U.S. patent application number 10/191885 was filed with the patent office on 2002-12-12 for method for culturing langerhans islets and islet autotransplantation islet regeneration.
Invention is credited to Yoon, Tai-Wook.
Application Number | 20020187551 10/191885 |
Document ID | / |
Family ID | 23659488 |
Filed Date | 2002-12-12 |
United States Patent
Application |
20020187551 |
Kind Code |
A1 |
Yoon, Tai-Wook |
December 12, 2002 |
Method for culturing langerhans islets and islet
autotransplantation islet regeneration
Abstract
A method for culturing Langerhans islets to obtain an amount
sufficient for transplant and autotransplant is disclosed The
islets are cultured in a culture serum (rat/human) medium which is
supplemented with radical scavengers, growth factors, a matrix
material, nerve growth factor, cell migrating/scattering factors
and anti-integrin .beta.1 antibody at proper the time during the
culturing process. The medium is supplemented with radical
scavengers and growth factors for the first time and then further
supplemented with matrix material, radical scavengers, nerve growth
factor and the growth factors around 12-24 hours after culturing.
Thereafter, the medium is supplemented with growth factors, cell
migrating/scattering factors and anti-integrin .beta.1 antibody at
4-5 days into the culturing process. The culturing process is
conducted for an extended period of time, so that any latent red
blood cells are eliminated from the islet culture. The islets then
continue to proliferate to produce an amount of islets which is
sufficient for transplantation into a diabetic patient, including a
human, to provide extended normoglycaemia and islet regeneration to
fully treat diabetes mellitus without the growth of
fibroblasts.
Inventors: |
Yoon, Tai-Wook; (Seoul,
KR) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Family ID: |
23659488 |
Appl. No.: |
10/191885 |
Filed: |
July 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10191885 |
Jul 8, 2002 |
|
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09418765 |
Oct 15, 1999 |
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Current U.S.
Class: |
435/384 ;
435/366; 435/389; 435/392 |
Current CPC
Class: |
C12N 5/0676 20130101;
C12N 2501/135 20130101; C12N 2501/11 20130101; C12N 2501/39
20130101; C12N 2501/585 20130101; C12N 2509/00 20130101; C12N
2500/25 20130101; C12N 2501/105 20130101; C12N 2501/13 20130101;
C12N 2500/38 20130101; C12N 2501/12 20130101; C12N 2500/90
20130101; A61K 35/12 20130101; C12N 2501/70 20130101 |
Class at
Publication: |
435/384 ;
435/389; 435/392; 435/366 |
International
Class: |
C12N 005/00; C12N
005/02; C12N 005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 17, 1998 |
KR |
98-43491 |
Claims
What is claimed is:
1. A method for in vitro culturing and proliferating isolated
Langerhans islets endocrine cells so as to be suitable for
transplantation comprising: providing viable Langerhans islets
endocrine cells including cells capable of differentiating into
insulin producing cells, providing a first culturing medium
comprising a basal medium supplemented with serum, at least one
radical scavenger selected from the group consisting of
nicotinamide, mannitol or superoxide dismutase, at least one growth
factor selected from the group consisting of: insulin transferring
selenite (ITS), epidermal growth factor (EGF), platelet derived
growth factor (PDGF), thrombin, Linoleic Acid-BSA, hydrocortisone
and progesterone; and at least one antinecrosis or antiapoptosis
factor selected from the group consisting of: insulin-like growth
factor-I (IGF 1) and -II (IGF 2), vascular endothelial growth
factor (VeGF); culturing the Langerhans islets endocrine cells
including cells capable of differentiating into insulin producing
cells for a period of about one day in the first culturing medium
to form a first culture growth, collecting the first culture growth
and incubating at room temperature in fresh Dulbecco modified Eagle
medium (DMEM) or serum free basal medium with anti-integrin .beta.1
antibody for 45.about.120 minutes to form a second culture growth;
suspending the second culture growth in a matrix material to
provide a 3 dimensional culture growth environment and adding a
second culturing medium comprising the supplemental basal medium
and further including, at least, another growth factor and then
culturing for 1 or 2 days to provide a third culture growth
dispersed in the matrix material; providing a third culturing
medium for culturing the third culture growth in the matrix
material, the third culturing medium comprising the supplemented
basal medium without VeGF, and optionally adding to the third
culturing medium nerve growth factor (NGF) and hepatocyte growth
factor (HGF) if the islets of the third culture growth appeared
thick and the center of the islets appeared dark, or optionally
adding to the third culturing medium NGF and anti-integrin .beta.1
antibody if the islets appeared too spread out and culturing for a
period of about one or two days to form a fourth culture growth;
collecting the islets from the matrix material and adding an enzyme
to the collected islets and to any adhering gel and incubating for
about 10 minutes to provide an incubated product; and aspirating
the incubated product back and forth numerous times causing the gel
acted on by the enzyme to be removed from the islets thereby
exposing the fibroblasts to the force created during the back and
forth aspiration causing the fibroblasts to become separated from
the surface of the islets to prepare fibroblast free islets.
