Biomedical subjects

Y Ikada

Publications and source records attributed to Y Ikada.

At least 307 records · Page 17Linked to original sources

PubMedDate not supplied

Application of a novel B cell line MIN6 to a mesh-reinforced polyvinyl alcohol hydrogel tube and three-layer agarose microcapsules: an in vitro study.

This study examines the function of a novel B cell line (MIN6) enclosed in hybrid bioartificial pancreas with mesh-reinforced polyvinyl alcohol hydrogel tube (MRPT) or with improved, three-layer agarose microcapsules. MIN6 was established from insulinomas obtained by targeted expression of the simian virus 40 T-antigen gene in transgenic mice. MIN6 retains the ability to secrete insulin in response to physiological glucose concentrations. The MRPT and the three-layer agarose microcapsules, which were developed to act as an artificial pancreas, were readily permeated by insulin, glucose, and other nutrients. Both can immunoisolate enclosed MIN6 cells from the recipient's humoral and cellular immunosystems, which causes a xenogeneic rejection response. MIN6 cells (5.0 x 10(6) or 1.5 x 10(6)) were enclosed in MRPT or in a hundred three-layer microcapsules and subjected to an in vitro perifusion study or a static incubation study to observe the insulin release from each bioartificial pancreas in response to glucose stimulation. In vitro study revealed that the insulin secretion in response to 16.7 mM glucose stimulation was twice that with 3.3 mM glucose stimulation with both MRPT and the three-layer agarose microcapsules. The present study demonstrates that MIN6 effectively functions as a bioreactor for the hybrid bioartificial pancreas. The application of MIN6 cells to the hybrid bioartificial pancreas may offer a solution to the current serious dearth of organs.

Animals↗

PubMedDate not supplied

Bone regeneration by basic fibroblast growth factor complexed with biodegradable hydrogels.

The objective of this study is to enhance the bone induction activity of basic fibroblast growth factor (bFGF) for reconstruction of skull bone defects which has been clinically recognized as almost impossible. For this purpose, we prepared biodegradable hydrogels from gelatin with an isoelectric point of 4.9 which is capable of polyionic complexing with basic bFGF. When implanted in rabbit skull defects of 6 mm in diameter (6 defects per experimental group), the gelatin hydrogels incorporating 100 microg of bFGF promoted bone regeneration at the defect in marked contrast to free bFGF of the same dose, finally closing the bone defects after 12 weeks of implantation as is apparent from histological examination. In dual energy X-ray absorptometry analysis, the bone mineral density at the skull defects enhanced by the hydrogels was significantly higher than that by free bFGF at doses ranging from 2 to 200 microg/defect (P < 0.05). The extent of bone regeneration induced by gelatin hydrogels incorporating 100 microg of bFGF increased with a decrease in their water content. Histological examination indicated that more slowly degrading hydrogels of lower water content prolonged the retention period of osteoblasts in the bone defects. This led to enhanced bone regeneration compared with faster degrading hydrogels of higher water content. It was concluded that this biodegradable hydrogel system was a promising surgical tool to assist self-reconstruction of the skull bone.

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PubMedDate not supplied

Mechanism of amide formation by carbodiimide for bioconjugation in aqueous media.

To study the mechanism of amide formation between carboxylic acid and amine in aqueous media using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC), hydrogels with two different types of carboxyl group locations were employed as substrates containing the carboxylic acid, while ethylenediamine and benzylamine were used as amine. In parallel, a study was undertaken with cyclizable carboxylic acids (maleic acid and poly(acrylic acid) and noncyclizable carboxylic acids (fumaric acid and poly(ethylene glycol) with the terminal carboxyl groups) to assess the reaction products by 13C-NMR and IR. EDC rapidly lost its activity in aqueous media of low pH, producing the corresponding urea derivative, but was very stable at neutral and higher pH regions. EDC could react with carboxyl groups at a relatively narrow low pH range such as 3.5-4.5. If carboxyl groups were cyclizable, they would react quickly with EDC producing carboxylic anhydrides, which formed the corresponding amides when amine compounds were present. On the other hand, a trace of amide was formed in the case of noncyclizable carboxylic acids. In addition, an excess of EDC caused an undesired side reaction to form stable N-acylurea, regardless of the special location of carboxylic acids.

Amides↗

PubMedDate not supplied

Usefulness of microspheres composed of gelatin with various cross-linking density.

