U.S. patent number RE40,555 [Application Number 10/979,527] was granted by the patent office on 2008-10-28 for preparation of agarose coated, solid agarose beads containing secretory cells.
This patent grant is currently assigned to The Rogosin Institute. Invention is credited to Kanti Jain, Albert L. Rubin, Barry Smith.
United States Patent |
RE40,555 |
Jain , et al. |
October 28, 2008 |
Preparation of agarose coated, solid agarose beads containing
secretory cells
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.
Inventors: |
Jain; Kanti (New York, NY),
Rubin; Albert L. (Englewood, NJ), Smith; Barry (New
York, NY) |
Assignee: |
The Rogosin Institute (New
York, NY)
|
Family
ID: |
22663563 |
Appl.
No.: |
10/979,527 |
Filed: |
November 1, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
10336442 |
Jan 2, 2003 |
Re. 39542 |
|
|
|
09345196 |
Jun 30, 1999 |
Re. 38027 |
|
|
|
08181269 |
Jan 13, 1994 |
|
|
|
Reissue of: |
08483728 |
Jun 7, 1995 |
05643569 |
Jul 1, 1997 |
|
|
Current U.S.
Class: |
424/93.7;
435/395; 435/356; 435/397; 435/382; 435/182; 435/178; 435/177 |
Current CPC
Class: |
C12N
11/04 (20130101); A61K 9/5036 (20130101); C12N
5/0012 (20130101); A61K 9/1652 (20130101); A61P
3/10 (20180101); A61P 5/00 (20180101); A61K
9/1658 (20130101); A61K 35/39 (20130101); C12N
5/0677 (20130101); A61K 38/00 (20130101); C12N
2533/76 (20130101); C12N 2533/54 (20130101); A61K
2035/126 (20130101) |
Current International
Class: |
A61K
35/12 (20060101); C12N 11/10 (20060101); C12N
11/02 (20060101); C12N 5/00 (20060101); C12N
5/08 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Brodelius, et al., FEBS Letters; 122(2): 312-316 (1980). cited by
examiner .
Iwata, et al., Journal of Biomedical Materials Research, vol. 26,
1992, pp. 967-977. cited by examiner .
Brodelius, et al., FEBS Letters, vol. 122, No. 1980, pp. 312-316.
cited by examiner.
|
Primary Examiner: Naff; David M
Attorney, Agent or Firm: Fulbright & Jaworski LLP
Parent Case Text
.Iadd.More than one reissue application has been filed for the
reissue of U.S. Pat. No. 5,643,569. .Iaddend.This application is a
.Iadd.divisional of reissue application Ser. No. 10/336,442, filed
Jan. 2, 2003, which is a divisional of reissue application Ser. No.
09/345,196, filed Jun. 30, 1999, now U.S. Reissue Pat. No. RE
38,027 E, which is a reissue of application Ser. No. 08/483,728,
filed Jun. 7, 1995, now U.S. Pat. No. 5,643,569, which is a
.Iaddend.continuation of application Ser. No. 08/181,269 filed Jan.
13, 1994, now abandoned.
Claims
What is claimed is:
.[.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..].
.[.2. The method of claim 1, wherein said secretory cells are
contained in pancreatic islets..].
.[.3. The method of claim 2, wherein said pancreatic islets are
human pancreatic islets, bovine pancreatic islets, rat pancreatic
islets or porcine pancreatic islets..].
.[.4. The method of claim 3, wherein said pancreatic islets are
human pancreatic islets..].
.[.5. The method of claim 1, wherein said secretory cells are
contained in from about 50,000 to about 700,000 pancreatic
islets..].
.[.6. An agarose coated, solid agarose-collagen bead containing
secretory cells prepared by the process of claim 1..].
.[.7. The agarose coated, solid agarose-collagen bead of claim 6,
wherein said secretory cells are contained in pancreatic
islets..].
.[.8. The agarose coated, solid agarose-collagen bead of claim 6
wherein said pancreatic islets are human pancreatic islets, bovine
pancreatic islets, rat pancreatic islets or porcine pancreatic
islets..].
.[.9. The agarose coated, solid agarose-collagen bead of claim 8
wherein said pancreatic islets are human pancreatic islets..].
