Survival of human islets in microbeads containing high guluronic acid alginate crosslinked with Ca2+ and Ba2+.

BACKGROUND: The main hurdles to the widespread use of islet transplantation for the treatment of type 1 diabetes continue to be the insufficient number of appropriate donors and the need for immunosuppression. Microencapsulation has been proposed as a means to protect transplanted islets from the host's immune system. METHODS: This study investigated the function of human pancreatic islets encapsulated in Ca(2+) /Ba(2+) -alginate microbeads intraperitoneally transplanted in diabetic Balb/c mice. RESULTS: All mice transplanted with encapsulated human islets (n = 29), at a quantity of 3000 islet equivalent (IEQ), achieved normoglycemia 1 day after transplantation and retained normoglycemia for extended periods of time (mean graft survival 134 ± 17 days). In comparison, diabetic Balb/c mice transplanted with an equal amount of non-encapsulated human islets rejected the islets within 2 to 7 days after transplantation (n = 5). Microbeads retrieved after 232 days (n = 3) were found with little to no fibrotic overgrowth and contained viable insulin-positive islets. Immunofluorescent staining on the retrieved microbeads showed F4/80-positive macrophages and alpha smooth muscle actin-positive fibroblasts but no CD3-positive T lymphocytes. CONCLUSIONS: The Ca(2+) /Ba(2+) -alginate microbeads can protect human islets from xenogeneic rejection in immunocompetent mice without immunosuppression. However, grafts ultimately failed likely secondary to a macrophage-mediated foreign body reaction.

Encapsulation of human islets in novel inhomogeneous alginate-ca2+/ba2+ microbeads: in vitro and in vivo function.

Microencapsulation may allow for immunosuppression-free islet transplantation. Herein we investigated whether human islets can be shipped safely to a remote encapsulation core facility and maintain in vitro and in vivo functionality. In non-encapsulated islets before and encapsulated islets after shipment, viability was 88.3+/-2.5 and 87.5+/-2.7% (n=6, p=0.30). Stimulation index after static glucose incubation was 5.4+/-0.5 and 6.3+/-0.4 (n=6, p=0.18), respectively. After intraperitoneal transplantation, long-term normoglycemia was consistently achieved with 3,000, 5,000, and 10,000 IEQ encapsulated human islets. When transplanting 1,000 IEQ, mice returned to hyperglycemia after 30-55 (n=4/7) and 160 days (n=3/7). Transplanted mice showed human oral glucose tolerance with lower glucose levels than non-diabetic control mice. Capsules retrieved after transplantation were intact, with only minimal overgrowth. This study shows that human islets maintained the viability and in vitro function after encapsulation and the inhomogeneous alginate-Ca(2+)/Ba(2+) microbeads allow for long-term in vivo human islet graft function, despite long-distance shipment.

Evaluation of the Performance of Dual Polyelectrolyte Systems on the Re-Flocculation Ability of Calcium Carbonate Aggregates in Turbulent Environment.

Flocculation can be used in turbulent environments resulting in floc breakage due to shearing. The degree of re-flocculation relates directly to product quality and process efficiency. This study aimed at looking for alternatives to improve the re-flocculation ability of aggregates when polyelectrolytes (PEL) are used as flocculation agents. Moreover, because branched PEL have proved previously to lead to high flocculation efficiencies, the work presented focus on the improvement of the re-flocculation ability of branched PEL. Thus, a selection of branched polymers were used primarily as flocculation aid and after flocs break up a linear polymer was added to the system in order to improve re-flocculation. Different mixtures were tested with the objective to try to induce, during re-flocculation, complementary flocculation mechanisms, favoring the patching mechanism. Re-flocculation improved significantly with this strategy. Laser Diffraction Spectroscopy was used to monitor the flocculation and re-flocculation processes supplying information about the floc size and structure. Since inorganic materials, namely bentonite, have been widely used to improve the re-flocculation capacity of polyelectrolytes, the results of using dual polyelectrolyte systems were compared with the effect of adding bentonite to the system.

Effect of microcapsule composition and short-term immunosuppression on intraportal biocompatibility.