The release rate of insulin, as a model peptide, from gelatin microspheres (GM) prepared with gelatin having various cross-linking densities in vitro was examined. The release of insulin from GM showed the burst effect, followed by a slow release phase regardless of the cross-linking density of gelatin. The total amount of insulin released in 2 weeks decreased with increasing cross-linking density of gelatin. The release rate of insulin within 6 h was well correlated with the cross-linking density of gelatin. The remaining amounts of both insulin and GM after injection of insulin incorporated in GM to mice femoral muscle tissue were also examined in vivo. Both insulin and GM rapidly disappeared from the injection site within 1 day, and thereafter slowly disappeared over 14 days. The time courses of the remaining amounts were fairly similar to each other. Furthermore, the remaining amount of insulin 1 day after administration was well correlated with the cross-linking density of gelatin. These data suggest that insulin was released from GM with the degradation of GM in mice muscular tissue and that the release rate of insulin can be controlled by modifying the cross-linking density of gelatin. In conclusion, the control of the release rate of insulin from GM can be achieved under both in vitro and in vivo conditions by gelatin through the alteration of cross-linking conditions.

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PubMedDate not supplied

Positively charged gelatin microspheres as gastric mucoadhesive drug delivery system for eradication of H. pylori.

Gastric mucoadhesive drug delivery systems are very promising for eradication of Helicobacter pylori (H. pylori), a spiral bacterium that resides in the gastric mucus layer and at the mucus-epithelial cell interface. New positively charged biodegradable microspheres were prepared using aminated gelatin by surfactant-free emulsification in olive oil, followed by a cross-linking reaction with glutaraldehyde. The amino group contents of the modified gelatin and the microspheres were determined using a 2,4,6-trinitrobenzenesulfonic acid method. With the increase of glutaraldehyde concentration, the amino group content of the microspheres decreased accordingly. The influence of glutaraldehyde concentration, cross-linking reaction time, drug-loading patterns, and type of release media on the in vitro release characteristics of amoxicillin from the microspheres was investigated. Amoxicillin release rate from the modified gelatin microspheres was significantly reduced compared with that from gelatin microspheres. Furthermore, the release was decreased with the increase of glutaraldehyde concentration and/or cross-linking time. On the other hand, a faster release was observed in a lower pH release medium and/or using a lower pH solution for amoxicillin loading. The gastric mucoadhesive properties of the microspheres were evaluated using RITC-labeled microspheres in an isolated rat stomach. The gastric mucoadhesion of the modified gelatin microspheres was markedly improved compared with that of gelatin microspheres. The modified gelatin microsphere proves to be a possible candidate delivery system for the effective eradication of H. pylori.

Adhesives↗

PubMedDate not supplied

Experimental studies on an artificial esophagus for the purpose of neoesophageal epithelization using a collagen-coated silicone tube.

An artificial esophagus was fabricated from a collagen-coated silicone tube 5 cm long, with an inner diameter of 2.5 cm and a wall thickness of 1 mm. The outer surface was coated with dry collagen to 5 mm thickness. The bilayered prosthesis was anastomosed to the cervical esophagus of adult mongrel dogs that were fed only by intravenous hyperalimentation (IVH) with 80 cal/kg/day and 60 ml water/kg/day for 3 weeks after the operation. Once the dogs began to consume food orally, the artificial esophagus dropped spontaneously into the stomach and formation of the neoesophagus was complete. Macroscopically, the neoesophagus showed no evidence of leakage or inflammation and had a smooth internal surface, but it developed slight segmental narrowing. Microscopically, layers of stratified squamous epithelium covered the neoesophagus within 3 to 4 weeks. The most characteristic feature of the artificial esophagus was that the replacement prosthesis did not remain in the esophagus after healing. Esophageal epithelization extended into the collagen layer coating the silicone tube and a new esophageal lumen was regenerated, thereby decreasing the chance of infection and leakage. However, neoesophageal stenosis was observed when the silicone tube was removed.

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PubMedDate not supplied

Efficiency and biocompatibility of a polyethylene glycol grafted cellulosic membrane during hemodialysis.

Dialytic efficiency and biocompatibility of a new modified cellulose membrane (NMC) were examined in vitro and clinically. NMC was obtained by grafting polyethylene-glycol (PEG) chains to the membrane surface of ordinary cellulose (OC), and it was expected that the random movement of PEG chains would prevent blood cells and large plasma proteins from coming into contact with the membrane surface, resulting in improving the biocompatibility and thrombogenicity of the membrane. Surface characteristics of NMC were rendered anionic and hydrophilic, however, the activations of complement and platelet systems were clearly suppressed in NMC. Minimum heparin requirement for hemodialysis was significantly lower with NMC than with OC dialyzer. No significant difference in solute and water removal was observed between the two dialyzers. These results indicate that NMC can provide increased biocompatibility and antithrombogenic effect while retaining the essential dialysis efficiency of OC.

