Wah Jun Tze

Studies with implantable artificial capillary units containing rat islets on diabetic dogs

SummaryAn implantable artificial endocrine pancreas consisting of a coiled single acrylic copolymer capillary surrounded by rat islets (1000/kg body weight) was implanted in 10 streptozotocin-alloxan diabetic dogs. About 5 h following implantation plasma glucose decreased from an initial mean value of 350 mg/dl to 150 mg/dl, and then to 100 mg/dl at 12 h. Plasma insulin increased to a mean of 39 mU/l (range 23–83 mU/l) at 5 h in the recipient animals. In addition a much improved plasma glucose disappearance rate (K = 1.9 ±0.3) with slightly delayed insulin responses was seen after intravenous glucose tolerance tests performed in 4 dogs at 7, 8, 10 and 18 h following implantation. These findings suggest that xenogeneic rat islets implanted as an artificial endocrine pancreas can improve glucose metabolism in the diabetic dog.

Implantable artificial capillary unit for pancreatic islet allograft and xenograft

SummaryImplantable artificial capillary units containing approximately 1,200 allogeneic rat islets, or approximately 3,000 xenogeneic rabbit or human islets as an implantable artificial endocrine pancreas (IAEP) were implanted in streptozotocin-induced (55 mg/kg) diabetic rats. A total of 26 rats received IAEP containing allogeneic islets. Twenty were short term experiments which lasted for 12–24 h. Four recipients survived between 1–3 days and the remaining two for 4 and 11 days respectively. Five diabetic rats received IAEP containing xenogeneic islets. The four recipients of IAEP containing rabbit islets survived up to 4 days while the recipient of IAEP containing human islets survived for 8 days. Following implantation, a decrease of plasma glucose from the initial value of 500 mg/dl to normoglycaemia and a corresponding increase in circulating levels of insulin up to 100 μU/ml were observed in the recipient animals. Furthermore the IAEPs were shown to produce a near normal plasma glucose and insulin response to an intravenous glucose tolerance test. These findings suggest the feasibility of achieving amelioration of diabetes with allogeneic or xenogeneic pancreatic islets implanted as an artificial endocrine pancreas unit in the experimental animals which has the potential of future clinical application in man.

Intracerebral allotransplantation of purified pancreatic endocrine cells and pancreatic islets in diabetic rats

Allogeneic pancreatic endocrine cells (PEC) and whole islets from inbred Lewis (AgB 1/1) and outbred Wistar rats were implanted intracerebrally (i.c.) into two designated areas of streptozotocin-induced diabetic ACI (AgB 4/4) rats across the major histocompatibility barrier. All the transplants of PEC from Lewis (n=12) and Wistar (n=7) donors remained functional for an observation period in excess of 200 days. In contrast, only 3/6 Lewis and 3/9 Wistar whole-islet transplants were able to maintain function for a prolonged period. Recipients with functional PEC or islet allografts had normalized nonfasting blood glucose (BG) in the 24-hr. BG profile, and they maintained a steady body weight gain. ACI recipients of PEC from Lewis rats had glucose disappearance K rates of 1.3±0.3 (mean ± SE) and a normal basal BG level in 4 hr following the i.v. glucose load. Histological section of the brain tissues with successful i.c. islet or PEC grafts up to a duration of 51/2 months revealed healthy endocrine cells in the cortex and the subarachnoid space. These grafts were permeated with capillaries but devoid of exocrine tissues or lymphoid cell infiltration. These observations suggest that the brain is an immunologically privileged site, and that it is a hospitable site for the pancreatic endocrine cell suspension. However, the immunological protection offered to allogeneic transplants by the brain is incomplete, and purified PEC must be employed to ensure consistent long-term allograft survival.

Successful Intracerebral Allotransplantation of Purified Pancreatic Endocrine Cells in Diabetic Rat

Single pancreatic endocrine ceils (PEC) have been demonstrated to restore normoglycemia when transplanted intracerebrally in diabetic rat recipients across a major histocompatibility barrier without immuno-suppression. Twelve out of twelve transplants of purified PEC have achieved long-term survival of over 176 days. This approach would provide a model to identify the specific cell(s) as inducer of graft rejection and an opportunity for transplantation study in spontaneous diabetic BB rats and larger diabetic animal models.

