation to the canine thymus and establish the feasibility of intrathymic islet transplantation in a phylogenetically advanced animal model. The ability of islets to survive within the thymic environment for a period of at least 8 weeks after transplantation suggests that the successful induction of specific unresponsiveness secondary to intrathymic transplantation will not be impaired or limited by the inability of a viable islet mass to survive within the thymus for a sufficient period.     

Transplantation of isolated islets of Langerhans in diabetic dogs. II. The influence of histocompatibility.

By means of collagenase digestion and the Ficoll gradient separation technique, viable islets of Langerhans could be isolated from dog pancreata. The isolated islets were capable of secreting insulin after glucose stimulation. These islets of Langerhans were transplanted into the livers of diabetic dogs. We transplanted islets isolated from I donor pancreas for 1 kg body wt of the recipients. The dogs were made diabetic by subtotal pancreatectomy (80%) and two injections of 25 mg streptozotocin/kg body wt. In our experiments, we selected donor-recipient pairs by means of the mixed lymphocyte reaction macrophage electrophoretic migration inhibition (MLR-EM) test. In the group with a depression in the MLR-EM test of more than 15% (strong histoincompatibility), the mean survival time of the transplanted dogs was 46 ± 16 days. Another group of recipients with a depression of macrophage migration of less than 8% (weak histocompatibility) had a mean survival time of 135 ± 49 days after intraportal islet transplantation, and the blood glucose values could be normalized in this group for a period of 70 days. We found significant differences between both the groups in the glucose tolerance tests and in the IRI concentrations in the portal vein and in the vena cava superior. In the recipients, which received weakly histoincompatible islets, the IRI concentration in the superior vena cava was 64% of the normal value in the vena cava superior 4 weeks after intrahepatic islet transplantation. By means of the MLR-EM test, we were able to correlate inhibition with signs of rejection of the transplanted islets. Our experiments demonstrated that an allogeneic islet transplantation in diabetic dogs across a weakly histocompatible barrier can improve the results in comparison to the islet transplantation across a strongly histocompatible barrier. However, this typing maneuver alone is not able to prevent the islet rejection.     

Exercise induces hypoglycemia in rats with islet transplantation

Recently, islet transplantation in patients with type 1 diabetes has had greater success than in the past, but the important question of whether the kinetics of islet secretion are able to accommodate the metabolic demands of special conditions such as exercise remains unanswered. Syngeneic rat islets (4,000 islet equivalents/rat) were transplanted into the liver, kidney, and peritoneal cavity (encapsulated or nonencapsulated) of rats with streptozocin-induced diabetes. Normoglycemic transplanted rats and age-matched controls were subjected to 30 min of moderate exercise on a treadmill 5 weeks after transplantation. Although control rats maintained near normoglycemia during and after exercise, the rats with islet transplants had significantly lower blood glucose levels. For the rats with islets in the liver, increased C-peptide levels were found at 30 min (790 +/- 125 and 1,450 +/- 250 pmol/l at 0 and 30 min, respectively; P < 0.01), whereas a decrease was found in controls and in rats with islets transplanted into the peritoneal cavity or under the kidney capsule. Moreover, increased glucagon levels were found after exercise in the rats with islets transplanted into the liver (62 +/- 6, 165 +/- 29, 155 +/- 27, and 97 +/- 13 pg/ml at 0, 30, 60, and 90 min, respectively; P < 0.05), whereas no changes in glucagon levels were observed in controls. In conclusion, moderate exercise caused hypoglycemia in rats with islet transplants in different sites including liver, kidney, and peritoneal cavity. C-peptide and glucagon responses to exercise were very different in rats with transplanted islets compared with controls. This islet dysfunction led to exercise-induced hypoglycemia.     