With higher nutrient and oxygen supply and close contact to blood, the portal vein is a possible alternative to the peritoneal cavity for transplantation of encapsulated cells. Data regarding intraportal biocompatibility of microcapsules are lacking. Microcapsules were built from five alginate types differing in their molar mass and mannuronic/guluronic acid ratios by complex formation with divalent cations (barium or calcium) or mixtures of divalent cations and polycations. They were injected in the portal vein of rats, and cellular and fibrotic pericapsular infiltration thickness was measured 3 and 7 days after implantation. Overgrowth was characterized using various stainings or immunohistochemistry (hematoxylin and eosin, Giemsa, ED-1 for monocyte/macrophage, alpha-actin for myofibroblasts, CD31 for endothelial cells). The impact of short-term immunosuppression (gadolinium-chloride IV 20 mg/kg/day on days--1 and 4 as well as 10 days of rapamycin PO 1 mg/kg/day, tacrolimus PO 3 mg/kg/day, or combinations of rapamycin/tacrolimus or gadolinium/tacrolimus) was further assessed 3, 7, and 42 days after implantation. Overall, overgrowth increased from day 3 to day 7 (p < 0.05). Three and 7 days after implantation, polycation-containing microcapsules induced more reaction than microbeads (p < 0.0001 and p < 0.01). Considering polycation-free beads, barium-alginate induced the weakest reaction. Biocompatibility of microbeads was independent of mannuronic/guluronic acid ratio and molar mass of the alginate. Infiltration was mainly a monocyte/macrophage-rich foreign body reaction, but an eosinophil-containing immunoallergic reaction was also observed. Short-term immunosuppression significantly reduced infiltration in all conditions and up to 42 days after implantation. Biocompatibility after intraportal infusion was best for barium-alginate microbeads and poorest for polycation-containing microcapsules. Short- and long-term overgrowth could be significantly reduced by short-term immunosuppression.

Liquid chromatography of polymers under limiting conditions of adsorption. IV. Sample recovery.

The high performance liquid chromatography of polymers under limiting conditions of adsorption (LC LCA) separates macromolecules, either according to their chemical structure or physical architecture, while molar mass effect is suppressed. A polymer sample is injected into an adsorption-active column flushed with an adsorption promoting eluent. The sample solvent is a strong solvent which prevents sample adsorption. As a result, macromolecules of sample elute within the zone of their original solvent to be discriminated from other, non-adsorbing polymer species, which elute in the exclusion mode. LC LCA sample recovery has been studied in detail for poly (methyl methacrylate)s using a bare silica gel column and an eluent comprised toluene (adsorli) and tetrahydrofuran (desorli). Sample solvent was tetrahydrofuran. It was found that a large part of injected sample may be fully retained within the LC LCA columns. The amount of retained polymer increases with decreasing packing pore size and with higher sample molar masses and, likely, also with the column diameter. The extent of full retention of sample does not depend of sample volume. An additional portion of the injected desorli sample solvent (a tandem injection) does not fully eliminate full retention of the sample fraction and the reduced recovery associated with it. The injected sample is retained along the entire LC LCA column. The reduced sample recovery restricts applicability of many LC LCA systems to oligomers and to discrimination of the non-adsorbing minor macromolecular components of complex polymer mixtures from the adsorbing major component(s). The full retention of sample molecules within columns may also complicate the application of other liquid chromatographic methods, which combine entropic and enthalpic retention mechanisms for separation of macromolecules.

Gastro retention using polymer cocoons.

A gastro-retentive capsule has been prepared which is retained in the stomach for a period of 24h, providing a vehicle for the controlled delivery to the upper intestines. These "gastro cocoons" can resist passage through the sphincter of the stomach, and can retain a high drug payload (30%). They are made from oppositely charged polyelectrolytes and can swell to twice their initial volume. They are strong and also can resist 550 N of compressive force. They are based on filled pharmaceutical capsules which are visible to X-rays. Using ambroxol hydrochloride as a model drug linear, zero-order, release curves were obtained.

Chitosan-gelatin sheets as scaffolds for muscle tissue engineering.

Hydrogels made of natural polymers [chitosan (CS) and gelatin (G)] have been prepared having mechanical properties similar to those of muscle tissues. In this study, the effect of polymer concentration and scaffold stiffness on the behavior of seeded muscle-derived cells (MDCs) on the CS-G hydrogel sheets has been evaluated. Both variables were found to be important in cell viability. Viability was assessed by observation of the cell morphology after 1 day as well as a 14-day MTT assay. The CS-G hydrogels were characterized using Fourier transform infrared (FTIR) analysis, which revealed evidences of strong intermolecular interactions between CS and G. Hydrogel samples with intermediate concentration of CS had suitable handling characteristics for surgical purposes as well as similar elasticity to muscle tissues. The sample with intermediate stiffness (22 ± 1kPa) exhibited the greatest attachment, expansion, and proliferation rate. Such CS-G hydrogels with intermediate stiffness may be considered as new candidates for muscle tissue engineering in the reconstructive field of urology.

