The activation of p38 mitogen-activated protein kinase (MAPK) has been shown to cause ischemia/reperfusion injury of several organs used for transplantation and also to play a significant role in primary islet graft nonfunction. Activation of p38 MAPK may also occur during islet cryopreservation and thawing. In this study, a p38 MAPK inhibitor (p38IH) was applied to human islet cryopreservation to improve islet yield and quality after thawing. Under serum-free conditions, human islets were cryopreserved, thawed and cultured using our standard procedures. Three types of solutions were tested: conventional RPMI1640 medium (RPMI), a newly developed islet cryopreservation solution (ICS), and ICS supplemented with a p38IH, SD-282 (ICS-p38IH). Activation or inhibition of p38 MAPK was demonstrated by the diminished phosphorylation of HSP27 substrate. Islet recovery on day 2 after thawing was highest with ICS-p38IH and islet viability was not significantly different in the three groups. beta Cell numbers and function were the highest in islets cryopreserved with ICS-p38IH. Glucose-stimulated human C-peptide levels were 86% of that of the nonfrozen islets when measured 4 weeks after transplantation into NODscid mice. This improvement may provide an opportunity to establish islet banks and allow the use of cryopreserved islets for clinical transplantation.

The ability of the fetal pancreatic islet cells to multiply rendered them a potential tissue for transplantation studies to cure diabetes. A bank of fetal islets could be created with proper storage in liquid nitrogen. The aim of this study is to evaluate the effect of thawing rate and post-thaw culture on the structural and functional integrity of isolated cryopreserved islets of rat fetuses. Fetal rat islets were isolated by the collagenase digestion, cultured for three days, and then cryopreserved using dimethylsulphoxide as cryoprotectant and the step-rate cooling to -40 degrees C before immersing them in liquid nitrogen. The islets were thawed by the slow or fast warming rates using hyperosmolar sucrose solution and then cultured for 1 or 2 days. Insulin and C-peptide contents of the slow thawed islets were higher than those of the control. In the fast thawed islets the contents were similar to those of the control. Insulin and C-peptide release in response to glucose for the slow thawed islets were lower than those of the control and in the fast thawed islets they were similar to that of the control. Histological examination showed irregular periphery and fragmented central part of the large slowly thawed islets, which showed also variable immunohistochemical reaction to anti-insulin serum, ranging from strongly positive reaction to markedly weak reaction. Fast thawed islets showed mostly regular periphery and their reaction to the anti-insulin serum was slightly weaker than that of the control islets. It was concluded that fast thawing and post-thaw culture is much better than slow thawing, as indicated by nearly normal insulin and C-peptide content and release and intact structural integrity.

Tissue from human fetal cadavers has long been used for medical research, experimental therapies, and various other purposes. Research within the last two decades has led to substantial progress in many of these areas, particularly in the application of fetal tissue transplantation to the treatment of human disease. As a result, clinical trials have now been initiated at centers around the world to evaluate the use of human fetal tissue transplantation for the therapy of Parkinson's disease, insulin-dependent diabetes mellitus, and a number of blood, immunological and, metabolic disorders. Laboratory studies suggest a much wider range of disorders may in the future be treatable by transplantation of various types of human fetal tissue. A combination of characteristics renders fetal tissue uniquely valuable for such transplantation, as well as for basic research, the development of vaccines, and a range of other applications. Although substitutes for human fetal tissue are being actively sought, for many of these applications there are at present no satisfactory alternatives. Important issues remain unresolved concerning the procurement, distribution, and use of human fetal cadaver tissue as well as the effects of such use on abortion procedures and incidence. These issues can be addressed by the introduction of appropriate guidelines or legislation, and need not be an impediment to legitimate research and therapeutic use of fetal tissue.

The radical treatment of type 1 diabetes by transplantation requires the extracorporeal storage of islets, and this has frequently been studied. Damage from ice formation, however, has prevented the development of any satisfactory method for preservation. We compared islet function after frozen storage with that after non-frozen storage. Isolated rat islets immersed in 10% dimethyl sulfoxide were kept at -2 degrees C (group A) and -196 degrees C (group B) for 7 days. After one day of culture, some of the islets were incubated in 3.3 and 16.7 mM glucose-containing Krebs--Henseleit bicarbonate buffer for 60 min. The other islets were incubated with 3H-leucine for 2 h. The radioactivity of whole-islet homogenate and the insulin extracted from it were measured. We also counted the number of islets before and after the 7-day storage. The islets thus preserved were transplanted into streptozotocin-induced diabetic rats and the fasting plasma glucose was determined weekly. Non-cooled islets were used as controls (group C). Insulin release in the presence of 16.7 mM glucose did not significantly differ between groups C and A, whereas it was lower in group B than in groups A or C. The islet uptake of 3H-leucine was lower in A and B compared with C, but the insulin synthesis was similar in all three groups. More islets were recovered from A than B. Fasting plasma glucose was lowered similarly in the diabetic rats after transplantation of islets from A and B. The relative ease of preservation at -2 degrees C, and the positive results of this experiment, favor this method of preservation.

