Biological agents such as secretory cells are encapsulated in a hydrophilic gel made of agarose or collagen-agarose and gelatin sponge-agarose combinations. In a preferred embodiment, semi-solid beads are formed from a suspension containing collagen, agarose and secretory cells such as pancreatic islets, the collagen is polymerized to form solid, agarose-collagen beads and the solid beads are coated with agarose. Coating is preferably by rolling the solid beads in about 5-10% agarose, contacting the rolled beads with mineral oil and washing oil from the beads. Beads containing secretory cells can be transplanted into a mammal to treat a condition caused by impaired secretory cell function.
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1. A method for preparing an agarose coated, solid agarose-collagen bead which contains secretory cells, comprising:
(a) suspending secretory cells in a collagen containing solution, (b) adding agarose to said solution, (c) forming a semi-solid bead of said collagen, agarose, and secretory cells, (d) polymerizing collagen in said semisolid bead to form a solid, agarose-collagen bead containing secretory cells, and (e) coating said solid, agarose-collagen bead containing secretory cells with agarose.
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11. Method for treating a mammal having a condition caused by impaired secretory cell function, comprising:
transplanting into said mammal a therapeutically effective amount of the agarose coated, solid agarose-collagen bead of
12. The method of
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This application is a
The present invention relates to macroencapsulation of biological agents, and preferably, secretory cells in a hydrophilic gel material, therapeutic methods employing the macroencapsulated biological agents, and preferably, secretory cells, and preserving the biological agents, preferably secretory cells by macroencapsulation. The hydrophilic gel material comprises agarose, and combinations of collagen-agarose and gelatin sponge-agarose. Gelatin sponge will hereinafter be referred to as gelfoam.
The term biological agent denotes a living organism and its products, e.g. proteins, enzymes, hormones, polypeptides, serum, antibodies, and antibiotics and also genetically engineered cells. Biological agents include enzymes, e.g., glucose oxidase, lactase complex, microorganisms, e.g., Klebsiella aerogenes for removal of ammonia and urea, trophic agents, including recombinantly produced trophic agents, e.g. recombinantly produced growth hormone, and cytotoxic agents.
The term secretory cell includes a pancreatic islet, although technically, a pancreatic islet is not a secretory cell, but mostly a cluster of secretory cells scattered throughout the pancreas and comprising its endocrine potion. In humans, they are composed of at least four different types of secretory cells: alpha cells which secrete the hyperglycemic factor, glucagon; beta cells which are the most abundant (70%-80%) and secrete insulin; delta cells which secrete somatostatin, and polypeptide cells which secrete polypeptide hormone.
As explained previously, transplanted material must be compatible with the host. Agarose has a long history of use in biological research, and its quality is well-controlled. 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. Gelfoam is non-immunogenic and has been used extensively in surgical procedures. It is also well-tolerated by secretory cells.
The biological agents, and preferably, secretory cells, are first isolated using procedures well known in the art. In a preferred embodiment, pancreatic islets are cultured at either 4°C C., 24°C C., or at 37°C C. before they are macroencapsulated. This method allows one to select only surviving islets after the isolation trauma. Also, the islets become less immunogenic resulting in the protection of macrobeads form fibrosis.
In one embodiment of the invention, a biological agent, preferably pancreatic islets, and more preferably about 50,000-700,000 pancreatic islets, are suspended in an aqueous solution of collagen, preferably about 0.5%-2% atello-collagen solution. Atellocollagen is obtained by treating collagen with pepsin, which removes antigenic telopeptides, responsible for intermolecular cross linkage of collagen. About 0.5%-5% of agarose, preferably about 1%, is then added to the suspended pancreatic islets to form pancreatic islets suspended in a mixture of collagen and agarose. The mixture containing the pancreatic islets is then transformed into a semisolid bead using techniques well known in the art, preferably by dropping the mixture onto mineral all or a Teflon® sheet. The semisolid bead is then transferred to an antibiotic medium, washed, and then incubated under standard conditions to polymerize the collagen, preferably at 37°C C. in a humidified 5% CO2 atmosphere, whereby a solid collagen-agarose macrobead is formed.
