MXPA96002765A - Secretor cells macroencapsula - Google Patents

Abstract

The present invention relates to the macroencapsulation of secretory cells in a hydrophilic gel material, to therapeutic methods employing macroencapsulated secretory cells, and to the preservation of secretory cells by macroencapsulation.

Description

MACROENCAPSULATED SECRETOR CELLS Field of the Invention The present invention relates to the macroencapsulation of secretory cells in a hydrophilic gel material, to therapeutic methods employing macroencapsulated secretory cells, and to the preservation of secretory cells by macroencapsulation.

Background of the Invention Secretory cells are cells that are characterized by secreting biological products, such as, but not limited to, hormones (eg, insulin), growth factors, cytokines, and so forth. Its role in biological processes is well known, and it is not necessary to expose it in the present. The number of diseases and pathological conditions are related to a failure of the secretory cells to work properly, such as a poor production of the secretory products, for example, hypothyroidism and stupid dwarfism, both due to the deficiency of the thyroid hormone, pituitary dwarfism due to deficiency of pituitary growth hormone, Lesch-Hyhan syndrome, due to deficiency of hypoxanthine-guanine fssforibosyltransferase, fulminant hepatic failure due to deficiency of hepatotrophic factor, extracellular matrix disease due to chondrocyte deficiency, and insulin-dependent diabetes due to insulin deficiency. One proposal to treat these conditions is to transplant the secretory cells in the patient. The transplanted material, to be clinically safe and effective, must (1) be non-immunogenic, non-thrombogenic, and stable and completely non-toxic to host cells and tissues, (2) maintain cell viability for a prolonged period of time, (3) allow the free passage of nutrients, secretagogue (a substance that stimulates secretion), and cellular products, (4) facilitate surgical implantation and cellular reinoculation and (5) be easily fixed in place and also removed. Pancreatic islet transplantation to treat insulin-dependent diabetes has been the subject of interest. renewed due to technological advances in the isolation of the islets of Langerhans. By way of background, the human pancreas contains islets of Langerhans (hereinafter referred to as "pancreatic islets") that are scattered throughout the exocrine pancreas with some concentrations near the pancreatic ducts. The pancreatic islets, taken together, can be thought of as an individual endocrine organ that occupies about 1% of the volume of the pancreas. Within the pancreas, small islets (up to a diameter of 160 um) tend to be distributed throughout the exocrine tissue. These small islets account for 75% of the islets in number, but only approximately 15% by volume. The islets with a diameter greater than 250 μm constitute only 15% of the total number of islets, only 60% of the volume. These islets are located near the major canals and blood vessels, and are not surrounded by acinar tissue. A human pancreas can contain over 1 million islets, and each islet typically consists of several thousand cells. Each islet is comprised of a central core of insulin-producing beta cells (B-cells) and a surrounding blanket of alpha cells (A-cells) containing glucagon, somatostatin-secreting delta cells (D-cells) and cells containing polypeptides , pancreatic (PP-cells). The B-producing insulin cells make up the majority of the cells, and comprise up to about 80% of the islets in the human. The clinical applications of pancreatic islet transplantation have been limited by the inability to prevent rejection of the allograft-islet xenograft, that is, a rejection of the transplanted pancreatic islets because the host's immune system attacks the transplanted pancreatic islets. To counteract the rejection, the pancreatic islets have been transplanted in combination with the administration of immunosuppressive agents. However, immunosuppressive therapy has proven to be a two-edged weapon, while reducing the risk of rejection, damaging the body's complete immune defenses. Several methods have been examined by many investigators to protect the transplanted tissue from the host's immune response. As discussed below, although temporary success has been reported (see Lacy, Diabetes Reviews 1 (1): 76 (1993)), effective long-term methods have not yet been achieved.The five main proposals to protect the transplanted tissue from the response The immune immunology of the host includes all isolating transplanted tissue from the host immune system.The immunoisolation techniques used to date include: extravascular diffusion chambers, intravascular diffusion chambers, intravascular ultrafiltration chambers, microencapsulation, and macroencapsulation. methods have failed, due to one or more of the following problems: a host's fibrotic response to the implant material, instability of the implant material, limited diffusion of nutrients through the semi-permeable membranes, permeability of the product and the secretagogues , and diffusion delay through the membrane barriers is For example, a microencapsulation procedure to enclose viable cells, tissues, and other labile biological membranes within a semipermeable membrane was developed by Lim in 1978 (Lim, Research report to Damon Corporation) Lim used microcapsules of alginate and poly L- lysine to encapsulate the islets of Langerhans. In 1980 the first successful live application of this new technique in diabetes research was reported ((Lim, et al., Science 210: 908 (1980)). The implantation of these islets of microencapsulated Langerhans resulted in sustaining of an euglycemic state in diabetic animals, however, other investigators repeated these experiments finding that the alginate causes a reaction to the tissue and were unable to reproduce the results of Lim et al. (Lamberti, et al., Applied Biochemistry and Biotechnology 10:10 ( 1984); Dupuy, et al., Jour. Bio ed. Material and Res. 22: 1061 (1988); Weber, et al., Transplatation 49: 396 (1990); and Soon-Shiong, et al., Transplantation Proceedings 22:; 754 (1990). The water solubility of these polymers is now considered to be responsible for the limited stability and biocompatibility of these microcapsules in vivo ((Dupuy et al., Supra, Weber, et al., Supra, Soon-Shiong, et al., Supra, and , Smidsrod, Faraday Discussion of Chemical Society 57: 263 (1974).) Recently, Iwata et al. (Iwata, et al., Biomedical Material and Res. 26: 967 (1992)) used agarose for the microencapsulation of pancreatic islets. and that it could be used as a medium for the preparation of microbolites In their study, 1500-2000 islets were individually microencapsulated in 5% agarose and implanted in diabetic mice induced by streptozotocin. period and the recipients remained in normoglycemia indefinitely.However, their method suffers a great number of disadvantages.It is difficult to handle and inaccurate for example, many pellets; partially coated and several hundred pellets in the form of an empty agarose. In this way, additional time is required to separate the encapsulated islets from the empty pellets. In addition, most of the implanted microbolites are concentrated in the pelvic cavity, and a large number of islets are required in the fully coated individual pellets to achieve normoglycemia. In addition, the transplanted pellets are difficult to recover, tend to be fragile, and will easily release islets in light damage. A macroencapsulation procedure has also been tried. Macrocapsules of different mixed materials, such as poly-2-hydroxyethyl-methacrylate, polyvinylchloride-co-acrylic acid, and cellulose acetate were made for the immunoisolation of islets of Langerhans. (see Altman, et al., Diabetes 35: 625 (1986), Altman, et al., Transplantation American Society of Artificial Internal Organs 30: 382 (1984); Ronel, et al., Jour Biomedical Material Research 17: 855 (1983); Klomp, et Al., Jour. Biomedical Material Research 17: 865-871 (1983)). In all these studies, only a transient normalization of glycaemia was achieved. Archer et al. Journal of Surgical Research, 28:77 (1980), used acrylic copolymer hollow fiber to temporarily prevent rejection of islet xenografts. They reported long-term survival of murine, neonatal, pancreatic grafts, dispersed in the hollow fibers that were transplanted into diabetic hamsters. Recently Lacy et al., Science 254: 1782-1784 (1991) confirmed their results, but found that the euglycemic state is in a transient phase. They found that when the islets are injected into the fiber, they are added into the hollow tube and result in necrosis in the central portion of the islet masses. Central necrosis prevented the extension of the graft. To solve this problem, they used alginate to disperse the islets in the fiber. Using this method they were able to achieve long-term graft survival, however, this experiment has not been repeated extensively, therefore, the role of the membrane as a means of transplantation of islets in humans is questionable. In this way there is a need to achieve the transplantation of secretory cells, and in particular the survival of the allograft and pancreatic islet xenograft without the use of chronic immunosuppressive agents. It has been surprisingly discovered that macroencapsulation of secretory cells in a hydrophilic gel material results in a non-immunogenic, functional macrobolite that can be transplanted into animals and can be stored for extended periods of time. The macroencapsulation of the secretory cells of the present invention provides a more effective and manageable technique for the transplantation of secretory cells. The macroencapsulation technique can also be used to macroencapsulate other biological agents such as enzymes, microorganisms, trophic agents that include recombinantly produced trophic agents, cytotoxic agents and chemotherapeutic agents. Macroencapsulated biological agents can be administered to treat known conditions because they respond to the biological agent.