Long-term survival of intratesticular porcine islets in nonimmunosuppressed beagles

BACKGROUND: The testis is an immunoprivileged organ, and at 37 degrees C, the intratesticular microenvironment supports the survival of allogeneic islets. The objective of this study was to determine whether the immunoprotection afforded by the intratesticular environment is potent enough to prevent the rejection of xenogeneic porcine islets in a large-animal model. METHODS: A bilateral cryptorchid condition was surgically created in sexually mature beagle dogs. Porcine islets were prepared from neonatal pigs by collagenase digestion and 9 days of culture, after which they were injected into each of the cryptorchid testes. Control dogs received liver subcapsular space transplants of porcine islets and autologous islets. After 100 days, the testes and relevant portions of liver were studied immunohistochemically for the presence of islet tissue. RESULTS: The testicular interstitial space of all dogs contained abundant islet tissue. No evidence of lymphocytic infiltration or inflammation was observed. In contrast, porcine islets transplanted to the liver subcapsular space do not survive, although autologous islets engraft well in that position. This occurs even though the recipient's serum contains preformed cytotoxic antibodies to porcine islets that persist after transplantation. CONCLUSIONS: These results demonstrate that the microenvironment existing within the surgically repositioned intra-abdominal testis supports the survival of xenogeneic tissue. The survival of xenogeneic tissue in the absence of immunosuppression in this large-animal model raises the possibility that xenogeneic porcine islet tissue will also survive in humans if transplanted into a similar environment.     

Rejection of islets versus immediately vascularized pancreatic allografts: a quantitative comparison

It has been suggested that free grafts of islets are rejected more vigorously than immediately vascularized intact organs grafts. However, the physiological manifestations of rejection depend, in part, upon the functional reserve of the transplanted tissue. If the number of islets transplanted is just adequate to maintain normoglycemia, the immune destruction of only a few islets will be manifested by hyperglycemia. Thus, differences in rejection time could be an artifact of the islet mass transplanted. We compared the onset of rejection of immediately vascularized segmental pancreatic grafts and of free grafts of islets under conditions in which the β cell mass transplanted, as determined by tissue insulin content, was equivalent. Lewis rats, made diabetic (plasma glucose > 400 mg/dl) by streptozotocin, received either free islet allografts by portal embolization or vascularized segmental pancreatic allografts derived from Fischer donors. Identical pancreatic segments that were not transplanted had a mean (± SE) total tissue insulin content of 33 ± 3 μg. The mean total insulin content of Fischer islets prepared by collagenase digestion in a quantity identical to that used for transplantation to single recipients was 35 ± 7 μg. Similar measurements were made in Fischer to Fischer and Lewis to Lewis isograft control groups. Recipients of both segmental pancreas and free islet grafts became normoglycemic after transplantation and this state was sustained indefinitely in recipients of syngeneic grafts. In rats receiving allografts, the day of rejection, defined as an elevation of plasma glucose to >200 mg/dl, occurred at a mean of 12.1 ± 0.3 days for recipients of pancreatic grafts (n = 17) and 5.2 ± 0.3 days in recipients of islet grafts (n = 17) (P < 0.001). The functional survival of free grafts of allogeneic islets is less than that of islets contained within immediately vascularized pancreatic grafts, even when the transplanted β cell mass is equivalent. However, this difference could still be due to nonimmunologic, quantitative factors that influenced the rate with which hyperglycemia occurred after initiation of the rejection process. The insulin content in the livers of islet isograft recipients showed that only 53 to 71% of the transplanted islets survived. Further experiments that compensate for this factor are needed to determine whether or not there are differences in susceptibility to rejection of the two types of grafts. Nevertheless, on the basis of the number of donors used per recipient, islet allotransplantation is less efficient than pancreas allotransplantation.     

Evidence of functional impairment of syngeneically transplanted mouse pancreatic islets retrieved from the liver

A drawback in pancreatic islet transplantation is the large number of islets needed to obtain insulin independence in patients with diabetes. This most likely reflects extensive posttransplantation islet cell death and functional impairment of the remaining endocrine cells. We aimed to develop an experimental method to retrieve transplanted islets from the mouse liver, which would enable comparisons of transplanted and endogenous islets and provide valuable information on functional changes induced by intraportal transplantation. Transplanted islets were obtained by retrograde perfusion of the liver with collagenase. The identity of retrieved tissue as transplanted islets was confirmed by intravital staining, immunohistochemistry, and electron microscopy. The retrieved islets, irrespective of whether they had resided in diabetic or nondiabetic recipients, had a markedly lower insulin content and glucose-stimulated insulin release when compared with isolated endogenous islets. The glucose oxidation rate was also markedly lower in the retrieved islets, suggesting mitochondrial dysfunction. These disturbances in insulin content, insulin release, and glucose oxidation rate were not reversed by a few days of culture after retrieval. The results implicate changes in islet function after intraportal transplantation. Such dysfunction may contribute to the high number of islets needed for successful transplantation in diabetic individuals.     