.[.10. The agarose coated, solid agarose collagen bead of claim 7,
comprising from about 50,000 to 700,000 islets..].
.[.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 claim 6..].
.[.12. The method of claim 11, wherein said condition is insulin
dependent diabetes..].
.[.13. The method of claim 12, wherein said bead contains cells
derived from a pancreatic islet..].
.[.14. The method of claim 13, wherein said pancreatic islet is a
human pancreatic islet..].
.[.15. The method of claim 13, wherein said pancreatic islet is a
bovine pancreatic islet..].
.[.16. The method of claim 13, wherein said pancreatic islet is a
porcine pancreatic islet..].
.[.17. The method of claim 11, wherein said bead is placed in the
peritoneal cavity of said mammal..].
.Iadd.18. A composition of matter comprising a sample of secretory
cells in a bead which comprises agarose, wherein said bead is
coated with a solution comprising from 5% to 10% agarose.
.Iaddend.
.Iadd.19. The composition of matter of claim 18, wherein said
secretory cells comprise pancreatic islet cells or pancreatic
islets. .Iaddend.
.Iadd.20. The composition of matter of claim 19, wherein said
pancreatic islet cells or pancreatic islets comprise human,
porcine, or bovine pancreatic islet cells or pancreatic islets.
.Iaddend.
.Iadd.21. The composition of matter of claim 19, comprising 1000
pancreatic islets. .Iaddend.
.Iadd.22. The composition of matter of claim 18, wherein said bead
is coated with a solution containing 5% agarose. .Iaddend.
.Iadd.23. A composition of matter comprising a sample of secretory
cells entrapped in a bead which comprises from 0.5% to 5% agarose,
wherein said bead is coated with a coating of agarose.
.Iaddend.
.Iadd.24. The composition of matter of claim 23, wherein said
secretory cells comprise pancreatic islet cells or pancreatic
islets. .Iaddend.
.Iadd.25. The composition of matter of claim 24, wherein said
pancreatic islet cells or pancreatic islets are human, bovine, or
porcine pancreatic islet cells or pancreatic islets. .Iaddend.
.Iadd.26. The composition of matter of claim 23, wherein said Bead
comprises about 1% agarose. .Iaddend.
.Iadd.27. The composition of matter of claim 23, wherein said
Coating comprises about 5% agarose. .Iaddend.
.Iadd.28. The composition of matter of claim 24 comprising 1000
pancreatic islets. .Iaddend.
.Iadd.29. A method for treating a subject having a condition caused
by impaired functioning of secretory cells, comprising
transplanting an amount of the composition of matter of claim 18
sufficient to alleviate said condition to said subject.
.Iaddend.
.Iadd.30. The method of claim 29, wherein said condition is
diabetes and said secretory cells comprise pancreatic islets or
pancreatic islet cells. .Iaddend.
.Iadd.31. A method for treating a subject having a condition caused
by impaired functioning of secretory cells, comprising
transplanting an amount of the composition of matter of claim 23
sufficient to alleviate said condition to said subject.
.Iaddend.
.Iadd.32. The method of claim 31, wherein said condition is Insulin
dependent diabetes and said secretory cells comprise pancreatic
islet cells or pancreatic islets. .Iaddend.
.Iadd.33. The method of claim 32, wherein said pancreatic islet
cells or pancreatic islets are human, bovine, or porcine pancreatic
islet cells or pancreatic islets. .Iaddend.
.Iadd.34. The method of claim 31, comprising transplanting said
amount of the composition of matter to the intraperitoneal cavity
of said subject. .Iaddend.
.Iadd.35. A composition of matter produced by combining a sample of
secretory cells and a solution which contains from 0.5% to 5%
agarose to form a suspension, treating said suspension to form a
bead, and coating said bead with agarose. .Iaddend.
.Iadd.36. The composition of matter of claim 35, wherein said
secretory cells comprise pancreatic islet cells or pancreatic
islets. .Iaddend.
.Iadd.37. The composition of matter of claim 36, wherein said
pancreatic islet cells or pancreatic islets are human, bovine or
porcine pancreatic islet cells or pancreatic islets. .Iaddend.
.Iadd.38. The composition of matter of claim 35, wherein said
solution contains about 1% agarose. .Iaddend.