Biocompatible Materials↗

PubMedDate not supplied

A new type of low blood flow ECCO2R using a hemodialysis system in apneic states.

To perform low blood flow extracorporeal CO2 removal (ECCO2R), the authors developed a device for extracorporeal circulation (ECC) equipped with a dialyzer for the elimination of CO2 as bicarbonate. The major problem with this method was the decrease in blood pH. To control blood pH and clarify the limit of CO2 elimination using this method, a study with apneic dogs was performed. Six anesthetized mongrel dogs were intubated and paralyzed with a muscle relaxant. Vascular access was achieved with a venovenous bypass. ECC was initiated under apneic oxygenation (100% O2, 10 cm H2O continuous airway pressure), and the CO2 concentration in the airway outlet was measured. The CO2 was converted to bicarbonate using systemic infusion of trihydroxy-methylamino methane (THAM), and the generated bicarbonate was removed by hemodialysis. Blood flow rate in the ECC was 15 ml/kg/min, and the duration of ECC was 5 hr. During ECC, the hemodynamic parameters of the dogs were stable, and the PaCO2 remained at about 90 mmHg with a PaO2 above 350 mmHg; CO2 elimination from the airway was negligible.

Acid-Base Equilibrium↗

PubMedDate not supplied

Experimental studies on an artificial trachea of collagen-coated poly(L-lactic acid) mesh or unwoven cloth combined with a periosteal graft.

Artificial tracheas were fabricated from poly(L-lactic acid) mesh or unwoven cloth coated with collagen in order to reconstruct the respiratory airway by self regeneration without retaining the prosthesis at the replacement site, thus preventing infection, air leakage, granulation tissue formation, and displacement of the prosthesis. Nine weeks after placing a periosteal autologous graft from the tibia around the cervical trachea of a rabbit, bone rings formed without infection. Two weeks after replacement of an 8 X 8 mm window-shaped section of the cervical trachea with autologous periosteum, the internal surface of the trachea was well epithelialized without local complications, but ossification was not seen. One week after cervical substitution with our 2 cm long artificial trachea combined with the periosteal graft, cartilage was produced around the mesh material but neither ossification nor epithelization was observed thereafter, probably due to local infection. On the other hand, cartilage, bone, and epithelium did not form around the unwoven cloth artificial trachea after the substitution, probably because of severe infection due to collapse of the tracheal lumen. However, the formation of bone rings around the trachea suggests preservation of the neotracheal lumen, which may have potential for the treatment of tracheomalacia and/or bronchomalacia.

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PubMedDate not supplied

The repair of tracheal defects using bioabsorbable mesh.

This study compares the use of synthetic bioabsorbable mesh with that of non-absorbable mesh in repairing tracheal defects. Two patch windows, each 1 cm x 1 cm, were created on the ventral wall of the cervical trachea in 12 adult mongrel dogs. One window was repaired using a bioabsorbable polyglycolic acid (PGA) mesh graft, and the other using a nonabsorbable Marlex mesh (polypropylene mesh; PP mesh) (C. R. Bard, Inc., Billerica, MA) graft. The dogs were killed at intervals from 1 to 35 weeks after surgery. Macroscopically, there was no difference between the two mesh groups until 3 weeks after surgery. At 6 weeks in the PP mesh group, the connective tissue over the mesh was remarkably thick, and after 9 weeks, constriction by scarlike tissue was evident, as compared with the PGA mesh group, which showed no constriction. Microscopically, epithelium covered the whole mesh in both groups at 6 weeks. However, in the PP mesh group, flat and poorly differentiated cells occupied a large area, and after 9 weeks the height of the epithelial cells in the PP mesh group was less than in the PGA mesh group. Furthermore, in the PP mesh group, nonciliated cells occupied a large area even at 15 weeks, and the submucous connective tissue layer was thicker than in the PGA mesh group after 6 weeks. The results indicate that, from the point of view of wound-healing, bioabsorbable mesh may be more adequate for repairing tracheal defects.

Absorption↗

PubMedDate not supplied

Experimental reconstruction of the intrathoracic trachea using a new prosthesis made from collagen grafted mesh.