Insulin-induced normoglycemia reduces islet number needed to achieve normoglycemia after allogeneic islet transplantation in diabetic mice

The Edmonton protocol established that insulin independence could be reached with the transplantation of an appropriate number of islet cells. However, to effect a cure, islets from two or three pancreases are needed. The aim of this study was to examine whether normoglycemia, with insulin treatment before and after transplantation, reduces the islet number needed to achieve normoglycemia in allogeneic islet transplantation. Swiss mice were used as donors and recipients. Diabetes was induced by i.p. administration of streptozotocin (180 mg/kg BW). Diabetic mice were transplanted with 300 (n = 16), 400 (n = 16), or 500 (n = 16) islets under the left kidney capsule. For every group, half the animals were kept normoglycemic with insulin treatment from day 4 before transplantation to day 10 after transplantation. At the end of the study, all normoglycemic mice were given an i.p. glucose tolerance test (IPGTT). For statistical analysis, paired or unpaired Student's t-test or ANOVA was used. Only insulin-treated mice achieved normoglycemia by the end of the study (37.5% of animals transplanted with 400 islets and 50% transplanted with 300 or 500 islets). At the end of the study, normoglycemic mice transplanted with 300 allogeneic islets showed better glycosylated hemoglobin (HbA1C) than did normoglycemic mice transplanted with 500 islets (300 islets: 2.7 +/- 0.2%; 500 islets: 3.6 +/- 0.2%; p < 0.05). After the IPGTT, insulin-treated mice transplanted with 500 islets showed abnormal glucose tolerance; however, insulin-treated mice transplanted with 300 or 400 islets showed normal glucose tolerance. Insulin treatment reduced the islet number needed to achieve normoglycemia in allogeneic islet transplantation. The HbA1C and IPGTT results suggest that transplanting smaller numbers of allogeneic islets improves beta-cell function; some studies suggest that this may be due to lower immunogenicity, hypoxia, and inflammation.     

Successful allotransplantation of encapsulated islets in pancreatectomized canines for diabetic management without the use of immunosuppression

BACKGROUND: Pancreatic islet transplantation has shown great success in the treatment of diabetic patients. However, the required immunosuppressive therapy exposes patients to serious side effects. METHODS: We have designed a novel five-component/three-membrane capsule and encapsulation system to protect the transplanted islet cells from immune system attack while allowing the influx of molecules and nutrients necessary for cell function/survival and efflux of the desired cellular product, specifically insulin, for making recipients healthy. RESULTS: We transplanted encapsulated canine pancreatic islets into the peritoneal cavity of pancreatectomized canines. Transplantation normalized fasting blood glucose levels in nine out of nine dogs for up to 214 days with a single transplantation. Retransplantation was assessed in three animals and encapsulated islets were effective in providing fasting glycemic control after the initial transplantation had run its course. No immunosuppression or anti-inflammatory therapy was used. CONCLUSION: This advancement in transplantation may lead to an alternative approach for islet transplantation treatment for diabetic patients. This approach may also benefit patients suffering from other hormone deficiency diseases including liver disease and Parkinson's disease.     

A review of piscine islet xenotransplantation using wild‐type tilapia donors and the production of transgenic tilapia expressing a “humanized” tilapia insulin

Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long‐term normoglycemia and provide human‐like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T‐cell‐dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co‐stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.     