Human fetal pancreatic islet tissue has several advantages as a transplant source for the amelioration of insulin deficiency in patients with Type I diabetes mellitus. It is now possible to obtain viable tissue, store and ship the tissue without adverse effects on the insulin secretory capacity, and transplant either minced tissue or isolated islet-like cell clusters following digestion and culture into animal models or man. A number of centers have undertaken studies of human fetal pancreatic allografts in man. Optimal results have occurred when pooled tissue from six to 20 donors has been implanted and a number of sites have been studied. The author's own experience in four recipients who did not receive immunosuppression has documented insulin secretion for up to 1 year in the absence of an anticytoplasmic islet cell antibody response on the part of the recipients. Nevertheless, the procedure has not resulted in insulin independence for the recipients and the implanted tissue has not secreted insulin in response to a glucose-amino acid challenge in a normal physiologic pattern. Thus, human fetal pancreatic transplantation for the treatment of Type I diabetes remains an experimental approach.

Human fetal pancreata (HFP) were obtained from dilatation and extraction aborted fetuses of 11-18 weeks' gestation. The pancreas was excised under sterile conditions and kept in culture medium at 4 degrees C, prior to stepwise digestion into 50- to 150-micron fragments. The fragmented pieces were allowed to sediment by gravity, then transferred to tissue culture for 24-48 h, and cryopreserved. The freeze-thaw protocol used stepwise equilibration with dimethyl sulfoxide, nucleation of the sample at -10 degrees C, and a slow cooling rate of 0.25 degrees C/min to -40 degrees C, followed by submersion in liquid nitrogen (-196 degrees C). Rapid thawing at 300 degrees C/min from -196 degrees C was employed. Both fresh and frozen-thawed HFP fragments appeared viable as judged by light and electron microscopy, and secreted insulin in a perifusion system upon stimulation with glucose (28 mM) and theophylline (10 mM) or glucose (2.8 mM) and theophylline (10 mM). Six patients with Type I insulin-dependent diabetes mellitus, already requiring immunosuppression for a kidney transplant, had intraportal injection of 20 cryopreserved-thawed and pooled HFP fragments. Up to the 1-year post-transplant follow-up, there has been no evidence of in vivo insulin or C-peptide production. The usefulness of cryopreserved human fetal pancreata as a source of insulin-producing tissue for diabetic patients, therefore, remains to be demonstrated.

Dispersed canine pancreatic grafts were cryopreserved and the in vivo function was studied following intrasplenic autotransplantation. Four protocols were employed, examining the effects of cooling and thawing rates and cryoprotectant (dimethylsulfoxide) concentration on graft survival. The degree of graft injury by each protocol was assessed by examining the requirement for exogenous insulin following transplantation. Cooling at 5 degrees C/min and thawing at 80 degrees C/min allowed three successful grafts from seven when thawed at 80 degrees C/min using 1.4 or 2 M Me2SO but only one success from eight when thawed at 8 degrees C/min. Of the seven experiments where successful preservation was achieved graft injury was estimated as less than 50% in four but for three it was probably greater than 50%. Each protocol exhibited considerable variability of islet survival. When sufficient islet mass was transplanted to restore fasting euglycaemia, graft function, as assessed by glucose-stimulated insulin release and intravenous glucose disposal, was identical to fresh grafts. Successful graft implantation, however, does not guarantee indefinite survival as six of seven grafts in this study became exhausted within 13 months of implantation.