Intrathoracic tracheal replacement was performed in dogs using a tracheal prosthesis we had constructed from mesh. The prosthesis consists of Marlex mesh (polypropylene) reinforced with a continuous polypropylene spiral, and is grafted and coated with pig collagen (Types I and III). Complete surgical resection of the mediastinal trachea was performed in seven adult mongrel dogs. In 1 dog, a 4 tracheal ring segment (2 cm) was resected and replaced with a 3 cm prosthesis, and in 6 dogs, a 7 to 8 tracheal ring segment (4 cm) was resected and replaced with a 5 cm prosthesis. In the latter six dogs, a silicone tube was temporarily inserted into the replacement, and removed by bronchoscopy one month after surgery. In one dog that received a 4 cm replacement, we added omentopexy around the reconstructed trachea. The prostheses in all dogs were promptly infiltrated by surrounding tissue and incorporated by the host trachea. No dehiscence or air leakage was observed after surgery. Mild luminal stenosis was evident in one dog, and partial exposure of the mesh (ulceration) was observed in five dogs within an observation period of 3 to 26 months. However, in the dog that received omentopexy after tracheal reconstruction, no stenosis or ulceration was observed, and the luminal surface seemed lustrous even after 6 weeks. Formation of respiratory epithelium, which lined the prosthetic lumen, was seen to various degrees: in the 2 dogs killed 12 months and 26 months after surgery, confluent epithelization was confirmed histologically from the upper to the lower anastomosis of the prosthesis. The tracheal prosthesis is useful for the repair of intrathoracic tracheal defects and shows promise for clinical application with further investigation.

Animals↗

PubMedDate not supplied

In vitro evaluation of metabolic functions of a bioartificial liver.

The purpose of this study is to develop a bioartificial liver (BAL) with such a simple structure that it can be prepared within several hours and through which whole blood can be perfused as in current hemodialyzers. Hepatocytes were isolated from 37 pigs; each liver weighed 300 to 400 g. The average yield of hepatocytes was 2.4 +/- 0.6 x 10(10) cells per liver, with a cell viability of 89.6 +/- 3.9%. To prepare a BAL device, a cartridge, composed of hollow fibers made of cellulose diacetate was used. Nominal cut-off molecular weight of the hollow fibers was 68 kDa, and the internal diameter was 195 microm. One hundred milliliters of hepatocyte suspension, containing 1 x 10(10) cells, was inoculated into the inner space of the hollow fibers, and both the inlet and outlet of the hollow fiber cartridge were closed. It took only 3 hrs from administration of the pig's anesthesia to the start of an in vitro evaluation of the prepared BAL device. To evaluate the functions of this BAL quantitatively, using a pharmacokinetic method, a mixture of fresh human blood and Dulbecco's modified Eagle medium was circulated in the shell space of the hollow fibers at 200 ml/min. Chemicals (lidocaine, ammonia, and galactose) were then loaded into the perfusion medium. The average intrinsic clearance of the BAL device was found to be 46 ml/min for lidocaine and 8.8 ml/min for ammonia. The galactose elimination capacity of the BAL device was 1.34 mg/min. The metabolic function of the BAL device decreased by 81%, 49%, and 64% of the initial function for lidocaine, ammonia, and galactose, respectively, after 10 days of in vitro circulation.

Animals↗

PubMedDate not supplied

Morphologic studies of hepatocytes entrapped in hollow fibers of a bioartificial liver.

A bioartificial liver cartridge was prepared by inoculating porcine hepatocytes into the inner space of hollow fibers of a hemodialyzer. The hepatocytes formed rod shaped cell aggregates during in vitro perfusion culture within 1 day. Morphologic examination was carried out on the aggregates by optical and electron microscopy. Each hepatocyte was in direct contact with adjacent cells and a bile canaliculus-like structure was occasionally seen between hepatocytes. High magnification observation showed that the canaliculus was separated from the remainder of the intercellular space by a tight junction. These facts suggest that the hepatocytes formed functionally associated cell aggregates with a compact morphology not unlike hepatocyte spheroids. These structures were well maintained for 7 days in culture, and then the amorphous area in the aggregates and the nonviable cell number increased with lengthening culture period. The bioartificial liver maintained the ability to metabolize lidocaine, ammonia, and galactose for 7 days and then deteriorated with time.

Ammonia↗

PubMedDate not supplied

Experimental corneal neovascularization by basic fibroblast growth factor incorporated into gelatin hydrogel.