In another embodiment of the invention, a biological agent, preferably pancreatic islets, and more preferably about 50,00-700,000 pancreatic islets, are spread onto the surface (3-5 cm) of a gelatin sponge. The gelatin sponge is then rolled into a sphere. Agarose, 3%-5% is poured onto the sphere to form a bead.
In yet another embodiment of the invention, a biological agents, preferably pancreatic islets, and more preferably about 50,000-700,000 pancreatic islets, are placed in an agarose solution ranging from about 0.5%-5% agarose, preferably about 1% agarose. The mixture is then transformed into a macrobead by contacting the mixture to mineral oil or teflon. The bead is then transferred to an antibiotic medium, washed, and incubated overnight, preferably at 37°C C. in a humidified 5% CO2 atmosphere.
In all the aforementioned embodiments, the macrobeads are uniformly coated with agarose, preferably by rolling the bead 3-4 times in a Teflon spoon containing about 500-2,000 μl of 5%-10% agarose. Similarly, the term biological agent macrobeads, as used herein, denotes macroencapsulated biological agents in the form of a bead.
The macrobeads may be used as a vehicle to deliver the biological agent to the body where the agent will perform its known function. More than one type of biological agent may be encapsulated in one bead. For example, a macrobead can contain multiple enzymes, such as hemoglobin and glucose oxidase. Such a bead can be administered to remove bilirubin. These beads can be used either for oral administration of digestive enzymes (lactase complex) or for selective removal of undesirable amino acids from the body. Encapsulation of the enzymes will also prevent the degradation of the enzyme in the tureen. Furthermore, recombinant gene products can be safely delivered using encapsulation as the medium. K. aerogenes gene, for example, can be macroencapsulated in macrobeads for urea and ammonia removal. Where the biological agent is immunogenic to the host, the macrobead allows the administration of the biological agent without the use of immunosuppressant or with decreased amounts of immunosuppressant.
The secretory macrobeads may be used to treat conditions caused by an impaired functioning of the secretory cells of the subject, e.g. insulin dependant diabetes, growth factor deficiency disorder, and hormonal disorders, by transplanting the secretory cell macrobeads into the subject. The macrobeads may be inserted into the appropriate location for that particular treatment. For example, macrobeads containing hepatocytes can be implanted into the abdominal cavity to treat diseases related to liver non-function. A preferred application is transplanting 5-10 pancreatic islet macrobeads, each containing 50,000-700,000 pancreatic islets, into a patient to treat insulin-dependant diabetes. The macrobeads can be inserted into the peritoneal cavity.
The secretory cell macrobeads are transplanted into a patient in an amount sufficient to treat the condition. An mount adequate to accomplish this is defined as a "therapeutically effective mount" or "efficacious amount". Amounts effective for this use will depend upon the severity of the condition, the general state of the patient, the route of administration, the placement of macrobeads, and whether the secretory cell macrobeads are being administered in combination with other drugs.
The secretory macrobeads can be used for allogeneic and xenogeneic transplantation in combination with immunosuppressants or preferably, without immunosuppressants. In a preferred embodiment, patients having chronic or acute insulin dependant diabetes arc treated by xenotransplantating animal pancreatic islets, e.g. porcine, bovine, marine, rat, picin, or any other suitable species into the patient without the use of immunosuppressants. The secretory cell macrobeads can also be administered in combination with other therapeutic agents, e.g. the commonly used triple drug therapy (cyclosporine, azathioprine, and hydrocortisone), rapamycin, deoxyspergualin or antibodies, to treat the condition.
The macrobeads can also be used as a means to store the biological agents, and preferably secretory cells, for extended periods of time. To maintain the viability of the biological agents, and preferably secretory cells, the biological agents, and preferably secretory cell macrobeads are incubated-until they are transplanted in the animal.
When the secretory cells are pancreatic islets, the pancreatic islet macrobeads are incubated at a temperature of 24°C C. or 37°C C.
