Effect of Over 10-Year Cryopreserved Encapsulated Pancreatic Islets Of Langerhans.

OBJECTIVES: Immunoisolation of pancreatic islets of Langerhans performed by the encapsulation process may be a method to avoid immunosuppressive therapy after transplant. The main problem related to islet transplant is shortage of human pancreata. Resolution of this obstacle may be cryopreservation of encapsulated islets, which enables collection of sufficient numbers of isolated islets required for transplant and long-term storage. Here, we assessed the ability of encapsulated islets to function after long-term banking at low temperature. MATERIALS AND METHODS: Islets of Langerhans isolated from rat, pig, and human pancreata were encapsulated within alginate-poly-L-lysine-alginate microcapsules. Cryopreservation was carried out using a controlled method of freezing (Kriomedpol freezer; Kriomedpol, Warsaw, Poland), and samples were stored in liquid nitrogen. After 10 years, the samples were thawed with the rapid method (with 0.75 M of sucrose) and then cultured. RESULTS: We observed that microcapsules containing islets maintained their shape and integrity after thawing. During culture, free islets were defragmented into single cells, whereas encapsulated islets were still round in shape and compact. After 1, 4, and 7 days of culture of encapsulated islets, the use of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tests showed increased mitochondrial activity. After they were thawed, the insulin secretion capacity was comparable with that obtained with fresh islets. CONCLUSIONS: Cryopreservation and storage of free and microencapsulated islets were possible for about 10 years, although only encapsulated islets retained viability and secretory properties.

Bmp-12 activates tenogenic pathway in human adipose stem cells and affects their immunomodulatory and secretory properties.

BACKGROUND: Cell-based therapy is a treatment method in tendon injuries. Bone morphogenic protein 12 (BMP-12) possesses tenogenic activity and was proposed as a differentiating factor for stem cells directed to transplantation. However, BMPs belong to pleiotropic TGF-ß superfamily and have diverse effect on cells. Therefore, the aim of this study was to determine if BMP-12 induces tenogenic differentiation of human adipose stem cells (hASCs) and how it affects other features of this population. RESULTS: Human ASCs from 6 healthy donors were treated or not with BMP-12 (50 or 100 ng/ml, 7 days) and tested for gene expression (COLL1, SCX, MKH, DCN, TNC, RUNX2), protein expression (COLL1, COLL3, MKH), proliferation, migration, secretory activity, immunomodulatory properties and susceptibility to oxidative stress. RT-PCR revealed up-regulation of SCX, MKH and RUNX2 genes in BMP-12 treated cells (2.05, 2.65 and 1.87 fold in comparison to control, respectively, p < 0.05) and Western Blot revealed significant increase of COLL1 and MHK expression after BMP-12 treatment. Addition of BMP-12 significantly enhanced secretion of VEGF, IL-6, MMP-1 and MPP-8 by hASCs while had no effect on TGF-ß, IL-10, EGF and MMP-13. Moreover, BMP-12 presence in medium attenuated inhibitory effect of hASCs on allo-activated lymphocytes proliferation. At the same time BMP-12 displayed no influence on hASCs proliferation, migration and susceptibility to oxidative stress. CONCLUSION: BMP-12 activates tenogenic pathway in hASCs but also affects secretory activity and impairs immunomodulatory potential of this population that can influence the clinical outcome after cell transplantation.

Surgical removal of the pancreas with one-step autotransplantation of isolated Langerhans islets into the hepatic portal system in the pig.

INTRODUCTION: Autotransplantation of isolated Langerhans islets is regarded as the only way to prevent iatrogenic diabetes in patients who had been scheduled for pancreatectomy due to painful chronic pancreatitis. A sufficient number of Langerhans islets capable of secretory activity need to be transplanted to maintain normoglycemia after the surgical procedure. In order to optimize all stages, including collection, storage, isolation and transplantation of pancreatic islets, a reproducible animal-based experimental model should be developed before a new method is introduced into clinical practice. OBJECTIVES: The aim of the present study was to develop a reproducible autogenic model-based method for collection, conservation and isolation of porcine pancreas so that transplantation of isolated pancreatic islets could be performed and postoperative normoglycemia achieved. MATERIAL AND METHODS: Pigs were subjected to total pancreatectomy with simultaneous splenectomy and without removal of the duodenum. The collected pancreas was stored in the University of Wisconsin solution with the addition of pentoxifylline (PTX) until the isolation procedure (<4 hours). Efficacy of isolation was evaluated based on the number, quality and viability of obtained islets. Following autotransplantation into the liver, secretory activity of the islets was assessed intravitally by serum glucose monitoring. RESULTS: The islet yield per gram of pancreas was 1452 (standard deviation [SD] +/- 125) for the PTX group and 384 (SD +/- 115) for the control non-PTX group (p<0.01). Viability of islets for individual isolations did not reveal any statistically significant differences between groups and was estimated at 85-93%. Three out of five animals demonstrated normoglycemia with features of neoangiogenesis in the islets transplanted into the liver, which was confirmed by histological examination. One animal developed hyperglycemia up to 430 mg/dl, and histological image showed intensive apoptosis and degranulation in the transplanted islets. CONCLUSIONS: Efficacy of the isolation method was confirmed by achieving normoglycemia after autotransplantation of pancreatic islets into the liver, while histological examination showed hepatic vascularization to be the most appropriate location for an autogenic graft. PTX presence in the preserving solution for the pancreas storage produced the cytoprotective effect, which directly correlated with the islet yield.