BRIEF DESCRIPTION OF THE INVENTION It is therefore an object of the present invention to provide a macrobolite of secretory cells that can be transplanted into animals to treat conditions caused by damaged functioning of the host secretory cells. It is a further object of this invention to provide a macrobolite of secretory cells that can be stored for extended periods of time. In achieving these and other objects, a method for producing a agarose-coated agarose-collagen secreting macrobolite has been provided according to one aspect of the present invention; a macrobolite of gel foam secreting cells, coated with agarose; and a macrobolite of secretory cells, agarose, coated with agarose.

In another aspect of the invention there is provided a method for treating a patient having a condition characterized by an insufficiency of a secretory cell product, which comprises transplanting in the patient a therapeutically effective amount of macrobolites from secretory cells selected from the group that consists of macrobolites of agarose-collagen secreting cells, coated with agarose; macrobolites of gel foam secreting cells coated with agarose; and macrobolites of secretory, agarose cells coated with agarose. In still a further aspect of the invention, there is provided a method for preserving secretory cells, comprising forming macrobolites selected from the group consisting of agarose-collagen-secreting cell macrobolites coated with agarose.; macrobolites of gel foam secreting cells, coated with agarose; and macrobolites of agarose-secreting agarose coated cells; and incubate the macrobolites of secretory cells. Other objects, features and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent. apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1 and 2 show pancreatic islet, collagen-agarose macrobolites coated with agarose.