Rejection of porcine islet xenografts mediated by CD4+ T cells activated through the indirect antigen recognition pathway

We have previously demonstrated that human T cells responding to porcine islets are primarily CD4+ and recognized porcine major histocompatibility complex class I molecules through the indirect pathway of antigen presentation. To determine whether this mechanism is responsible for rejection of adult porcine islets xenografts, porcine islets from adult pigs were transplanted under the kidney capsule of streptozotocin-treated CD4-knockout (KO), CD8-KO, Ig-KO and normal C57BL/6 mice. Islet xenografts were acutely rejected with similar kinetics when transplanted into normal C57BL/6 (MST=17.6 +/- 3.5 days) and Ig-KO (MST=19.0 +/- 1.7 days) mice. Interestingly, islet xenografts were rejected significantly earlier when transplanted into CD8-KO mice as compared with normal C57BL/6 (MST=7.0 +/- 0.01 days, P=2 x 10-4). Histopathological analysis revealed classical acute cellular rejection with severe diffuse interstitial cellular infiltrates in all rejected islet xenografts. In contrast, islet xenografts were not rejected when transplanted into CD4-KO mice (MST >/= 100 days, P=1 x 10-9). Histopathological analysis revealed no cellular infiltrates and intact islet xenografts. CD4+ T cells from both normal C57BL/6 and CD8-KO xenograft recipients showed detectable proliferative responses to porcine islets in the presence but not in the absence of syngeneic antigen-presenting cells. In addition, the anti-islet proliferative responses observed in normal C57BL/6 mice were significantly lower than those observed in CD8-KO mice. IgG anti-porcine antibodies were readily detected in C57BL/6 and CD8-KO xenograft recipients but not in Ig-KO or CD4-KO recipients. These results indicate that indirectly activated CD4+ T cells mediate acute rejection of adult porcine islet xenografts and that xenoreactive CD8+ T cells and antibodies are not necessary in this process.     

Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature pancreas digestion.

BACKGROUND: Frequent success in human islet isolation is prevented by the large variability of scarce organ donors; this favors the future utilization of pigs as donors for clinical islet xenotransplantation. Porcine-specific difficulties of islet isolation are attributed to the intrinsic fragility of islets during pancreas digestion. METHODS: To preserve islet integrity during efficient pancreas dissociation, porcine pancreata (n=48) were distended after cold storage with cold University of Wisconsin solution containing Liberase HI and digested at 24-28 degrees C using digestion-filtration. Pancreata distended with University of Wisconsin solution containing well-proven crude collagenase and digested at 32-34 degrees C served as controls (n=46). Monolayer Ficolldiatrizoate gradient purification was performed in a Cobe 2991. RESULTS: Purified yield of islet equivalents per pancreas (mean+/-SEM) was almost doubled by Liberase HI compared with crude collagenase (526,480+/-46,560 vs. 270,270+/-19,420; P < 0.0001) and also significantly increased comparing islet equivalents per gram of pancreas (4,210+/-320 vs. 2,640+/-245; P=0.0004). Islet integrity was better preserved during Liberase HI digestion compared with crude collagenase digestion as indicated by isolation index (2.1+/-0.1 vs. 1.4+/-0.1; P<0.0001). Purity, viability, and in vitro function of islets did not differ between experimental groups. Preserved in vivo function of islets isolated by Liberase HI was demonstrated after subcapsular transplantation into 16 diabetic nude rats. CONCLUSIONS: If the problems related to xenograft rejection and xenosis could be solved, low-temperature digestion of porcine pancreata using Liberase HI could serve as an essential prerequisite for successful 1:1 xenotransplantation of pig islets into type 1 diabetic human recipients.     

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

Abstract: The use of lower concentrations of collagenase (0.8 mg/ml as compared to 1.25 and 1.5 mg/ml) resulted in isolation of porcine pancreatic islets retaining their original compact, wholesome appearance. In in vitro experiments, 100% of islet batches isolated with the low collagenase concentration responded to glucose stimulation, while only 63.1% of islet batches isolated with the high concentration of the enzyme did. The response to high glucose challenge of islets isolated with the low enzyme concentration was considerably higher compared to high collagenase concentration islets.
In in vivo experiments, intraperitoneal transplants of microencapsulated islets isolated using 0.8 mg/ml of collagenase reversed diabetes in diabetic BALB-c mice in a significantly larger proportion of recipients (87.5%) as compared to 33.3% of recipients grafted with 1.5 mg/ml collagenase islets.
The results of this study demonstrate that the use of a low collagenase concentration is conducive to isolation of islets of a superior morphology, purity, viability, and functioning ability.     