The present study was undertaken to define optimal conditions for cryopreservation of fetal rat liver tissue fragments. First, various cooling rates (1, 4, 10, 20 and 30 degrees C/min) and preserving temperatures (-20, -80 and -196 degrees C) were examined. Next, various concentrations of Me2SO (5, 10, 20 and 30 per cent vol/vol) were examined by freezing at a rate of 4 degrees C/min to -80 degrees C before transfer to -196 degrees C. All samples were preserved for at least one week. After recovery from cryopreservation, the fragments were transplanted into the spleens of syngeneic rats. Histological assessment of the grafts was made one month after transplantation. Consequently, the optimal cryopreserving conditions for fetal rat liver fragments were defined as follows: cooling rate was 1 to 10 degrees C/min, preserving temperature was at -196 degrees C, concentration of Me2SO was 20 per cent (final concentration: 10 per cent). In the long term observation, the stored liver fragments could be differentiated into adult hepatocytes and the surviving hepatocytes showed little difference from the nonfrozen controls, either histochemically or ultrastructurally. The surviving hepatocytes in the stored transplants were less numerous than in the nonfrozen ones and hepatic cell plates and sinusoids were nil.

Mechanically prepared isolated islets of Langerhans were cryopreserved in liquid nitrogen for a period of 4 days. Intraportal autotransplantation studies were performed on two groups of six pigs rendered diabetic by total pancreatectomy (group 2) or by partial pancreatectomy combined with streptozotocin (group 4) and compared with two control groups (groups 1 and 3, respectively). The pigs were assessed for survival, weight gain, glycosuria, polyuria, systemic blood sugar and insulin, and, in selected pigs, intravenous glucose tolerance tests. Results showed that partial pancreatectomy with streptozotocin was the better tolerated experimental diabetes. Variable control of hyperglycemia was obtained over an experimental period of 3 months. Random blood glucose returned to normal in one of six pigs in the totally pancreatectomized group and three of six pigs in the partial pancreatectomy and streptozotocin group. Despite these normal circulating glucose levels, imperfect glucose homeostasis was achieved as shown by the response to glucose tolerance testing. These results report blood glucose control after cryopreserved islet autotransplants in diabetic pigs but further study is still necessary to achieve consistency.

Fetal rat pancreases, cultured for 8 days in RPMI 1640, were successively frozen to -196 degrees C. The samples, defrosted at different intervals (0, 5, 1, 2, 7, and 15 days), were examined by TEM and SEM. The effects of culture, various cooling times, warming rates, thawing procedures, dimethyl sulfoxide concentration, and ultrastructural features of cellular elements were analyzed.

When using methods of perfusion to preserve the rat pancreas, we found that perfusion into the celiac axis was the most effective. The viability of the cadaver pancreas from decapitated rats preserved by perfusion into the celiac axis for 6 hours under various conditions of hypothermia, hyperbaria and oxygenation was investigated. The condition of perfusion affected the ratio of degenerative islets/normal islets in the pancreas. The ratio, under conditions of hypothermia and oxygenation was the lowest, while that under conditions of hyperbaria was the highest. Insulin-releasing activity of islets from 6-hour-perfusion-pancreas, under conditions of hypothermia and oxygenation was 86.5 per cent or more of that of the control. Stainings with fluorescent antibody and peroxidase antiperoxidase, revealed a large number of A and B cells in the islets of the pancreas, in cases of up to 6 hours of perfusion.

(1) The freezing protocol we have devised for rat islets results in normal clinical indices and almost normal glucose tolerance in diabetic recipients of the same inbred strain. (2) Cryopreservation of canine islet-containing pancreatic tissue required a higher temperature than rat islets during exposure to the protective agent. (3) Because of the similar compactness of the pancreas in man and dog, we consider this canine model useful for formulating optimal cryopreservation techniques for the human pancreas. (4) Cryopreservation may partially purify islet-containing tissue of its exocrine content.

Cryopreservation of pancreatic islets of Langerhans offers the possibility of storage of sufficient quantities of this tissue for transplantation in the treatment of certain forms of diabetes, as well as providing a means of precise histocompatibility matching. In these studies, the effects of dimethylsulfoxide and various cooling rates on islet function are examined. These studies demonstrate that islets treated with 1.4 M dimethylsulfoxide and slowly cooled at a rate of 0.3 degrees C/min release insulin biphasically upon glucose challenge. In addition, this stimulated release is significantly improved (P less than 0.05) by increasing the duration of post-thaw culture. After thawing, these cryopreserved islets also retain the capacity to synthesize insulin. Islets frozen at faster cooling rates (3, 14, and 48 degrees C/min) exhibit varying degrees of glucose-induced insulin release, indicative of freeze-induced damage. These manifestations of freeze-induced damage include high basal (nonstimulatory) insulin release rates, little or no increase in the stimulated rate versus the nonstimulated rate, and failure of the stimulated release to return to basal levels when the glucose concentration is reduced.