Isolation, banking, encapsulation and transplantation of different types of Langerhans islets.

INTRODUCTION: The discovery of a cure for diabetes is a dream of many medical researchers. The transplantation of Langerhans islets is a potential treatment of choice for patients with type 1 diabetes as a source of endogenous insulin for the recipient. OBJECTIVES: The aim of the experiment was to transplant Langerhans islets without immunosuppression. To protect the grafts against transplant rejection, semipermeable membranes could be used. MATERIAL AND METHODS: Langerhans islets were isolated from rats and pigs and immunoisolated by encapsulation in alginate-protamine-heparin (APH) or alginate-poly-L-lysine-alginate (APA) membranes. Islets were pooled in a controlled manner. Tests for cryopreservation and biocompatibility were also performed. RESULTS: The capsules coated with APH are more resistant than the capsules coated with APA. After transplantation of the islets immunoisolated with APA, euglycemia is maintained longer than after transplantation of the islets immunoisolated with APH. Microencapsulation protects the islets from destruction by the host. CONCLUSIONS: It is feasible to treat experimental diabetes by transplantation of encapsulated Langerhans islets without immunosuppression.

TransEndoscopic Gastric SubMucosa Islet Transplantation (eGSM-ITx) in pigs with streptozotocine induced diabetes - technical aspects of the procedure - preliminary report.

BACKGROUND: Islets and pancreas transplantation have become standard treatments of patients with diabetic complications. However pancreas transplantation is associated with high incidence of complications and the long-term results of islet transplantation are still unsatisfactory. Loss of pancreatic islets grafts is caused not only by immunological reactions but also due to the site of grafting and IBMIR. Gastric submucosal space could be an alternative site for transplantation. The aim of this study was to assess the possibility of endoscopic islets transplantation into the gastric submucosa-its efficacy and potential complications.
MATERIAL/METHOD: 20 Landrace pigs weighing 19-24 kg were obtained for the study. Seven animals were controls (C-group) and 13 formed the transplantation group (TX group). In both groups diabetes was induced by streptozotocine (stz) infusion at a dose of 200 mg/kg. At 7 days post stz infusion pigs of both groups underwent endoscopy-in group C to assess the feasibility of gastroscopic examination under general anaesthesia in pigs with diabetes and to study the influence of basiliximab infusion on pigs, in the Tx-group to perform endoscopic submucosal islet transplantation (eGSM-ITx). Immunosuppression consisted of tacrolimus 0.2 mg/kg and sirolimus 6 mg/m(2). At 7 days post transplantation, control gastroscopy was performed to assess the gastric mucosa and to obtain biopsies for histopathology. 10 to 30 days after eGSM-ITx, magnetic resonance (MRI) scan was performed. Stomach and pancreas were obtained at autopsy for histopathology. Glycemia was assessed twice daily during the experiment. For 10 days after diabetes induction (up to three days after eGSM-ITx) in both groups, insulin was given to reach glycemia between 150-200 mg/dl, after that period insulin was given only when glycemia exceeded 600 mg/dl.
RESULTS: There were no differences in insulin requirement and glycemia up to the day of eGSM-ITx between the groups. Tx-group animals received a mean of 6000+/-3170 IEQ/kg. Tx-group animals had a significantly lower insulin requirement and significantly lower mean glycemia since the first day post transplantation. C-group animals all required insulin once daily to keep glycemia below 600 mg/dl. There were no signs of perforation, ulceration or bleeding after eGSM-ITx on gastroscopy and histopathological examination. MRI scans revealed unspecific thickening of gastric wall at sites of islet deposition.
CONCLUSIONS: Transendoscopic islets transplantation into gastric submucosa is feasible and a safe procedure in an experimental animal setting. Its potential for clinical application in human subjects needs further studies.

The influence of immune system stimulation on encapsulated islet graft survival.

INTRODUCTION: The aim of this study was to determine the influence activating of the recipient immune system on the function of microencapsulated islet xenografts. MATERIAL/METHODS: The skin of WAG or Fisher rats and WAG free or encapsulated (APA) Langerhans islets were transplanted to healthy or to streptozotocin diabetic BALB/c mice. Skin grafts were performed following the method of Billingham and Medawar. Rat islets were isolated from pancreas by the Lacy and Kostianovsy method and encapsulated with calcium alginate-poly-L-lysine-alginate according to the 3-step coating method of Sun. RESULTS: The transplantation of encapsulated WAG islets, despite activation of the host immune system, restored euglycemia for over 180 +/-100 days. A subsequent skin graft taken from the same donor was rejected in the second set mode, but euglycemia persisted. In diabetic recipients, impaired immune response was corrected by successful encapsulated islet transplantation. In diabetic mice, strong stimulation with 2-fold skin transplantation induced primary non-function of grafted islets despite their encapsulation. CONCLUSIONS: The survival of an islet xenograft depends on the level of activation of the recipient immune system. The immune response of diabetic mice was impaired, but increased after post-transplant restitution of euglycemia. Microencapsulation sufficiently protected grafted islets, and remission of diabetes was preserved. However, after strong specific or non-specific stimulation of the host immune system, non-function of xenografted islets developed despite their encapsulation. Therefore, islet graft recipients should avoid procedures which could stimulate their immune systems. If absolutely necessary, the graft should be protected by exogenous insulin therapy at that time.