Immunoisolation of murine islet allografts in vascularized sites through conformal coating with polyethylene glycol

Islet encapsulation may allow transplantation without immunosuppression, but thus far islets in large microcapsules transplanted in the peritoneal cavity have failed to reverse diabetes in humans. We showed that islet transplantation in confined well‐vascularized sites like the epididymal fat pad (EFP) improved graft outcomes, but only conformal coated (CC) islets can be implanted in these sites in curative doses. Here, we showed that CC using polyethylene glycol (PEG) and alginate (ALG) was not immunoisolating because of its high permselectivity and strong allogeneic T cell responses. We refined the CC composition and explored PEG and islet‐like extracellular matrix (Matrigel; MG) islet encapsulation (PEG MG) to improve capsule immunoisolation by decreasing its permselectivity and immunogenicity while allowing physiological islet function. Although the efficiency of diabetes reversal of allogeneic but not syngeneic CC islets was lower than that of naked islets, we showed that CC (PEG MG) islets from fully MHC‐mismatched Balb/c mice supported long‐term (>100 days) survival after transplantation into diabetic C57BL/6 recipients in the EFP site (750‐1000 islet equivalents/mouse) in the absence of immunosuppression. Lack of immune cell penetration and T cell allogeneic priming was observed. These studies support the use of CC (PEG MG) for islet encapsulation and transplantation in clinically relevant sites without chronic immunosuppression.     

Advantages of combined fetal and adult porcine islets as donor tissue for transplantation

Abstract: The molecular similarity between human and porcine insulin is the primary reason for selecting the pig as a donor for islet xenotransplantation in order to treat type I diabetes. Porcine islets can be prepared from adult, newborn, or fetal pigs. However, each of these islet types possesses advantages and disadvantages due to their anatomical, developmental, and physiological characteristics. We have evaluated both adult and fetal porcine islets for their ability to reverse diabetes using an allograft model. Based on our accumulated data, we recommend transplantation of combined adult and fetal porcine islets as a prospective approach for pig to man islet xenotransplantation. The advantages of combined adult and fetal islet transplantation are as follows: 1) Adult porcine islets can reverse hyperglycemia soon after transplantation; 2) well controlled blood glucose at the critical developmental stage of fetal islets will provide the time and environment necessary to achieve the optimal proliferation, growth and maturation of fetal β cells; 3) after 2 to 3 months, fetal islets will functionally mature and begin to share the insulin-producing load previously carried by adult islets, while new islets will continuously emerge and proliferate from stem cells; and 4) islets from fetal pigs will subsequently play a primary role in controlling hyperglycemia, and provide long-lasting insulin independence.     

Chemotaxis activation of peritoneal murine macrophages induced by the transplantation of free and encapsulated pancreatic rat islets

The present study concerns the influence of the transplantation of free and encapsulated (AN69 membrane, Hospal) islets on the chemotaxis of peritoneal macrophages. Fifty free or encapsulated rat islets, cultured for 24 h, were transplanted in the peritoneal cavity of mice (n = 12). Three days after transplantation, the chemotaxis of peritoneal murine macrophages was tested towards formyl-methionyl-leucyl-phenylalanine (fMLP) and a culture medium conditioned for 3 days by free rat islets isolated from the same rat donor. In response to fMLP, the chemotactic indexes of macrophages from mice transplanted with free or encapsulated islets were 8.09 +/- 2.10 and 9.45 +/- 2.76, respectively. These values were significantly higher than those obtained when macrophages from untreated mice were tested (2.42 +/- 0.23; p < 0.01). In response to culture medium conditioned by free islets, the transplanted encapsulated islets failed to enhance macrophage chemotaxis (2.41 +/- 0.53) compared to transplanted free islets (7.00 +/- 2.63; p < 0.01). Thus, encapsulation decreased the specific chemotactic activity of peritoneal macrophages induced by free islet transplantation, probably by prohibiting the diffusion of chemoattractants.