Figure 3 shows the glucose levels of diabetic mice transplanted with pancreatic collagen-agarose islet macrobolites, coated with agarose.

Figure 4 shows the glucose levels of diabetic mice transplanted with agarose-coated pancreatic islet macrobolites of gel foam.

Figure 5 shows the glucose levels of diabetic mice transplanted with agarose coated agarose macrobolites.

Figure 6 shows the glucose levels of diabetic mice transplanted with free pancreatic islets.

Figure 7 shows the glucose levels of diabetic mice transplanted with agarose-coated collagen-agarose macrobolites and free pancreatic islets.

Figure 8 shows a glucose tolerance test for normal mice, diabetic mice, induced by streptozotocin, diabetic mice induced by streptozotocin receive the alotransplant in the kidney capsule "(KCT mice)", and diabetic mice induced with streptozotocin that receive pancreatic islet macrobolites, agarose-collagen coated with agarose; pancreatic islet macrobolites, gel foam coated with agarose; and agarose coated pancreatic islet macrobolites (collectively referred to as "BEAD mice").

Detailed description of the invention The present invention relates to the macroencapsulation of biological agents and, preferably, secretory or secretory cells in a hydrophilic gel material, to therapeutic methods that employ macroencapsulated biological agents, and preferably, secretory cells, and to the preservation of biological agents, preferably secretory cells by macroencapsulation. The hydrophilic gel material comprises agarose, and combinations of collagen-agarose and gelatin-agarose sponge. The gelatin sponge will hereinafter be referred to as gel foam. The term "biological agent" denotes a living organism and its products, for example, proteins, enzymes, hormones, polypeptides, serum, antibodies, and antibiotics and also genetically engineered cells. Biological agents include enzymes for example, glucose oxidase lactase complexes, microorganisms, for example, Klebsiella aerogenes for the removal of ammonia and urea, trophic agents, which include trophic agents produced recombinantly, for example, the growth hormone produced in a manner recombinant, and cytotoxic agents.

The term secretory cell includes a pancreatic islet, although technically, a pancreatic islet is not a secretory cell, but mainly a multitude of secretory cells scattered throughout the length of the pancreas and comprising its endrocrine portion. In humans, they are composed of at least four different types of secretory cells; the alpha cells that secrete the hyperglycemic factor, glucagon; the beta cells that are the most abundant (70% - 80%) and secrete insulin; the delta cells that secrete somatostatin; and the polypeptide cells that secrete the polypeptide hormone. As previously explained, the transplanted material must be compatible with the host. Agarose has a long history of use in biological research, and its quality is well proven. Collagen is the most abundant protein in mammals, provides firm mechanical support and serves as the biological space for cell replication, differentiation, organogenesis, individual growth and wound repair. Collagen also has good biocompatibility. Gel foam is non-immunogenic and has been used extensively in surgical procedures. It is also well tolerated by secretory cells.

The biological agents and preferably, the secretory cells, are first isolated using methods well known in the art. In a preferred embodiment, the pancreatic islets are cultured either at 4 ° C, 24 ° C or at 37 ° C before they are macroencapsulated.; this method allows to select only the surviving islets after the isolation trauma; also the islets become less immunogenic resulting in the protection of the fibroblast macrobolites. In one embodiment of the invention, a biological agent preferably pancreatic islets and more preferably approximately 50,000-700,000 pancreatic islets are dispersed in an aqueous solution of collagen, preferably a solution of atelocollagen at 0.5% -2%. Atelocollagen is obtained by treating collagen with pepsin, which removes the antigenic telopeptides responsible for the intermolecular cross-linking of collagen. Approximately 0.5% -5% agarose, preferably about 1%, is then added to the islets. pancreatic cells dispersed to form pancreatic islets dispersed in a mixture of collagen and agarose. The mixture containing the pancreatic islets is then transformed into a semisolid pellet using techniques well known in the art, preferably by dropping the mixture in mineral oil or a Teflon® sheet. The semisolid pellet is then transferred to an antibiotic medium, washed, and then incubated under normal conditions to polymerize the collagen, preferably at 37 ° C in a 5% C02, moisturized atmosphere, whereby a macrobolite is formed. Collagen-agarose, solid. In another embodiment of the invention, a biological agent, preferably pancreatic islets, more preferably approximately 50,000-700,000 pancreatic islets are spread on the surface (3-5 cm) of a gelatin sponge. The gelatin sponge is then stirred into a sphere. Agarose, 3% -5%, is poured into the sphere to form a ball. In yet another embodiment of the invention, a biological agent, preferably pancreatic islets, and more preferably approximately 50,000-700,000 pancreatic islets, are placed in an agarose solution ranging from about 0.5% -5% agarose, preferably approximately 1% agarose. The mixture is then transformed into a macrobolite by contacting the mixture with mineral oil or Teflon. The pellet is then transferred to an antibiotic medium, washed and incubated overnight, preferably at 37 ° C in a 5% C02, moisturized atmosphere.