.Iadd.39. The composition of matter of claim 35, wherein said bead
is coated with a solution containing about 5% agarose.
.Iaddend.
.Iadd.40. The composition of matter of claim 35, comprising 1000
pancreatic islets. .Iaddend.
.Iadd.41. The composition of matter of claim 35, wherein said
coating comprises from about 5% to 10% agarose. .Iaddend.
.Iadd.42. A composition of matter comprising a sample of secretory
cells contained in a bead which comprises agarose and collagen,
wherein said bead is coated with a coating of agarose.
.Iaddend.
.Iadd.43. The composition of matter of claim 42, wherein said
secretory cells comprise pancreatic islet cells or pancreatic
islets. .Iaddend.
.Iadd.44. The composition of matter of claim 42, wherein said
pancreatic islet cells or pancreatic islets comprise human,
porcine, or bovine pancreatic islet cells or pancreatic islets.
.Iaddend.
.Iadd.45. The composition of matter of claim 42, comprising 1000
pancreatic islets. .Iaddend.
.Iadd.46. The composition of matter of claim 42, wherein said bead
is coated with a solution comprising from 5% to 10% agarose.
.Iaddend.
.Iadd.47. A method for treating a subject having a condition caused
by impaired functioning of secretory cells, comprising
transplanting an amount of the composition of matter of claim 42
sufficient to alleviate said condition to said subject.
.Iaddend.
.Iadd.48. The method of claim 47, wherein said condition is
diabetes and said secretory cells comprise pancreatic islets or
pancreatic islet cells. .Iaddend.
.Iadd.49. A method for preserving secretory cells, comprising: (a)
forming an agarose coated, agarose containing bead, or an agarose
coated bead containing both agarose and collagen, with secretory
cells entrapped therein, and, (b) maintaining the secretory cell
containing bead to preserve the secretory cells therein. .Iaddend.
Description
FIELD OF THE INVENTION
The 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 INVENTION
Secretory 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 hypoxanthine-guanine
phosphoribosyltransferase deficiency, fulminant hepatic failure due
to the hepatotrophic factor deficiency, extracellular matrix
disease due to chrondrocyte deficiency, and insulin dependent
diabetes due to insulin deficiency.
One approach to treat such conditions is to transplant the
secretory cells into the patient. The transplanted material, in
order to be clinically safe and effective, must (1) be
non-immunogenic, non-thrombogenic, bio-stable, and completely
non-toxic to cells and tissues of the host, (2) maintain cell
viability for an extended period of time, (3) permit free passage
of nutrients, secretagogues (a substance that stimulates
secretion), and cell products, (4) facilitate surgical implantation
and cell reseeding, and (5) be easily fixed in place and, likewise,
removed.
Pancreatic islet transplantation to treat insulin-dependant
diabetes has been the subject of renewed interest due to
technological advances in isolating islets of Langerhans. By way of
background, the human pancreas contains islets of Langerhans
(hereinafter "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
a single endocrine organ occupying around 1% of the volume of the
pancreas. Within the pancreas, small islets (up to 160 .mu.m
diameter) tend to distribute throughout the exocrine tissue. These
small islets represent 75% of the islets in number but only about
15% in volume. Islets greater than 250 .mu.m diameter constitute
only 15% of the total number of islets but 60% of the volume. These
islets are localized near larger ducts and blood vessels, and are
not surrounded by acinar tissue. A human pancreas may 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 mantle of
glucagon containing alpha cells (A-cells), somatostatin secreting
delta cells (D-cells) and pancreatic polypeptide containing cells
(PP-cells). Insulin producing B-cells makeup the majority of the
cells, and comprise up to about 80% of the islets in a human.
The clinical application 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. To counteract the rejection, the pancreatic
islets have been transplanted in combination with the
administration of immunosuppressive agents.
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. Various methods of protecting the
transplanted tissue from the host immune response have been
explored by many investigators. 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.
The five major approaches to protecting the transplanted tissue
from the host's immune response all involve attempts to isolate the
transplanted tissue from the host's immune system. The
immunoisolation techniques used to date include: extravascular
diffusion chambers, intravascular diffusion chambers, intravascular
ultrafiltration chambers, microencapsulation, and
macroencapsulation. All of these methods have failed, however, due
to one or more of the following problems; a host fibroptic 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.