2. The method of claim 1, further providing a fourth culturing
medium comprising the basal medium supplemented with serum,
insulin-transferrin-sodium selenite (ITS), Linoleic Acid-BSA,
thrombin, EGF, nicotinamide, VeGF, IGF-1, IGF-2, superoxide
dismutase and mannitol; culturing the fibroblast free islets for
about an 8-12 hours to provide a fifth culture growth; providing a
fifth culturing medium comprising DMEM with anti-integrin .beta.1
antibody and culturing the fifth culture growth for 45.about.120
minutes at room temperature to form a sixth culture growth;
suspending the sixth culture growth in a matrix material to provide
a 3 dimensional culture growth environment and adding a sixth
culturing medium comprising the supplemented basal medium and
further including, at least, another growth factor and then
culturing for 1 or 2 days to provide a seventh culture growth
dispersed in the matrix material; providing a seventh culturing
medium for culturing the seventh culture growth dispersed in the
matrix material, the seventh culturing medium comprising the
supplemented basal medium without VeGF, and optionally adding to
the third culturing medium NGF and HGF if the islets of the third
culture growth appeared thick and the center of the islets appeared
dark, or optionally adding to the third culturing medium NGF and
anti-integrin .beta.1 antibody if the islets appeared too spread
out and culturing for a period of about one or two days to form an
eighth culture growth; and collecting the islets from the matrix
material and adding an enzyme to the collected islets and to any
adhering gel and incubating for about 10 minutes to provide an
incubated product; and aspirating the incubated product back and
forth numerous times causing the gel acted on by the enzyme to be
removed from the islets thereby exposing the fibroblasts to the
force created during the back and forth aspiration causing the
fibroblasts to become separated from the surface of the islets to
prepare an increased number of fibroblast free islets.
3. The method of claim 1 wherein the serum is obtained from the
same species as that of the Langerhans islets
4. The method of claim 1 wherein the Langerhans islets are either
from a rat and the serum used is about 10% rat serum or are from a
human and the serum used is about 10% human serum.
5. The method of claim 1 wherein the growth factor added to the
second culturing medium is pituitary extract.
6. The method of claim 2 wherein the growth factor added to the
sixth culturing medium is pituitary extract.
7. The method of claim 1 wherein the viable Langerhans islets
endocrine cells including cells capable of differentiating into
insulin producing cells for proliferation are derived from a
patient for autotransplantation.
8. A method for removing fibroblasts growing from the surface of in
vitro proliferated Langerhans islets, comprising: providing a
plurality of proliferated islets having fibroblasts growing
therewith and collected from a gel proliferation matrix; adding an
enzyme to the collected islets and to any gel adhering to the
surface of the collected islets and incubating to provide an
incubated product; and aspirating the incubated product back and
forth numerous times causing the gel acted on by the enzyme to be
removed from the islets thereby exposing the fibroblasts to the
force being created during the back and forth aspiration causing
the fibroblasts to become separated from the surface of the islets
to prepare fibroblast free islets
9. The method of claim 8 wherein the enzyme is dispase
10. A culture product of proliferated fibroblast free islets
produced by the method of claim 1.
11. Use of the proliferated fibroblast free islets produced by the
method of claim 1 in treating diabetes mellitis by transplanting
the proliferated Langerhans islets endocrine cells into a patient
suffering from diabetes mellitis.
12. Use of the proliferated fibroblast free islets produced by the
method of claim 7 to treat diabetes mellitis and to regenerate
islets by autotransplanting the proliferated Langerhans islets
endocrine cells into a patient suffering from diabetes mellitis.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a method for culturing the
Langerhans islets suitable for transplantation. More particularly,
the present invention relates to a culturing method by which the
Langerhans islets can be proliferated in volume, and the fact that
proliferated islet autotransplantation can stimulate islet
regeneration via islet replication and neogenesis, leads to a
perfect diabetes cure.
[0003] 2. Description of the Prior Art
[0004] Diabetes mellitus (usually referred to simply as diabetes)
is a complex disease characterized by a grossly abnormal pattern of
carbohydrate metabolism resulting from impaired insulin secretion
and/or effectiveness The incidence of diabetes in industrialized
countries is about 10%. Indeed, diabetes is the most common serious
metabolic disease in the world, it affects hundreds of millions
[0005] Diabetes may be classified as insulin-dependent diabetes or
noninsulin-dependent diabetes. An absence of or insufficient
intrinsic insulin is a characteristic of insulin-dependent
diabetes. Some diabetics have a normal or even higher than normal
level of insulin in their blood, but they are quite unresponsive to
the hormone. This form of the disease, known as
non-insulin-dependent diabetes, typically develops later in life
than does the insulin-dependent form. However, the diabetes-causing
mechanism with which these two types can be discriminated has yet
to be revealed.