In all the modalities mentioned above, the macrobolites are uniformly coated with agarose, preferably by stirring the pellet 3-4 times in a Teflon spoon containing approximately 500-2,000 μl of 5% -10% agarose. Similarly, the term "biological agent macrobolites" as used herein denotes macroencapsulated biological agents in the form of a pellet. Macrobolites can be used as a vehicle to distribute the biological agent to the body where the agent will perform its known function. They can be encapsulated in a pellet more than one type of biological agents. For example, a macrobolite can contain multiple enzymes, such as hemoglobin and glucose oxidase. This pellet can be administered to remove bilirubin. These pellets can be used either for the oral administration of digestive enzymes (lactase complexes) or for the selective removal of undesirable amino acids from the body. The encapsulation of the enzymes will also prevent the degradation of the enzyme in the lumen. Additionally, recombinant genetic products can be safely distributed using the encapsulation as the medium. For example, the K. aerogenes gene can be macroencapsulated in macrobolites for the removal of urea and ammonia. Where the biological agent is immunogenic to the host, macrobolite allows the administration of the biological agent without the use of the immunosuppressant or with decreased amounts of the immunosuppressant. The secretory macrobolites can be used to treat conditions caused by a damaged functioning of the subject's secretory cells, for example, insulin dependent diabetes, growth factor deficiency disorder, and hormonal disorders, by transplanting the macrobolites of secretory cells in the subject. Macrobolites can be inserted in the appropriate location for that particular treatment. For example, macrobolites containing hepatos can be implanted in the abdominal cavity to treat diseases related to non-function of the liver. A preferred application is the transplantation of 5-10 pancreatic islet macrobolites, each containing 50,000-700,000 pancreatic islets, in a patient to treat diabetes, dependent on insulin. Macrobolites can be inserted into the peritoneal cavity. The macrobolites of secretory cells are transplanted into a patient in an amount sufficient to treat the condition. An amount adequate to achieve this is defined as a "therapeutically effective amount" or "effective amount". The effective amounts for this use will depend on the severity of the condition, the general condition of the patient, the route of administration, the placement of the macrobolites, and whether the macrobolites of secretory cells are being administered in combination with other drugs. The secretory macrobolites can be used for allogeneic and xenogeneic transplantation in combination with immunosuppressants or, preferably, without immunosuppressants. In a preferred embodiment, patients having insulin-dependent, chronic or acute diabetes are treated by xenotransplanting animal pancreatic islets, eg, porcine, bovine, murine, rat, picin, or any other suitable species in the patient without the use of immunosuppressants. The secretory cell macrobolites can also be administered in combination with other therapeutic agents, for example, triple drug therapy, commonly used (cyclosporine, azithioprine, and hydrocortisone) rapamycin, deoxyspergualin, or antibodies, to treat the condition. Macrobolites can also be used as a means to store biological agents, and preferably secretory cells, for extended periods of time. To maintain the viability of the biological agents, and preferably the secretory cells, the biological agents and preferably the secretory cell macrobolites are incubated until they are transplanted into the animal. When secretory cells with pancreatic islets, pancreatic islet macrobolites are incubated at a temperature of 24 ° C or 37 ° C.

EXAMPLES Example I Isolation of Pancreatic Islets Pancreatic islets were isolated from rats by a modification of the method described in Gotoh et al Transplantation 40: 437 (1985). Injected into the pancreas via the common bile duct collagenase solution (collagenase type XI, Sigma Chemical, St, Louis, MO; 1 mg / ml containing 2 mg / ml bovine serum albumin type V, Sigma 1 mg / ml of Ca C12). (Gotoh et al., Transplantation 40: 437 (1985), Supra). The pancreas was removed and collected in a flask kept on ice. Once the pancreas of four rats were collected, the flask was placed in a 38 ° C water bath for 30 minutes. The resulting digested tissue was washed four times cold (8 ° C) HBSS Hank's Balanced Salts Solution). Undigested tissue, large lymph nodes and other foreign material were removed by repeated tissue mobilization, followed by removal of the supernatant. The purified islets were isolated in a discontinuous Ficoll gradient, consisting of 25%, 23%, 21% and 11% Ficoll layers, prepared in the Euro-Collins solution (Frescenius AG, Gluchen Steinweg, Homburg VDH) and centrifuge at 200 rpm for 16 minutes. The islets were collected from the interfacial zone between 11% and 21% and the interfacial zone between 21% and 23% of the Ficoll layers. The islets of each fraction were combined and washed four times in HBSS solution containing 10% fetal calf serum. The combined islet cells were then transferred into Petri dishes containing the RPMI complete medium, ie the RPMI 1640 complete medium (GIBCO, Grand Island, NY), supplemented with heat inactivated fetal bovine serum, 25 mM HEPES (10%). ), and antibiotic-antifungal solution (1 ml / 100 ml) containing: 100 μg / ml penicillin, 100 μg / ml streptomycin, and 25 μg / ml amphotericin B. Any lymphatic or ductular vascular nodes are identified, acinar, not of islet with the help of a dissecting microscope, and carefully removed with a sterile, fine-tipped pipette. The final purity is assessed by dyeing the islet preparation with diphenylthiocarbazone. After isolation, the islets were incubated in bacteriological plastic boxes (100 mm) containing 10 ml of RPMI medium, at 37 ° C in a humidified atmosphere having 5% C02, for 4 days. The medium was changed every day, and the islets were then transplanted either directly or macroencapsulated.