For example, a microencapsulation procedure for enclosing viable
cells, tissues, and other labile biological membranes within a
semipermeable membrane was developed by Lira in 1978. (Lim,
Research report to Damon Corporation (1978)). Lim used
microcapsules of alginate and poly L-lysine to encapsulate the
islets of Langerhans. In 1980, the first successful in vivo
application of this novel technique in diabetes research was
reported ((Lim, et al., Science 210:908 (1980)). The implantation
of these microencapsulated islets of Langerhans resulted in
sustaining a euglycemic state in diabetic animals. Other
investigators, however, repeating these experiments, found the
alginate to cause a tissue reaction and were unable to reproduce
Lira et al's results (Lamberti, et al., Applied Biochemistry and
Biotechnology 10:101 (1984); Dupuy, et al., Jour. Biomed. Material
and Res. 22:1061 (1988); Weber, et al., Transplantation 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. Jour. Biomedical Material
and Res. 26:967 (1992)) utilized agarose for microencapsulation of
allogeneic pancreatic islets and discovered that it could be used
as a medium for the preparation of microbeads. In their study,
1500-2000 islets were microencapsulated individually in 5% agarose
and implanted into streptozotocin-induced diabetic mice. The graft
survived for a long period of time, and the recipients maintained
normoglycemia indefinitely.
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. Additional
time is thus required to separate encapsulated islets from empty
beads. Moreover, most of the implanted microbeads gather in the
pelvic cavity, and a large number of islets are required in
completely coated individual beads to achieve normoglycemia.
Furthermore, the transplanted beads are difficult to retrieve, tend
to be fragile, and will easily release islets upon slight
damage.
A macroencapsulation procedure has also been tested. Macrocapsules
of various different materials, such as
poly-2-hydroxyethyl-methacrylate, poly vinylchloride-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
transitory normalization of glycemia 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 dispersed
neonatal murine pancreatic grafts in hollow fibers which were
transplanted into diabetic hamsters. Recently hey et al., Science
254:1782-1784 (1991) confirmed their results, but found the
euglycemic state to be a transient phase. 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. The central necrosis precluded prolongation 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
extensively repeated. Therefore, the membrane's function as an
islet transplantation medium in humans is questionable.
Thus, there exists a need for achieving secretory cell
transplantation, and in particular, pancreatic islet allograft and
xenograft survival without the use of chronic immunosuppressive
agents.
The inventors have surprisingly discovered that macroencapsulating
secretory cells in a hydrophillic gel material results in a
functional, non-immunogenic, macrobead that can be transplanted
into animals and can be stored for long lengths of time. The
macroencapsulation of the secretory cells of the present invention
provides a more effective and manageable technique for secretory
cell transplantation. The macroencapsulation technique can also be
used to macroencapsulate other biological agents, such as enzymes,
microorganisms, trophic agents including recombinantly produced
trophic agents, cytotoxic agents, and chemotherapeutic agents. The
macroencapsulated biological agents can be administered to treat
conditions known to respond to the biological agent.
SUMMARY OF THE INVENTION
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 impaired 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.
In accomplishing these and other objects, there has been provided,
in accordance with one aspect of the present invention a method of
producing an agarose coated, agarose-collagen secretory cell
macrobead; an agarose coated, gel-foam secretory cell macrobead;
and a agarose coated, agarose secretory cell macrobead.
In another aspect of the invention, there is provided a method of
treating a patient having a condition characterized by an
insufficiency in a secretory cell product, comprising transplanting
into said patient a therapeutically effective amount of secretory
cell macrobeads selected from the group consisting of agarose
coated, agarose-collagen secretory cell macrobeads; agarose coated,
gelfoam secretory cell macrobeads; and agarose coated, agarose
secretory cell macrobeads.
In yet a further aspect of the invention, there is provided a
method for preserving secretory cells, comprising forming
macrobeads selected from the group consisting of agarose coated,
agarose-collagen secretory cell macrobeads; agarose coated, gelfoam
secretory cell macrobeads; and agarose coated, agarose secretory
cell macrobeads; and incubating said secretory cell macrobeads.