Example I
Pancreatic Islet Isolation
Pancreatic islets were isolated from rats by a modification of the method disclosed in Gotbh et al., Transplantation 40:437 (1985).
Collagenase solution (collagenase Type XI, Sigma Chemical., St. Louis, Mo.; 1 mg/ml containing 2 mg/ml of Sigma, Type V, bovine serum albumin and 1 mg/ml CaCl2) was injected into the pancreas via the common bile duct. (Gotoh et al., Transplantation 40:437 (1985), Supra). The pancreas was removed and collected in a flask maintained on ice. Once pancreata from 4 rats had been collected, the flask was placed in a waterbath, at 38°C C., for 30 minutes. The resulting digested tissue was washed 4 times in cold (8°C C.) HBSS (Hank's Balanced Salts Solution).
Undigested tissue, large lymph nodes, and older extraneous material were removed by repeated mobilization of the tissue, followed by removal of the supernatant. Purified islets were isolated on a discontinuous Ficoll gradient, consisting of 25%, 23%, 21%, and 11% Ficoll layers, prepared in Euro-Collies solution (Frescenius A. G., Gluehen Steinweg, Hornburg V. D. H.) and centrifuged at 2000 r.p.m. for 16 minutes. The islets were collected from the interface between 11% and 21% and the interface between 21% and 23% Ficoll layers. Islets from each fraction were pooled and washed four times in HESS solution containing 10% fetal calf serum.
The pooled islets cells were then transferred to petri dishes containing RPMI complete medium, i.e., cold RPMI 1640 medium (GIBCO, Grand Island, N.Y.), supplemented with 25 mM HEPFS, heat inactivated fetal bovine serum (10%), and antibiotic-antimycotic solution (1 ml/100 ml) which contains: 100 μg/ml of penicillin, 100 μg/ml of streptomycin sulfate, and 25 μg/ml of amphotericin B. Any remaining non-islet acinar; vascular, ductular, or lymph node tissue was identified with the aid of a dissecting microscope, and carefully removed with a fine-tip sterile pipette. Final purity was assessed by staining the islet preparation with diphenylthiocarbazone.
After isolation, the islets were incubated in bacteriological plastic dishes (100 mm) containing 10 ml of RPMI medium, at 37°C C., in a humidified atmosphere having 5% CO2, for 4 days. The medium was changed every day, and the islets were then either directly transplanted or macroencapsulated.
Example II
A. Preparation of Agarose Coated, Agarose-Collagen Pancreatic islet Macrobeads
1000 pancreatic islets obtained by the method of Example I were washed four times in RPMI complete medium as described in Example I, less fetal calf serum. The pancreatic islets were then added to a tube containing 50 μl of 1% atelocollagen solution in phosphate buffered saline, to suspend the pancreatic islets, 100 μl of 1% low viscosity agarose (Sigma Type XII) solution, prepared either in RPMI or in MEM (minimal essential medium), maintained at 60°C C., was then added to the collagen-pancreatic islet suspension. The contents of the tube were then transferred immediately, as a single large drop; either onto sterilized mineral oil, maintained at room temperature, or onto a Teflon® sheet. After one minute, the drop became a semisolid macrobead which was then transferred to RPMI antibiotic medium, at 37°C C. The macrobeads were washed three times with the same medium to remove all oil. Finally, they were rinsed twice with complete medium (37°C C.) and incubated overnight, at 37°C C., in a humidified atmosphere having 5% CO2 During this period, the collagen polymerized and the pancreatic islets rested on the collagen fiber.