Example II A. Preparation of the macrobolites or macrospheres of Pancreatic Islets, of Agarose-Collagen Coated with Agarose. 1000 pancreatic islets obtained by method I were washed 4 times in the RPMI complete medium as described in example I, minus fetal calf serum. The pancreatic islets were then added to a tube containing 50 μl of 1% atelocollagen solution in phosphate buffered saline to disperse the pancreatic islets. Then, 100 μl of 1% low viscosity agarose solution (Sigma type XII) prepared either in RPMI or in MEM (minimum essential medium), maintained at 60 ° C, was added to the suspension of pancreatic islets-collagen. The contents of the tube are then transferred immediately, as a single large drop in either sterilized mineral oil, maintained at room temperature, or a Teflon® sheet. After one minute the drop becomes a semisolid macrobolite that is then transferred to the RPMI antibiotic medium at 37 ° C. The macrobolites are then washed three times with the same medium to remove all the oil. Finally, they are rinsed twice with the complete medium (37 ° C) and incubated overnight, at 37 ° C in a humidified atmosphere that has C02 at 5%, during this period, the collagen polymerizes and the pancreatic islets rest on the collagen fiber. The next day, the solid macrobolites were transferred to a Teflon® spoon containing approximately 1 ml of 5% agarose of RPMI medium or in MEM. The solid macrobolites were then stirred in this solution 2-3 times in order to uniformly coat them. Before the agarose solidifies, the macrobolites were transferred to mineral oil in a Teflon® box to obtain smooth surface macrobolites. After 60 seconds, the macrobolites were removed from the mineral oil and washed 3 times with the RPMI antibiotic medium, and then twice with the RPMI complete medium. They were then incubated overnight, at 37 ° C in a humid atmosphere having 7% C02. Figures 1 and 2 show pancreatic islet macrobolites, agarose-collagen coated with agarose.

B. Preparation of the Macrobolites of Pancreatic Islets of Gelatin Sponge, Coated with Collagen.

A small piece of gelatin sponge (gel foam), 3 mm2 was first soaked in the RPMI complete medium. The medium was removed with difficulty and the gel foam was left to rest for one minute. One thousand pancreatic islets, prepared according to example 1, were washed four times with the RPMI antibiotic medium. Then they were dispersed in 10 μl of the RPMI antibiotic medium. They were transferred by a thin-tipped plastic pipette and spread on the surface of the gel foam. After 20 seconds, the gel foam was stirred in a small sphere. 50 μl of 5% agarose was poured onto the surface of the sphere to create a pancreatic islet macrobolite. In order to uniformly cover the macrobolite with 5% agarose, 500 μl of 5% agarose was added to the macrobolite in a Teflon® spoon and stirred 3-4 times. Before the agarose will solidify, the macrobolite was transferred to the mineral oil, and the box was stirred to obtain a smooth surface in the macrobolite. The macrobolite was washed 3-4 times in RPMI antibiotic medium and then rinsed twice with RPMI complete medium. It was incubated overnight before it will be used for transplantation C. Preparation of Agarose Pancreatic Islet Macrobolites, Coated with Agarose.

About one thousand pancreatic islets obtained by the method of Example 1 were first washed 4 times in the RPMI antibiotic medium. The pancreatic islets were transferred to a tube containing 50 μl of the RPMI antibiotic medium and dispersed therein. Then, 100 μl of 1% agarose solution was added to the tube. The entire contents of the tube were transferred immediately, as a large, single drop, has either sterilized mineral oil or a Teflon® sheet. After 1 minute, the drop solidified to a macrobolite. The macrobolite was transferred to the RPMI antibiotic medium, maintained at 37 ° C. The oil was then removed by washing the macrobolite 3 times with the same medium, and then by rinsing twice with the RPMI medium. The pellets were incubated overnight at 37 ° C in a humidified atmosphere having 5% C02.

