Islet Suspension Research Articles

Intraperitoneal injection of cell-engineered pancreas in rats with experimental type i diabetes (preliminary results)

Creation of a bioartificial pancreas, including a cell-engineered construct (CEC) formed from pancreatic islets (islets of Langerhans) and a biocompatible matrix mimicking the native microenvironment of pancreatic tissue, is one of the approaches to the treatment of type 1 diabetes mellitus (T1D).Objective: to conduct preliminary in vivo studies of the functional efficacy of intraperitoneal injection of a cell-engineered pancreatic endocrine construct and a suspension of rat pancreatic islets in an experimental T1D model.Materials and methods. Tissue-specific scaffold was obtained by decellularization of human pancreatic fragments. The viability and functional activity of rat islets isolated with collagenase were determined. Experimental T1D was modeled by intraperitoneal injection of low-dose streptozotocin and incomplete Freund’s adjuvant into rats. The rats were intraperitoneally injected twice with pancreatic CEC (n = 2) or islet suspension (n = 1). Glucose levels in the blood and urine of the rats were assessed. Histological examination of organs (pancreas and kidneys) of the experimental animals was carried out.Results. After the first injection, blood glucose levels gradually decreased in all animals by more than 47% of the initial values; by follow-up day 24, the glucose level rose to the initial hyperglycemic values. After repeated administration, a 63.4% decrease in glycemic level was observed in the rats with pancreatic CEC and a 47.5% decrease in the one with islet suspension. At week 5 of the experiment, blood glucose levels gradually increased in all animals. At the same time, the glycemic index of the rat with injected pancreatic CEC was 62% lower than the glycemic index of the rat with injected islets.Conclusion. Allogeneic pancreatic islets in pancreatic CEC increase the duration of stable glycemic level in T1D rats.

A scalable device-less biomaterial approach for subcutaneous islet transplantation

The subcutaneous space has been shown to be a suitable site for islet transplantation, however an abundance of islets is required to achieve normoglycemia, often requiring multiple donors. The loss of islets is due to the hypoxic conditions islets experience during revascularization, resulting in apoptosis. Therefore, to reduce the therapeutic dosage required to achieve normoglycemia, pre-vascularization of the subcutaneous space has been pursued. In this study, we highlight a biomaterial-based approach using a methacrylic acid copolymer coating to generate a robust pre-vascularized subcutaneous cavity for islet transplantation. We also devised a simple, but not-trivial, procedure for filling the cavity with an islet suspension in collagen. We show that the pre-vascularized site can support a marginal mass of islets to rapidly return streptozotocin-induced diabetic SCID/bg mice to normoglycemia. Furthermore, immunocompetent Sprague Daley rats remained normoglycemia for up to 70 days until they experienced graft destabilization as they outgrew their implants. This work highlights methacrylic acid-based biomaterials as a suitable pre-vascularization strategy for the subcutaneous space that is scalable and doesn't require exogenous cells or growth factors.

Early efficacy of islet transplantation in the treatment of adult advanced diabetes

Objective: To evaluate the safety and efficacy of islet transplantation for patients with advanced diabetes. Methods: Five cases of islet allotransplantation were performed on 4 adult recipients. The same blood type adult brain-dead pancreas donors were selected and the islets were prepared in GMP laboratory. The prepared islet suspension was slowly injected into the liver of the recipients within 30-60 minutes. The immunosuppressive regimen was a combination of basiliximab, tacrolimus and mycophenolate mofetil and TNF-alpha monoclonal antibody was used to reduce the post-transplant inflammatory response. Insulin was temporarily applied to control blood glucose after surgery, and the dosage of insulin was adjusted to decrease according to the blood glucose level until it was discontinued. Results: A total of 5 islet transplants were performed in 4 patients, including 1 patient who received the second islet transplantations. All operations were succeed and the blood glucose and portal pressure were stable during the operation. Exogenous insulin was continued to keep blood glucose level stable (4-12 mmol/L) after surgery. Four cases (including the one who received two islet transplantation) started to stop using insulin at the third to fourth week, and the insulin dosage of the other case was 74% lower than that before the operation, and no hypoglycemic reaction occurred in all patients after islet transplantation. The C-peptide level in 3 patients reached the normal range, and the level in one patient with type I diabetes (without insulin release) remained at 0.45-0.6 μg/L (0.15-0.2 nmol/L). In addition, one patient showed a rise in blood glucose again and continued to use insulin half a year after insulin discontinuation. Then, he was performed the second islet transplantation which showed good effect and stopped taking insulin in 10 days after surgery. There were 3 cases of liver puncture bleeding after opeation, of which 2 cases were treated with ultrasound radiofrequency ablation to stop bleeding, 1 case stopped spontaneously, and no other complications were found. Conclusions: Islet transplantation is effective in the treatment of advanced diabetes patients with small trauma and high safety, which is worthy of more promotion. Long-term efficacy and maintenance therapy still need further investigation.