History, challenges and perspectives of cell microencapsulation.

Cell microencapsulation continues to hold significant promise for biotechnology and medicine. The controlled, and continuous, delivery of therapeutic products to the host by immunoisolated cells is a potentially cost-effective method to treat a wide range of diseases. Although there are several issues that need to be addressed, including capsule manufacture, properties and performance, in the past few years, a stepwise analysis on the essential obstacles and limitations has brought the whole technology closer to a realistic proposal for clinical application. This paper summarizes the current situation in the cell encapsulation field and discusses the main events that have occurred along the way.

Islet auto-transplantation into an omental or splenic site results in a normal beta cell but abnormal alpha cell response to mild non-insulin-induced hypoglycemia.

The present studies were designed to determine if totally pancreatectomized dogs that underwent islet auto-transplantation retained a functional pancreatic counterregulatory response to mild non-insulin-induced hypoglycemia. Six dogs underwent total pancreatectomy followed by islet auto-transplantation to spleen or omentum. The animals recovered and fasting plasma glucose and insulin levels were normal. Each study consisted of a 40-min control and 2-h test period. At the onset of the test period, a glycogen phosphorylase inhibitor was administered to create mild hypoglycemia. Plasma glucose in the transplanted dogs fell from 120 +/- 4 to 80 +/- 3 mg/dL, similar to the minimum in control dogs without islet auto-transplantation (108 +/- 2 to 84 +/- 5 mg/dL). The fall in plasma insulin was similar in both groups. Glucagon, however, rose in response to hypoglycemia in the control dogs (Delta24 +/- 7 pg/mL; p < 0.05), but failed to rise significantly in the transplanted dogs (Delta9 +/- 6 pg/mL). In fact, only 1 of 7 control dogs failed to increase plasma glucagon by at least 25%, whereas 4 of 6 transplanted dogs failed to do so. In conclusion, in conscious dogs with successfully auto-transplanted islets, the beta cell response to mild non-insulin-induced hypoglycemia was normal, whereas the alpha cell response was not.

Improving cell encapsulation through size control.

Capsules based on the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride have previously shown important advantages for cell encapsulation. However, in vivo long-term applications require capsule features that are well suited for the functionality of encapsulated cells. These should be targeted to the site of implantation with an appropriate size, a relative stability, and suitable diffusion properties. This study shows the effect of capsule size reduction, from 1 mm to 400 microm, on capsule quality control, mechanical stability, diffusion properties, and in vitro activities of the encapsulated cells. Following a controlled preparation, it was determined that the capsule mechanical stability was largely dependent on the volume ratio of the capsule over the membrane. The molecule diffusion time was related to the surface/volume ratio of the capsule even for the capsules exhibiting an identical cut-off towards the proteins and the dextran molecules. Finally, the in vitro cellular activities, for both primary cultures of rat islets and murine hepatocytes, were improved for cells encapsulated into the 400 microm capsules compared with those in the 1 mm capsules. All of these findings suggest that the smaller capsules present better properties for future clinical applications, at the same time widening the choice of implantation site, and strengthen the notion that slight changes in the capsular morphological parameters can largely influence the graft function in vivo.

Intra-portal injection of 400- microm microcapsules in a large-animal model.

To date, encapsulated grafts have usually been implanted in the peritoneal cavity. This site is, however, not ideal, mainly because of its low blood supply. We have investigated the feasibility of intra-portal injection of (400 microm) microcapsules in the pig. Ten-thousand microcapsules per kilogram body weight were injected into six Large White pigs. Portal pressure, various biological tests, portographies and liver histology were recorded before and at various time points after injection. As a result, portal pressure increased after injection (15+/-2.3 vs 8.7+/-1.7 mmHg) but remained within an acceptable range (<20 mmHg) and returned to normal values at 3 months (8.5+/-3.7 mmHg). During the 3-month follow up, liver function and liver tests remained stable. Portographies showed a homogenous implantation of the capsule, with the portal flow always directed to the liver. At histological examination after 3 months the capsules demonstrated various degrees of fibrosis. We can thus conclude that these results demonstrate that intra-portal injection of microcapsules is feasible in a large-animal model. Hemodynamic, biological and radiological results are similar to those observed in clinical free-islet transplantation.