The next day these pellets or spheres were transferred in a Teflon® spoon containing 1 ml of 5% agarose in either RPMI or MEM medium to uniformly coat the macrobolites with agarose, the pellets were then gently stirred in agarose 2- 3 times. They were then transferred to mineral oil, in a Teflon® box, before the agarose solidified. After 60 seconds, the pellets were removed from the mineral oil and washed 3 times in the RPMI antibiotic medium and 2 times in the RPMI complete medium. The pellets were then incubated overnight.

Example III - Transplant of Pancreatic Islet Microbolites in Mice.

A. Mice Receptors and Mice Donors The mice used were the C57BL / 6 and BALB / c strains, males. The recipient mice were diabetic by an i.v. individual streptozotocin (170-200 mg / kg). Plasma glucose levels, not fasting, were determined before the induction of diabetes.

All blood sugar levels in the recipient mice were inspected via blood samples from the tail vein with an ExacTech Pen Sensor. Only those mice with serum glucose level > 400 mg / dl on the day of transplant. Wistar Furth rats were used as donors for xenotransplantation.

B. Xenotransplantation of Pancreatic Islet Macrobolites in the Peritoneal Cavity.

At the time of xenotransplantation, the pancreatic islet macrobolites of Example II (A), II (B), and III (C), respectively, were gently transferred to separate the plates containing the RPMI antibiotic medium. To remove all the proteins from the serum, the medium was changed three times. The diabetic receptor mice were anesthetized with avertine. A midline incision is made to introduce a single pancreatic islet macrobolite into the free peritoneal cavity. A two-layer closure of the incision was made with an absorbent suture. The control mice received either an empty macrobolite i.p. (intraperitoneally), i.p. free pancreatic islets, or an empty macrobolite together with free pancreatic islets of the donor.

After transplantation, each recipient's blood glucose was checked daily or each day different until the normal range was reached; subsequently, blood glucose was checked only 2-3 times each week. The transplants were considered technically successful if the serum glucose was < 200 mg / dl and remained there during consecutive bleeding. It was considered that a transplant was rejected if the concentration of glucose in the serum rose above 200 mg / dl after a period of transient normoglycemia. Transplants were considered to have failed or to become "non-functional primary" if blood glucose never becomes normal (ie, consistently remained> 200 mg / dl).

C. Intraperitoneal Glucose Tolerance Test Glucose tolerance tests were performed approximately 70-84 days post-implantation. Glucose solution (1.0 g / kg body weight) was injected intraperitoneally in mice that have been fasting for 6 hours (9 a.m.-3 p.m.). In both pre- and post-injection (0, 30, 60 and 120 minutes), blood samples were taken to determine plasma glucose levels using the ExacTech Pen Sensors.

For comparison, glucose tolerance tests were performed in normal mice C57BL / 6 and BALB / c, in C57BL / 6 and BALB / c mice induced with streptozocin in which no pancreatic islets were transplanted, and in diabetic BALB / c mice induced with streptozocin, in which free pancreatic islets have been transplanted into the kidney capsule ("KCT" mice). Control experiments were carried out to ensure that the euglycemic state was being achieved via the macroencapsulated pancreatic islets and not the macrobolites themselves. Therefore, agarose-coated, agarose-coated, empty agarose and gel foam macrobolite, coated with agarose, were prepared in the same manner as the pellets of Example II (A) and (B).

D. Results of Intraperitoneal Xenotransplantation and Glucose Tolerance Test.

In the implant of pancreatic islet macrobolites, the observed changes in the level of glucose in the non-fasting plasma of C57BL / 6 mice induced with STZ diabetic estropzotocin are shown in Figures 3 and 4. The macrobolite receptors of pancreatic islets of agarose-collagen, coated with agarose, and pancreatic islet macrobolites, gel foam, coated with agarose maintained in a normoglycemic state for more than 60 days, and during this period, the body weight of these mice was increased 3 grams on average. When the agarose-coated pancreatic pancreatic islet macrobolites were transplanted, 2 of 6 animals became normoglycemic after 21-33 days post-transplant (Figure 5) and subsequently remained euglycemic. All the other transplanted animals failed to achieve an euglycemic state. The empty macrobolites (n = 6) did not affect blood glucose. When intraperitoneally free pancreatic islets were transplanted, 6 of 7 transplanted animals became normoglycemic 1 day after transplantation; however, they remained in this state for only 3-10 days (Figure 6). When free pancreatic islets were transplanted with empty pellets made of agarose-collagen macrobolites, coated with agarose, or gel foam macrobolites, coated with agarose, all animals became normoglycemic in the space of 24 hours and thus remained more 10 days (Figure 7). Subsequently, all animals became hyperglycemic. The animals containing the empty non-excited macrobolites were followed for the tissue reaction during the 90 days. The results obtained after performing the glucose tolerance tests are presented in the Figure 8 in BALB / c and C57BL / 6 mice and "KCT" mice, plasma glucose maximized at 30 minutes and returned to baseline levels for 120 minutes. Similar results were obtained when macroencapsulated pancreatic islets and unencapsulated pancreatic islets transplanted into kidney capsules were tested. The results of these experiments demonstrate that the agarose-collagen macrobolites, coated, with agarose; macropanites of pancreatic islets, agarose-gel foam, coated with agarose; and agarose-coated agarose islet macrobolites exhibit the properties required for a hybrid artificial organ. Although all three of these types successfully secreted insulin, agarose-coated, agarose-coated, agarose-coated, and gel-agarose-foam-coated macrobolites are more suitable as artificial biohybrid organs due to the uniformity of the results obtained in the number minimum number of transplanted animals. In addition, all three types of pellets showed no adverse objects. The macrobolites remained free in the peritoneum showing neither rejection of the tissue, nor any addition to any organ. In this way, these pancreatic biohybrid islands perform their function as efficiently in the macroencapsulated pellets as in their natural habitat, of the pancreas. In all mice, plasma glucose maximized at 30 minutes and returned to baseline levels for 120 minutes.

