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Preparation of agarose coated, solid agarose-collagen beads containing secretory cells

a technology of agarose and agarose gel, which is applied in the field of macroencapsulation of secretory cells, can solve the problems of inability to achieve effective long-term methods, limited clinical applications of pancreatic islet transplantation, and inability to achieve immunological defenses

Inactive Publication Date: 2003-03-11
THE ROGOSIN INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

It is therefore an object of the present invention to provide a secretory cell macrobead that can be transplanted into animals to treat conditions caused by an unpaired functioning of the host's secretory cells.
It is a further object of this invention to provide a secretory cell macrobead that can be stored for long lengths of time.

Problems solved by technology

The clinical applications of pancreatic islet transplantation have been limited by the inability to prevent islet allograft-xenograft rejection, i.e., a rejection of the transplanted pancreatic islets due to the host's immune system attacking the transplanted pancreatic islets.
Immunosuppressive therapy, however, has proved to be a double-edged sword; while reducing the risk of rejection, it impairs the body's overall, immunological defenses.
As discussed below, although temporary success has been reported (See hey, Diabetes Reviews 1 (1):76 (1993), effective long-term methods have yet to be achieved.
All of these methods have failed, however, due to one or more of the following problems; a host fibrotic response to the implant material, instability of the implant material, limited nutrient diffusion across semi-permeable membranes, secretagogue and product permeability, and diffusion lag-time across semi-permeable membrane barriers.
Their method, however, suffers from a number of drawbacks.
It is cumbersome and inaccurate for example, many beads remain partially coated and several hundred beads of empty agarose form.
Furthermore, the transplanted beads arc difficult to retrieve, tend to be fragile, and will easily release islets upon slight damage.
They found that when the islets are injected into the fiber, they aggregate within the hollow tube and result in necrosis in the central portion of the islet masses.
However, this experiment has not been extensively repeated.
Therefore, the membrane's function as an islet transplantation medium in humans is questionable.

Method used

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  • Preparation of agarose coated, solid agarose-collagen beads containing secretory cells
  • Preparation of agarose coated, solid agarose-collagen beads containing secretory cells
  • Preparation of agarose coated, solid agarose-collagen beads containing secretory cells

Examples

Experimental program
Comparison scheme
Effect test

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 CaCl.sub.2) 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.degree. C., for 30 minutes. The resulting digested tissue was washed 4 times in cold (8.degree. 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% Fi...

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 .mu.l of 1% atelocollagen solution in phosphate buffered saline, to suspend the pancreatic islets, 100 .mu.l of 1% low viscosity agarose (Sigma Type XII) solution, prepared either in RPMI or in MEM (minimal essential medium), maintained at 60.degree. 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.RTM. sheet. After one minute, the drop became a semisolid macrobead which was then transferred to RPMI antibiotic medium, at 37.degree. C. The macrobeads were washed three times with the same medium to remove all oil....

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 in...

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Abstract

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.

Description

FIELD OF THE INVENTIONThe present invention relates to macroencapsulation of secretory cells in a hydrophilic gel material, therapeutic methods employing the macroencapsulated secretory cells, and preserving the secretory cells by macroencapsulation.BACKGROUND OF THE INVENTIONSecretory cells are cells that are characterized by secreting biological products, such as, but not limited to, hormones (e.g., insulin), growth factors, cytokines, and so forth. Their role in biological processes is well known, and need not be set forth here. A number of diseases and pathological conditions are related to a failure of the secretory cells to work properly, such as a deficient production of the secretory products, e.g. hypothyroidism and cretin dwarfism, both due to thyroid hormone deficiency, hypophysial dwarfism due to pituitary growth hormone deficiency, Lesch-Hyhan Syndrome due to hypozanthine-guanine phosphoribosyltransferase deficiency, fulminant hepatic failure due to the hepatotrophic fa...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K35/39A61K35/37A61K9/16A61K9/50C12N11/04C12N5/00C12N11/00C12N5/06A61K35/12A61K38/00A61K35/28A61K47/26A61K47/36A61P3/10C12N5/07C12N5/071C12N11/10
CPCA61K9/1652A61K9/1658A61K9/5036A61K35/39C12N5/0012C12N5/0677C12N11/04A61K38/00C12N2533/76C12N2533/54A61K2035/126A61P5/00A61P3/10A61K47/26
Inventor JAIN, KANTIRUBIN, ALBERT L.SMITH, BARRY
Owner THE ROGOSIN INST
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