Other objects, features and advantages of the present invention
will become apparent from the following detailed description. It
should be understood, however, that the detailed description and
the 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 to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1 and 2 show agarose coated, collagen-agarose pancreatic
islet macrobeads.
FIG. 3 shows the glucose levels of diabetic mice transplanted with
agarose coated, collagen-agarose pancreatic islet macrobeads.
FIG. 4 shows the glucose levels of diabetic mice transplanted with
agarose coated, gelfoam pancreatic islet macrobeads.
FIG. 5 shows the glucose levels of diabetic mice transplanted with
agarose coated, agarose macrobeads.
FIG. 6 shows the glucose levels of diabetic mice transplanted with
free pancreatic islets.
FIG. 7 shows the glucose levels of diabetic mice transplanted with
agarose coated, collagen-agarose macrobeads and free pancreatic
islets.
FIG. 8 demonstrates a glucose tolerance test for normal mice,
streptozotocin-induced diabetic mice, streptozotocin-induced
diabetic mice receiving allotransplant in the kidney capsule "(KCT
mice)", and streptozotocin-induced diabetic mice receiving agarose
coated, agarose-collagen pancreatic islet macrobeads; agarose
coated, gelfoam pancreatic islet macrobeads; and agarose coated,
agarose pancreatic islet macrobeads (collectively referred to as
"bead mice").
DETAILED DESCRIPTION OF THE INVENTION
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 hose. 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.degree. C.,
24.degree. C., or at 37.degree. 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% atellocollagen 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 technique well known in the art,
preferably by dropping the mixture onto mineral oil or a
Teflon.RTM. 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.degree. C.
in a humidified 5% CO.sub.2 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
50,000-700,000 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.degree. C. in a humidified 5% CO.sub.2 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 .mu.l 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 in 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 xenogenic
transplantation in combination with immunosuppressants or
preferably, without immunosuppressants. In a preferred embodiment,
patients having chronic or acute insulin dependent diabetes are
treated by xenotransplantating animal pancreatic islets, e.g.
porcine, bovine, murine, 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.degree. C. or
37.degree. C.
EXAMPLES
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 other 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-Collins 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 HBSS 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 HEPES,
heta-inactivated fetal bovine serum (10%), and
antibiotic-antimycotic solution (1 ml/100 ml) which contains: 100
.mu.g/ml of penicillin, 100 .mu.g/ml of streptomycin sulfate, and
25 .mu.g/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.degree. C., in a humidified atmosphere having 5% CO.sub.2, 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 .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 semi-solid
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. Finally, they were rinsed twice with
complete medium (37.degree. C.) and incubated overnight, at
37.degree. C., in a humidified atmosphere having 5% CO.sub.2.
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.RTM. 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.RTM. dish to obtain smooth-surface 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.degree.
C., in a humidified atmosphere having 5% CO.sub.2, Agarose coated,
agarose-collagen pancreatic islet macrobeads are shown in FIGS. 1
& 2.
B. Preparation of Agarose Coated, Gelatin Sponge Pancreatic Islet
Macrobeads
A small piece of gelatin sponge (gelfoam), 3 mm.sup.2 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 .mu.l
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 .mu.l 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
.mu.l of 5% agarose was added to the macrobead in a Teflon.RTM.
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 .mu.l RPMI
antibiotic medium and suspended thereon. 100 .mu.l 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.degree. 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.degree. C. in a humidified atmosphere
having 5% CO.sub.2.
The next day, these beads were transferred onto a Teflon.RTM. 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 have 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 FIGS. 3 & 4.
The recipients of agarose coated, agarose-collagen pancreatic islet
macrobeads and agarose coated, gelfoam pancreatic islet macrobeads
maintained a normoglycemic state for more than 60 days and, during
this period, the body weight of these mice increased an average of
3 grams. When agarose covered, agarose pancreatic islet macrobeads
were transplanted, 2 of 6 animals became normoglycemic after 21-33
days post transplantation (FIG. 5) and remained euglycemic
thereafter. All other transplanted animals failed to achieve a
euglycemic state. Empty macrobeads (n=6) did not affect blood
glucose.
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 12 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 more 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 in the peritoneum showing neither tissue reaction, nor any
adhesion to any organs. 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.
* * * * *