Example IV Prolonged Storage Life of Pancreatic Islets The macroencapsulated pellets prepared according to Examples I (A), (B) and (C), which were incubated for 4 weeks at 37 ° C in complete RPMI medium, were tested for their long-term preservation properties in alive and in vitro. It was found that macroencapsulated pancreatic islets that were incubated for 4 weeks were functionally similar to those that were incubated for 1 day.

This example demonstrates that the macroencapsulation method according to the present invention can be used for the conservation of secretory cells, and preferably, the preservation of pancreatic islets.

It is noted that in relation to this date, the best method known by the applicant to carry out the present invention is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (50)

1. A method for producing a macrobolite or macrosphere of secretory agarose-collagen cells coated with agarose, characterized in that it comprises: (a) dispersing secretory cells in a solution containing collagen, (b) adding agarose to the secretory cells dispersed from the passage (a) to form secretory cells dispersed in a mixture of agarose and collagen, (c) to form a semisolid collagen-agarose macrobolite from the secretory cells dispersed from step (b), (d) to treat the semisolid collagen macrobolite. agarose from step (c) to polymerize the collagen contained in this semisolid macrobolite, whereby a solid collagen-agarose macrobolite is formed, (e) coating the solid macrobolite from step (d) with agarose to obtain a macrobolite of secretory cells , agarose-collagen, coated, agarose.

2. The method according to claim 1, characterized in that it comprises stirring the solid macrobolite from step (d) in 5% agarose, contacting the stirred solid macrobolite with mineral oil, and washing the stirred macrobolite to obtain the macrobolite of secretory cells. , agarose-collagen, coated with agarose.

3. The method in accordance with the claim 1, characterized in that the secretory cells are pancreatic islets.

4. The method according to claim 3, characterized in that the pancreatic islets are human pancreatic islets.

5. The method according to claim 3, characterized in that the pancreatic islets are bovine pancreatic islets.

6. The method according to claim 3, characterized in that the pancreatic islets are porcine pancreatic islets.

7. The method in accordance with the claim 3, characterized in that the macrobolite contains from about 50,000 to about 700,000 pancreatic islets.

8. A method for producing a macrobolite of secretory cells, of gel foam, coated with agarose, characterized in that: (a) dispersing secretory cells in gel foam, (b) stirring gel foam containing a secretory cells dispersed in a sphere, (c) coating the sphere with agarose to obtain a macrobolite of secretory cells, gel foam, coated with agarose.

9. The method according to claim 8, characterized in that step (c) comprises: (1) pouring agarose on the sphere surface to form a macrobolite, (2) stirring the macrobolite in 5% agarose, (3) putting in contact the stirred macrobolite produced in step (2) with mineral oil, (4) and wash the macrobolite from step (3) to form a macrobolite of gel-foam-secreting cells, coated with agarose.

10. The method according to claim 8, characterized in that the secretory cells are pancreatic islets.

11. The method according to claim 10, characterized in that the pancreatic islets are human pancreatic islets.

12. The method according to claim 10, characterized in that the pancreatic islets are bovine pancreatic islets.

13. The method according to claim 10, characterized in that the pancreatic islets are porcine pancreatic islets.

14. The method according to claim 10, characterized in that the macrobolite contains from about 50,000 to about 700,000 pancreatic islets.

15. A method for producing a macrobolite of secretory cells, agarose coated with agarose; characterized in that it comprises: (a) dispersing the secretory cells in agarose, (b) forming a macrobolite from the dispersed secretory cells of step (a), (c) incubating the macrobolite from step (b) in moist air, (d) ) coating the macrobolite from step (c) with agarose to form a macrobolite of secretory, agarose-coated, agarose cells.