Pancreatic Islet Autotransplantation After Completion Pancreatectomy for Pancreatic Fistula After Hemipancreatoduodenectomy for Carcinoma

ObjectivePancreatic islet autotransplantation (IAT) has a potential to prevent brittle diabetes in patients after total pancreatectomy. Because of the fear of tumor spread, IAT has rarely been used in case of malignancy. We report our experience with patients who underwent hemipancreatoduodenectomy for carcinoma and later completion pancreatectomy for pancreatic fistula with islet autotransplantation at our institution. MethodsFrom August 2007 to December 2012, 5 patients underwent IAT after completion pancreatectomy for pancreatic fistula after hemipancreatoduodenectomy for carcinoma. Islets were isolated from the pancreatic tail with the use of digestion with collagenase. Nonpurified islet suspension was infused into the portal vein during surgery. ResultsThe median number of islets transplanted was 175,000 islet equivalents (range, 70,000–365,000). One patient died after surgery for reasons unrelated to IAT. Another 3 patients had stable diabetes with partial graft function (fasting C-peptide levels 0.23, 0.41, and 0.61 nmol/L and HbA1c 4.8%, 4.6%, and 6.9% at 24, 24 and 9 months after IAT, respectively). The 1st patient, with pancreatic head carcinoma, was alive 28 months after IAT with lymph node and liver recurrence since 18 months after IAT. The 2nd patient, with gall bladder and distal bile duct carcinoma, died 47 months after IAT with tumor recurrence. The 3rd patient, with ampullary carcinoma, died 12 months after IAT with local recurrence and solitary liver metastasis. The last patient had been off insulin 9 months after IAT without tumor recurrence (fasting C-peptide, 0.89 nmol/L; HbA1c, 4.2%). ConclusionsAutotransplantation of pancreatic islets isolated from the residual pancreatic tissue in patients who previously underwent hemipancreatoduodenectomy for cancer may provide stable glucose control and thus improve quality of life. In this small series we did not observe early development of multiple liver metastases caused by islet suspension contamination with malignant cells. Oncologic outcome of the patients was not worse than what would be expected without IAT.

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Islet Transplantation in a Subarachnoid Cavity Surrounding Olfactory Bulb of Diabetic Rats

Immobilization of urokinase on the islet surface by amphiphilic poly(vinyl alcohol) that carries alkyl side chains

Transplantation of islets of Langerhans (islets) is a promising method to treat insulin-dependent diabetes mellitus (type I diabetes). However, insulin independence is typically realized for only ∼30% of transplant recipients, even with sufficient numbers of islets from multiple donors. Innate immunological reactions triggered by blood coagulation play a key role in the loss of islets at the early stage. Here we propose a method to inhibit blood coagulation on the islet surface. A plasminogen activator, urokinase, was immobilized on the islet surface via a poly(vinyl alcohol) (PVA) derivative that carries alkyl chains and thiol groups. When the PVA derivative was added to an islet suspension, the alkyl side chains spontaneously anchored into the lipid bilayer membranes of islet cells. The surfaces of islets were covered with the PVA derivative. Urokinase modified with maleimide groups could be immobilized onto the islet surface by thiol/maleimide bonding with the layer of PVA derivatives. Urokinase-immobilized islets exhibited fibrinolytic properties, indicating that blood coagulation can be controlled on the islet surface. Urokinase immobilization on islets, which does not impair insulin release, represents a promising method to reduce early graft loss after intraportal islet transplantation.