The next day, the solid macrobeads were transferred to a Teflon® spoon which contained approximately 1 ml of 5% agarose in RPMI or in MEM medium. The solid macrobeads were then rolled in this solution 2-3 times in order to uniformly mat them. Before the agarose solidified, the macrobeads were transferred to mineral oil in a Teflon® dish to obtain smooth-surfaced macrobeads. After 60 seconds, the macrobeads were removed from mineral oil and washed 3 times with RPMI antibiotic medium, and then two times with RPMI complete medium. They were then incubated overnight, at 37°C C., in a humidified atmosphere having 5% CO2. Agarose coated, agarose-collagen pancreatic islet macrobeads are shown in
B. Preparation of Agarose Coated, Gelatin Sponge Pancreatic Islet Macrobeads
A small piece of gelatin sponge (gelfoam), 3 mm2 was first soaked in RPMI complete medium. The medium was squeezed out and the gelfoam was allowed to rest for 1 minute. One thousand pancreatic islets, prepared according to Example I, were washed four times with RPMI antibiotic medium. They were then suspended in 10 μl of RPMI antibiotic medium. They were transferred by a fine-tipped plastic pipette and spread onto the surface of the gelfoam. After 20 seconds, the gelfoam was rolled into a small sphere. 50 μl of 5% agarose was poured onto the surface of the sphere to create an pancreatic islet macrobead.
In order to uniformly cover the macrobead with 5% agarose, 500 μl of 5% agarose was added to the macrobead in a Teflon® spoon and was rolled 3-4 times. Before the agarose solidified, the macrobead was transferred to mineral oil, and the dish was rotated to obtain a smooth surface on the macrobead. The macrobead was washed 3-4 times in RPMI antibiotic medium and then rinsed 2 times with RPMI complete medium. It was incubated overnight before being used for transplantation.
C. Preparation of Agarose Coated, Agarose Pancreatic Islet Macrobeads
One thousand pancreatic islets obtained by the method of Example I were first washed 4 times in RPMI antibiotic medium. The pancreatic islets were transferred to a tube containing 50 μl RPMI antibiotic medium and suspended thereon. 100 μl of 1% agarose solution was then added to the tube. The entire contents of the tube was immediately transferred, as a single large drop, to either sterilized mineral oil or a teflon sheet. After 1 minute, the drop solidified to a macrobead, The macrobead was transferred to RPMI antibiotic medium, maintained at 37°C C. The oil was then removed by washing the macrobead 3 times with the same medium, and then by rinsing 2 times with RPMI complete medium. The beads were incubated overnight at 37°C C. in a humidified atmosphere having 5% CO2.
The next day, these beads were transferred onto a Teflon® spoon containing 1 ml of 5% agarose in either RPMI or in MEM medium. To uniformly coat the macrobeads with agarose, the beads were then gently rolled in agarose 2-3 times. They were then transferred to mineral oil, in a teflon dish, before the agarose solidified. After 60 seconds, the beads were removed from the mineral oil and washed 3 times in RPMI antibiotic medium and 2 times in RPMI complete medium. The beads were then incubated overnight.
Example III--Transplantation of the Pancreatic Islet Macrobeads Into Mice
A. Recipient Mice & Donor Rats
The mice used were male C57BL/6 and BALB/c stains. Recipient mice were made diabetic by a single i.v. injection of streptozotocin (170-200 mg/kg).
Non-fasting plasma glucose levels were determined before the induction of diabetes. All blood sugar levels in the recipient mice were monitored via tail vein blood samples with an ExacTech Pen Sensor. Only those mice with serum glucose level >400 mg/dl on the day of transplantation were used.
Wistar Furth rats were used as donors for xenotransplantation.
B. Xenotransplantation of Pancreatic islet Macrobeads Into the Peritoneal Cavity
At the time of xenotransplantation, pancreatic islet macrobeads of Example II(A), II(B), and II(C), respectively, were transferred gently to separate plates containing RPMI antibiotic medium. To remove all serum proteins, the medium was changed three times. Diabetic recipient mice were anesthetized with avertin. A midline incision was made to introduce a single pancreatic islet macrobead into the free peritoneal cavity. A two-layer closure of the incision was done with an absorbent suture. Control mice received either an empty macrobead i.p. (intrapefitoneally), free pancreatic islets i.p., or an empty macrobead together with free donor pancreatic islets.