16. The method according to claim 13, characterized in that step (e) comprises stirring the solid macrobolite from step (c) in 5% agarose, contacting the stirred solid macrobolite with mineral oil and washing the stirred macrobolite to form the macrobolite of agarose secreting cells, coated with agarose.

17. The method according to claim 15, characterized in that the secretory cells are pancreatic islets.

18. The method according to claim 17, characterized in that the pancreatic islets are human pancreatic islets.

19. The method according to claim 17, characterized in that the pancreatic islets are bovine pancreatic islets.

20. The method according to claim 17, characterized in that the pancreatic islets are porcine pancreatic islets.

21. The method according to claim 17, characterized in that the macrobolite contains from about 50,000 to about 700,000 pancreatic islets.

22. A macrobolite of agarose-collagen secreting cells, coated with agarose.

23. A macrobolite of agarose-collagen secreting cells, coated with agarose. according to claim 22, characterized in that the secretory cell is a pancreatic islet.

24. A macrobolite of agarose-collagen secreting cells, coated with agarose, according to claim 23, characterized in that the pancreatic islet is a human pancreatic islet.

25. A macrobolite of agarose-collagen secreting cells, coated with agarose. according to claim 23, characterized in that the pancreatic islet is a bovine pancreatic islet.

26. A macrobolite of agarose-collagen-secreting cells coated with agarose, according to claim 23, characterized in that the pancreatic islet is a porcine pancreatic islet.

27. A macrobolite of gel foam secreting cells, coated with agarose.

28. A macrobolite of gel foam secreting cells, coated with agarose according to claim 27, characterized in that the secretory cell is a pancreatic islet.

29. A macrobolite of agarose-collagen secreting cells, coated with agarose according to claim 28, characterized in that the pancreatic islet is a human pancreatic islet.

30. A macrobolite of agarose-collagen secreting cells, coated with agarose according to claim 28, characterized in that the pancreatic islet is a bovine pancreatic islet.

31. A macrobolite of agarose-collagen secretory cells coated with agarose. according to claim 28, characterized in that the pancreatic islet is a porcine pancreatic islet.

32. A macrobolite of agarose secreting cells, coated with agarose.

33. A macrobolite of agarose-secreting cells, coated with agarose according to claim 32, characterized in that the secretory cell is a pancreatic islet.

34. A macrobolite of agarose-collagen secretory cells coated with agarose. according to claim 33, characterized in that the pancreatic islet is a human pancreatic islet.

35. A macrobolite of agarose-collagen secretory cells coated with agarose. according to claim 33, characterized in that the pancreatic islet is a bovine pancreatic islet.

36. A macrobolite of agarose-collagen secretory cells coated with agarose. according to claim 33, characterized in that the pancreatic islet is a porcine pancreatic islet.

37. A method for treating a patient having a condition caused by a damaged function of the secretory cells; characterized in that it consists of: transplanting in the patient a therapeutically effective amount of macrobolites from secretory cells selected from the group consisting of agarose-coated collagen-agarose-secreting cells macrobolite; macrobolite of gel foam secreting cells, coated with agarose; and macrobolite of agarose secreting cells, coated with agarose.

38. The method according to claim 37, characterized in that the condition is insulin dependent diabetes.

39. The method according to claim 38, characterized in that the secretory cell is a pancreatic islet.

40. The method according to claim 39, characterized in that the pancreatic islet is a human pancreatic islet.

41. The method according to claim 39, characterized in that the pancreatic islet is a porcine pancreatic islet.

42. The method according to claim 39, characterized in that the pancreatic islet is a bovine pancreatic islet.

43. The method according to claim 39, characterized in that the secretory cell macrobolites are placed in the intraperitoneal cavity

44. The method according to claim 39, characterized in that about 5 to about 10 macrobolite are inserted, each macrobolite containing from about 50,000 to about 700,000 pancreatic islets.

45. The method according to claim 39, characterized in that the secretory cell macrobolites are macrobolites containing agarose-coated secretory agarose-collagen cells.

46. The method according to claim 39, characterized in that the secretory cell macrobolites are macrobolites of gel foam secreting cells, coated with agarose.

47. The method according to claim 39, characterized in that the macrobolites of secretory cells are macrobolite of agarose-secreting cells, coated with agarose.

48. A method for preserving secretory cells, characterized in that it comprises: (a) forming macrobolite selected from the group consisting of agarose-coated collagen-agarose secreting cell macrobolites; macrobolites of secretory cells, of gel foam coated with agarose; and macrobolites of secretory, agarose cells, coated with agarcsa; and (b) incubating the macrobolites of secretory cells.

49. The method according to claim 48, characterized in that the secretory cell is a pancreatic islet.

50. The method according to claim 49, characterized in that the pancreatic islet is incubated at 24 ° C or 37 ° C.