Islet-encapsulation in ultra-thin layer-by-layer membranes of poly(vinyl alcohol) anchored to poly(ethylene glycol)–lipids in the cell membrane

The microencapsulation of islets of Langerhans (islets) in a semipermeable membrane, i.e., the creation of a bioartificial pancreas, has been studied as a safe and simple technique for islet transplantation without the need for immunosuppressive therapy. The total volume of the implant tends to increase after enclosure of the islets in the semipermeable membrane, which limits transplantation sites. Thus, ultra-thin membranes are required for clinical applications. Here, we propose a novel method to encapsulate islets in an ultra-thin membrane of poly(vinyl alcohol) (PVA) anchored to a poly(ethylene glycol) (PEG)–phospholipid conjugate bearing a maleimide group (Mal–PEG–lipids, PEG Mw: 5000) in the cell membranes of islets. When Mal–PEG–lipids were added to an islet suspension, they spontaneously formed a thin layer on cells of the outer layer of islets. The PEG–lipid layer on the islets was covered by a PVA monolayer, and the PVA membrane was further reinforced by using the layer-by-layer method with thiol/disulfide exchange reactions. No practical volume increase in islets was observed after microencapsulation by this method. In addition, encapsulation of the islet surface in PVA membranes did not impair insulin release in response to glucose stimulation.

Encapsulation of islets with ultra-thin polyion complex membrane through poly(ethylene glycol)-phospholipids anchored to cell membrane

The microencapsulation of islets of Langerhans (islets) has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. However, thinner membranes are desired, because the increased total volume of the implant led to limited transplantation sites. Here, we propose a novel method for microencapsulation by polyion complex membrane formation on islets. Amino group-terminated poly(ethylene glycol)-conjugated phospholipids (PEG-lipids, M(w): 5000) spontaneously formed a thin layer on cells existing in the outer layer of islets when they were added to islet suspension. This layer-by-layer membrane could be further formed on the PEG-lipid layer through polyion complex formation between amino groups at the end of PEG chains, sodium alginate and poly(l-lysine). Islets could be microencapsulated by this method without volume increase. Encapsulation of the islet surface with PEG-lipids and polyion complex membranes did not impair the insulin release function in response to glucose stimulation. Our method is promising to encapsulate islets without affecting cell viability or increasing volume.

Viability Analysis of Cryopreserved Rat Pancreatic Islets Using Laser Scanning Confocal Microscopy

We have developed a digital image analysis technique to assay the viability of frozen–thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18–24 h and then brought to a 2Mconcentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (−10°C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10, and 30°C/min to −70°C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 μm.

Three-dimensional distribution of damaged cells in cryopreserved pancreatic islets as determined by laser scanning confocal microscopy.

The technique of serial optical sectioning by confocal microscopy, in conjunction with off-line digital image analysis, was used to quantify the radial distribution of damaged cells in rat pancreatic islets following cryopreservation. The process consists of imaging frozen-thawed islets of Langerhans using laser scanning confocal microscopy (LSCM). The three-dimensional (3-D) distribution and analysis of the two populations of viable and damaged cells was visualized via acridine orange/propidium iodide (AO/PI) fluorescent staining. In preparation for cryopreservation, isolated and cultured rat pancreatic islets were brought to a 2 M concentration of dimethyl sulphoxide (DMSO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at -10 degrees C, and individual specimens were frozen to -70 degrees C at cooling rates of 1, 3, 10 and 30 degrees C/min in a programmable bulk freezer and subsequently stored in liquid nitrogen. After rapid thawing and serial dilution to remove DMSO, individual islets were prepared with AO/PI stains for imaging on the LSCM. Serial sections of the islets, 2-7 microns in thickness, were obtained and processed to obtain high-contrast images. Analysis algorithms consisted of template masking, grey-level thresholding, median filtering and 3-D blob colouring. The radial distribution of damaged cells in the islets was determined by isolating the cell and computing its distance from the centroid of the 3-D islet volume. An increase in the number of blobs corresponding to single and/or aggregates of damaged cells was observed progressively with distance from the centre towards the periphery of the islet. This pattern of freeze-induced killing of cells within the islet was found to occur consistently in the numerous individual specimens processed.