Long-term Survival of Adult Rat Islets of Langerhans in Artificial Capillary Culture Units

Pancreatic islets from adult rats were cultured in artificial capillary culture units that were perfused with nutrient medium. In this in-vitro culture system, islets remain functional for at least 97 days. As determined by insulin radioimmunoassay of the culture medium, islets continue to secrete insulin. Furthermore, islets responded to high glucose concentration in the perifusion medium by increased insulin release throughout the duration of the culture period. A normal appearance of individual epithelioid islet cells and the presence of aldehyde-fuchsin-positive granules were observed in islets fixed in medium. As indicated by the prolonged maintenance of tissue-specific function, the present simple technique for organ culture of intact islets should be useful for providing new information on the long-term effects in vitro of various factors on islet function and a means for the preservation of islets for transplantation.

A diabetic rabbit model for pig islet xenotransplantation.

An alloxan-diabetic rabbit model was established for the testing of the function of discordant xenogeneic pig islets isolated and purified from adult pig pancreata. The functional state of the pig islet transplants and immunological state of the rabbit recipients were assessed. Intraportal transplantation of 0.47±0.01 ml of pig islets with estimated 57418±5020 in number containing an estimated insulin content of 33.93±2.97 units (n=7; mean ± SEM) resulted in the normalization of blood glucose with a corresponding rise in insulin levels in the diabetic rabbit recipients for 2 days. An intravenous glucose tolerance test performed in 4 recipients during the normoglycemic period resulted in an improved K rate (2.5±0.4) over the diabetic controls, but this was significantly lower than the normal control animals (K rate = 4.5±0.4; n=8). In vitro studies demonstrated that the preformed antibodies detected in the rabbit recipients were cytotoxic to the pig islet cells and lymphocytes. Heat treatment at 56°C and mercaptoeth-anol treatment markedly reduced the cytotoxic activities of the sera. These findings implicated involvement of complement and IgM class antibodies in the killing of the pig islet cells. Furthermore, pig islet transplants at the kidney capsule site were coated with IgM class antibodies. This study has demonstrated that pig islets can be successfully isolated and purified in sufficient numbers for xenotransplantation studies in alloxan-diabetic rabbit. The porcine islet-to-alloxan diabetic rabbit combination can serve as a highly stringent and useful discordant model for assessing the effectiveness of various immunomodulation and immunosuppressive regimens. The finding of an optimal approach to immuno-rejection would potentially be applicable to actual clinical islet xenotransplantation in diabetic patients.

Successful intracerebral allotransplantation of pancreatic endocrine cells in spontaneous diabetic BB rats without immunosuppression

An approach for pancreatic-islet transplantation in the spontaneous diabetic Bio-Breeding (BB) Wistar rat is described in which purified allogeneic pancreatic endocrine cells (PEC) are inoculated intracerebrally in the diabetic recipients. Without immunosuppression, 12/12 of the recipient animals responded to the transplanted PEC with no further need of insulin administration. Also, 8/12 showed normalization of nonfasting blood glucose (BG), 24-hour urine output, and gradual increase in body weight (BW) for a mean observation period of over 122.1 +/- 13.6 days. They had a glucose disappearance K rate of 1.7 +/- 0.3 (mean +/- SE), following intravenous (IV) glucose load. The grafted PEC remain functional and apparently are not affected by the usual immunorejection and/or original disease process during the whole period of observation. This achievement of long-term allograft function without immunosuppression would provide an opportunity to study the recurrence of diabetes and the effect of islet-cell transplantation in chronic diabetic complication in the spontaneous diabetic BB rat model which closely resembles human type 1 diabetes mellitus.