The study was designed to investigate the feasibility of using an acidic gelatin hydrogel as a biodegradable vehicle for basic fibroblast growth factor (bFGF). bFGF was incorporated by polyion complexation into a biodegradable hydrogel prepared by cross-linking acidic gelatin with the isoelectric point of 4.9. The dried hydrogel (sized to 2x1 mm) was hydrated with bFGF aqueous solution including different doses of bFGF (20, 50, 125, 250 and 500 ng) and implanted into a rabbit corneal pocket (2.5x2 mm). As a control group, the gelatin hydrogel without bFGF or bFGF alone (500 ng) was used. Corneal angiogenesis was evaluated by biomicroscopy, corneal fluorescein angiography and histology for 21 days. Photographs were taken and corneal angiogenesis was evaluated by image analysis. The hydrogel degraded with time after its implantation into the corneal pocket. Experimental eyes receiving the hydrogel containing more than 50 ng of bFGF demonstrated significant corneal angiogenesis. Control eyes and eyes receiving the hydrogel containing 20 ng of bFGF showed no corneal angiogenesis. Corneal angiogenesis, which occurred on the 3rd or 4th day after implantation, reached maximal growth on about day 7 and regressed from day 10 after implantation. The area of angiogenesis showed a dose-dependency on bFGF. The gelatin hydrogel itself induced neither angiogenesis nor inflammation. These results suggested that acidic gelatin hydrogel releases bioactive bFGF with its biodegradation, resulting in corneal neovascularization.

Absorbable Implants↗

PubMedDate not supplied

Modified subcutaneous tissue with neovascularization is useful as the site for pancreatic islet transplantation.

The success rate of subcutaneous transplantation of pancreatic islets has been extremely low. Insufficient oxygen supply to the grafted islets is one possible major obstacle to the preservation of graft function. This study attempted to use basic fibroblast growth factor (bFGF) in subcutaneous transplantation to induce neovascularization and a sufficient blood flow around the space formed for grafted islets in the subcutaneous tissues. A bFGF-releasing device was designed enclosing bFGF in a polyethylene terephthalate mesh bag coated with polyvinylalcohol hydrogel. In the vascularized group (n = 5), two bFGF-releasing devices were implanted bilaterally into the subcutaneous tissue of the back of streptozotocin-induced diabetic Lewis rats. One week after implantation, isolated rat islets (5000) were syngeneically transplanted subcutaneously after the removal of the devices. In the control group (n = 5), no devices were implanted and the same number of rat islets was transplanted directly. One week after the implantation of the devices into the test animals, a thick, well-vascularized capsule was observed in the subcutaneous site. All vascularized recipient rats showed significant decreases in nonfasting blood glucose and maintained normoglycemia for more than 1 month after islet transplantation. However, in the control group, all rats failed to achieve normoglycemia after transplantation. This study provides evidence that the subcutaneous tissue is a promising site for pancreatic islet transplantation, offering convincing advantages in acceptability for diabetic recipients. Establishment of this subcutaneous islet transplantation technique will afford some new perspectives on successful clinical islet transplantation.

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PubMedDate not supplied

Subcutaneous xenotransplantation of hybrid artificial pancreas encapsulating pancreatic B cell line (MIN6): functional and histological study.

The biohybrid artificial pancreas is designed to enclose pancreatic tissues with a selectively permeable membrane that immunoisolates the graft from the host immune system, allowing those endocrine tissues to survive and control glucose metabolism for an extended period of time. The pancreatic B cell line MIN6 is established from a pancreas B cell tumor occurring in transgenic mice harbouring the human insulin promoter gene connected to the SV40 T-antigen hybrid gene. It has been proven that glucose-stimulated insulin secretion in MIN6 cells retains a concentration-dependent response similar to that of normal islets. In this study, we performed the histological and functional examination of three-layer microbeads employing MIN6 cells after subcutaneous xenotransplantation to evaluate this device as bioartificial pancreas. MIN6 cells were microencapsulated in three-layer microbeads formulated with agarose, polystyrene sulfonic acid, polybrene, and carboxymethyl cellulose. Microbeads were xenogenically implanted in the subcutaneous tissue of the back of Lewis rats with streptozotocin-induced diabetes. One week after implantation, microbeads were retrieved and cultured for 24 h before the static incubation. There was no evidence of adhesion to the graft and the fibrosis in the transplantation site as determined by gross visual inspection. Microscopic examination demonstrated that retrieved microbeads maintained normal shape, containing intact MIN6 cells. Histological study showed that these MIN6 cells in the microbeads appeared to be viable without cellular infiltration within or around the microbeads. Immunohistochemical analysis of the microbeads clearly revealed the intense staining of insulin in the cytoplasm of encapsulated MIN6 cells. Insulin productivity of MIN6 cells in the microbeads is strongly suggested to be preserved. In response to 16.7 mM glucose stimulation, static incubation of microbeads 1 wk after implantation caused the 2.3 times increase in insulin secretion seen after 3.3 mM glucose stimulation (84.3 +/- 10.0 vs. 37.4 +/- 10.7 microU/3 x 10(6) cells/hr, n = 5 each, p < 0.01). This study demonstrates that three-layer microbeads encapsulating MIN6 cells retain excellent biocompatibility and maintain good insulin secretion even after subcutaneous xenotransplantation, suggesting the possible future clinical application of this unique bioartificial pancreas to subcutaneous xenotransplantation.