After transplantation, each recipient's blood glucose was checked daily or every other day until it reached the normal range; thereafter blood glucose was checked only 2-3 times every week. Transplants were considered technically successful if the serum glucose was <200 mg/dl and remained there for consecutive bleedings. A transplant was considered to have been rejected if the serum glucose concentration rose above 200 mg/dl after a period of transient normoglycemia. Transplants were considered to have failed or to have become `primary nonfunctional` if the blood glucose never became normal (i.e., consistently remained 200 mg/dl).
C. Intraperitoneal Glucose Tolerance Test
Approximately 70-84 days post implantation, glucose tolerance tests were performed. Glucose solution (1.0 g/kg body weight) was intraperitoneally injected into mice who had been fasting for 6 hours (9 am-3 pm). 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 on normal C57BL/6 and BALB/c mice, on streptozotocin induced C57BL/6 and BALB/c mice in which no pancreatic islets had been transplanted, and on streptozocin induced diabetic BALB/c mice in which free pancreatic islets had been transplanted into the kidney capsule ("KCT" mice).
Control experiments were conducted to ensure that the euglycemic state in diabetic mice was being achieved via the macroencapsulated pancreatic islets and not the macrobeads themselves. Empty agarose coated, agarose-collagen macrobeads and agarose coated, gelfoam macrobeads were, therefore, prepared in the same manner as the beads of Examples II(A) and (B).
D. Results of the Intraperitoneal Xenotransplantation and Glucose Tolerance Test
Upon implantation of pancreatic islet macrobeads, the changes observed in the non-fasting plasma glucose level of STZ-diabetic streptozotocin induced C57BL/6 mice are shown in
When free pancreatic islets were transplanted intraperitoneally, 6 of 7 transplanted animals became normoglycemic 1 day after transplantation; however, they maintained this state for only 3-10 days (FIG. 6). When free pancreatic islets were transplanted with empty beads made of agarose coated, agarose-collagen macrobeads or agarose coated, gelfoam macrobeads, all the animals became normoglycemic within 24 hours and remained so for more than 2 days (FIG. 7). Subsequently, all animals became hyperglycemic. Animals which contained empty macrobeads excited no tissue reaction for the 90 days they were followed.
The results obtained after performing the Glucose Tolerance Tests are presented in FIG. 8. In normal BALB/c and C57BL/6 mice and "KCT" mice, plasma glucose peaked at 30 minutes and returned to baseline levels by 120 minutes.
Similar results were obtained when macroencapsulated pancreatic islets and non-encapsulated pancreatic islets transplanted in the kidney capsule were tested.
The results of these experiments demonstrate that the agarose coated, agarose-collagen islet macrobeads; agarose coated, agarose-gelfoam pancreatic islet macrobeads; and agarose coated, agarose islet macrobeads display the properties required for a hybrid artificial organ. Although all three types successfully secrete insulin, agarose coated, agarose-collagen and agarose coated, agarose-gelfoam macrobeads are mare suitable as biohybrid artificial organs due to the uniformity of results obtained in the minimum number of transplanted animals. Moreover, all the three types of beads showed no adverse effects. The macrobeads remained free is the peritoneum showing neither tissue reaction, nor any adhesion to any organ. Thus, these biohybrid pancreatic islets perform their function as efficiently in the macroencapsulated beads as in their natural habitat, the pancreas.
In all of the mice, plasma glucose peaked at 30 minutes and returned to baseline levels by 120 minutes.
Example IV
Extended Storage Life of Pancreatic islets
Macroencapsulated beads prepared according to Examples I(A), (B), and (C), which were incubated for 4 weeks at 37C in complete RPMI medium, were tested for their long-term preservation properties in vivo and in vitro. It was found that the macroencapsulated pancreatic islets which were incubated for 4 weeks were functionally similar to those which were incubated for 1 day.
This example demonstrates that the method of macroencapsulation according to the present invention can be used for secretory cell preservation, and preferably, pancreatic islet preservation.
Jain, Kanti, Rubin, Albert L., Smith, Barry
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