Transplantation of discordant pig islet xenografts in diabetic rats

Xenotransplantation of pig islets under the kidney capsule (KC) of diabetic rats was performed. Natural preformed ACI rat anti-pig leukocytotoxicity, leukoagglutination and hemagglutination antibody titers ranged from Neat-1: 16, 1:8-1:32 and 1:128-1:256, respectively (n = 14). Normal ACI sera were non-toxic to pig islets during short term incubation. Pig islet xenograft survival times in the nonimmunosuppressed ACI rats, ACI rats immunosuppressed with antithymocyte serum (ATS) or cyclosporin A were 3.8 +/- 0.4 (mean +/- SE; n = 5), 10.4 +/- 0.7 (n = 13) and 6.0 +/- 1.0 (n = 5) days, respectively. Pig islets implanted in the abdominal testis of ACI recipients immunosuppressed with 5 doses ATS survived for a mean of 6.4 +/- 1.0 days (n = 7). The mean K rate following an intravenous glucose tolerance test (IVGTT) in ACI rats 1 week after transplantation with pig islet under the KC was 2.2 +/- 0.4 (n = 10) compared to that of 2.91 +/- 0.30 found in normal control rats (n = 8). Peak insulin at 1 min was 60.1 +/- 3.9 microU/ml (n = 4). Histological and immunohistochemical examination showed that the xenograft from recipients treated with 5 doses of ATS still contained well-preserved islet tissue with many insulin- and glucagon-containing cells on the day of graft removal when blood glucose had returned to hyperglycemic level. Both CD4 and CD8 positive cells were in the vicinity of the graft tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Allotransplantation of Dispersed Single Pancreatic Endocrine Cells in Diabetic Rats

Dispersed pancreatic endocrine cells (PECs) were recently shown to survive indefinitely in the brain of allogeneic diabetic rat recipients across the major histocompatibility barrier without immunosuppression. Purified PECs (2−3 × 106 cells) prepared from Wistar rat islets were transplanted in 10 different sites in streptozocin-induced diabetic ACI rats. Blood glucose and body weight changes were monitored throughout the study. PEC isografts (n = 5) and allografts (n = 6) were nonfunctional when transplanted in intramuscular, intravenous, and intrahepatic sites. When transplanted intraportally (n = 6) and intraperitoneally (n = 6), similar grafts were functional but had a short survival period (6.6 ± 1.5 and 15.3 ± 16.6 days, respectively). Prolonged graft survival in some recipients was observed when kidney capsule [5, 9, 10, 240, 282, and 300 (2 rats) days], omentum pocket (7, 8, 10, 90, 105, 154, 155, and 157 days), and testis (7, 8, 12, 150, 230, and 234 days) were the transplantation sites. Permanent graft survival was achieved in 20 of 20 recipients with intracerebral transplantation and 21 of 22 recipients with intrathecal transplantation. These findings confirm that dispersed single PECs can be transplanted as a permanent functional graft and can normalize the hyperglycemia of the diabetic recipients. The duration of PEC graft survival is variable and depends on the transplantation site. Both the cerebral cortex and subarachnoid space, which are immunologically privileged sites, have provided the best allograft protection.

Successful banking of pancreatic endocrine cells for transplantation

To determine optimal freezing and thawing conditions for rat pancreatic endocrine cells (PEC) and insulinoma cells, five different cryopreservation protocols were compared in this study. PEC and insulinoma cells were cooled at rates of between -0.3 degrees C/min and -5 degrees C/min to -70 degrees C in the presence of 10%, 15%, or 20% dimethylsulfoxide (DMSO) with a programmable temperature controller and then transferred to liquid nitrogen for storage. Frozen cells were thawed by either rapid (in 37 degrees C water bath) or slow (in air) thawing procedure. One hour after the thawing process, cellular viability was determined by trypan blue dye exclusion. The viability results for PEC and insulinoma cells were similar and showed that a slow cooling rate at -0.3 degrees C/min in combination with a rapid thawing in 37 degrees C water bath gave the best results, with up to 80% cellular viability. Cryoprotectant DMSO used at 10% concentration was the most effective among the three concentrations tested. Later, transplantation studies were performed with PEC cryopreserved with the best protocol, which is -5 degrees C/min to 4 degrees C, held for 3 minutes, -0.3 degrees C/min to -7 degrees C, held for 3 minutes, -0.3 degrees C/min to -40 degrees C, and -5 degrees C/min from -40 degrees C to -70 degrees C in 10% DMSO with a programmable temperature controller then transferred to liquid nitrogen for storage.(ABSTRACT TRUNCATED AT 250 WORDS)