Animals↗

PubMedDate not supplied

Prolonged effect of troglitazone (CS-045) on xenograft survival of hybrid artificial pancreas.

Troglitazone (CS-045), a thiazolidinedione derivative, is a new oral antidiabetic agent that enhances insulin sensitivity and improves insulin responsiveness. In this study we examined the effects of CS-045 on the survival of xenografted bioartificial pancreas. Isolated rat islets were microencapsulated with three-layer agarose microcapsules (polybrene, carboxymethyl cellulose, and an agarose-polystyrene sulfonic acid mixture). Diabetes was induced by intraperitoneal injection of streptozotocin 220 mg/kg. Recipient diabetic mice were separated into two groups. In the CS-045 treated group, the recipient mice were given feed mixed with CS-045 (0.2% w/w) starting from 1 wk before transplantation up to graft failure. The mice in the control group had feed without CS-045. Three hundred microencapsulated rat islets were xenotransplanted into the intraperitoneal cavity of each recipient mouse in both groups. One month after xenotransplantation, IVGTT was performed for all recipients. Xenotransplantation of 300 rat islets in microcapsules decreased the nonfasting blood glucose levels of both groups within 2 days. In the CS-045-treated group (n = 3), the normoglycemic period lasted for more than 1 mo without administration of immunosuppressive drugs (45 +/- 4.3 days). However, in the control group (n = 4), the blood glucose levels of all recipients were already elevated on day 4. In the IVGTT study, the glucose assimilation was markedly and significantly better in the CS-045-treated group than in the control group (K = 1.7 +/- 0.1 vs. 0.7 +/- 0.28 respectively, p < 0.01). This study demonstrates that a newly developed oral antidiabetic agent, CS-045 could favorably ameliorate the diabetic state of the recipients xenotransplanted with the bioartificial pancreas, leading to an improved glucose tolerance and longer xenograft survival.

Animals↗

PubMedDate not supplied

Gelatin density gradient for isolation of islets of Langerhans.

Isolation of islets of Langerhans (islets) has been performed by means of collagenase digestion of the pancreatic tissue combined with density gradient separation of islets from unwanted exocrine tissues. An enormous number of islets are necessary for clinical islet transplantation. The density gradient used for isolation of a large number of islets should satisfy several requirements in addition to those for the conventional density gradients, such as high viscosity for creating fine interfaces with a large area, easy sterilization, and low cost. This study is concerned with the development of a new density gradient made of low-molecular-weight gelatin. We isolated islets from the hamster pancreatic tissue using the gelatin density gradients. The yield and purity of islet and its insulin release function were compared with those of islets isolated using Ficoll and Ficoll-Conray density gradients that have been conventionally used. The new gelatin density gradient can separate islets from the unwanted exocrine tissue as effectively as the Ficoll density gradient and more effectively than the Ficoll-Conray density gradients. The islets collected using the gelatin gradient retain ability of insulin release increase in response to glucose stimulation, similar to those isolated by the Ficoll-Conray gradient and more than those collected by the Ficoll gradient. In addition, the gelatin effectively inhibited enzyme activities, that is, collagenase and proteolytic enzymes released from the exocrine tissue, and thus it can inhibit overdigestion of islets during their density gradient isolation. The gelatin gradient satisfies most of the additional requirements for islet isolation from the pancreatic tissue of large animals mentioned above. Although several factors, such as molecular weight of gelatin, osmolality of the gradient, and centrifugal conditions, still remain to be optimized, our results suggest that the gelatin gradient has potentiality to isolate islets from the pancreatic tissue of a large animal.

Animals↗