[0006] For treatment, insulin-dependent diabetics should continue
to receive exogenous insulin because their capacity of producing
insulin is greatly lowered. However, it is virtually impossible to
continuously and properly provide insulin in response to patient's
physiological demands What is more difficult, the body has an
insulin concentration gradient such that the insulin concentration
is decreased in order of: the hepatic portal vein, the liver, the
hepatic vein, the aorta and the muscle, but an injection of
exogenous insulin does not result in such a concentration gradient,
which then causes side effects.
[0007] The .beta.-cells of the Langerhans islets secrete insulin
and 11 other materials. Thus, an injection of only insulin can
decrease the blood glucose level, but cannot prevent glucopenia and
other complications. Since one of the objectives in the treatment
of diabetes is to lower the blood glucose level, blood glucose
lowering agents are often employed. These lowering agents, however,
should not be prescribed for an extended period of time because
they result in resistance. Moreover, blood glucose lowering agents
were found to cause serious side effects.
[0008] Insulin, as mentioned above, is able to lower blood glucose
level as well as gives much lower resistance than do blood glucose
lowering agents. However, the necessary amount of insulin varies
with a patient's conditions so that it is very difficult to timely
administrate proper dosage of insulin Upon improper administration
of insulin, anti-insulin antibodies may be formed, making diabetes
worse
[0009] For curing diabetes, tissue transplantation has recently
been of great interest. For example, the pancreas or Langerhans
islets are transplanted into a patient who suffers from diabetes to
provide a controlled amount of insulin which is necessary for the
patient.
[0010] In such cases, however, immune rejection is always
problematic and must be considered. When the immune rejection
occurs, immune suppressors are administered to the patients. In
addition, the number of donors are not sufficient relative to the
demand.
[0011] The treatment of diabetes by insulin administration was
first conducted in 1921 by Banting and Best, but they failed to
cure the disease because of a diabetic complication. In 1966,
Lillihei of Minnesota University first transplanted a portion of
the pancreas into a diabetic patient By 1977, 57 patients had been
subjected to the transplantation. However, less than 10% of them
survived for one year or more. Recent development of immune
suppressors has increased the survival rate of pancreas or kidney
transplant recipients up to 70% For Langerhans islet
transplantation, the survival rate amounts up to 90%
[0012] The first thing into which account is taken is the
histocompatibility between donor and recipient. If tissue
transplantation is performed between two persons who have different
histocompatibility, an immune rejection occurs, leading to the
destruction of the transplanted islets at the worst. Generally, 50
donors are needed to discover the necessary histocompatibility for
one recipient. If fresh islets are transplanted, a large quantity
of fibrous tissues grow out from freshly isolated Langerhans islets
and divide and surround them if transplanted, so that the ability
of the .beta.-cells to secrete insulin in response to a stimulus
declines greatly.
SUMMARY OF THE INVENTION
[0013] In approaching the present invention, the present inventors
considered the following:
[0014] First, in order for cells or cell groups to proliferate in
vitro, they must contain stem cells or progenitor cells therein and
be in undifferentiated states. Fortunately, since many stem cells
or progenitor cells exist in Langerhans islets, it is highly
possible to proliferate undifferentiated Langerhans islets in
vitro.
[0015] Next, MHC class II antigens, which cause immune rejection,
must be absent in the proliferated Langerhans islets and thus, if
the blood cells, rich in MHC class II antigens, are eliminated from
Langerhans islets, the immune rejection can be greatly reduced in
the islet allotransplantation. The longer the islets remain in the
culture and proliferate, decreases the immune rejection
response.
[0016] Finally, fibrous tissues are developed from the crude islets
and must be able to be easily removed from the in vitro
proliferated islet.
[0017] Taking advantage of the above three points, the present
inventors tried to proliferate in vito the Langerhans islets with
the aim of preparing them so as to be easily and successfully
transplanted in the host for the long term treatment of
diabetes.
[0018] When the Langerhans islets were grown in a monolayer culture
method, they proliferated at a rate of, at most, 80% However, they
poorly secreted insulin so that it was impossible to control the
level of the blood glucose. The islets should proliferate at least
5-fold for successful transplantation.
[0019] Intensive and thorough research repeated by the present
inventors resulted in the discovering that upon in vitro culture in
media containing various biochemical materials, the Langerhans
islets isolated from rats proliferate at high rates sufficient to
be applied for transplantation, release the blood cells from
themselves so as to greatly reduce the immune rejection, and
function well enough so as to successfully continue to secrete
insulin after transplantation according to the present
invention.
[0020] Therefore, it is an object of the present invention to
provide a method for proliferating the Langerhans islets in a
suitable state for transplantation, whereby a greatly enhanced
treatment effect for diabetes can be brought about.
[0021] In accordance with the present invention, there is provided
a method for proliferating the Langerhans islets, in which a
culture medium is supplemented with radical scavengers, growth
factors, a matrix material, nerve growth factor, cell
migrating/scattering factors (such as HGF) antinecrosis factors or
antiapoptosis factors (such as IGF 1, IGF 2, VeGF) and a
cytoskeleton activator (anti-integrin .beta.1 antibody) at proper
culture times and the proliferation is conducted for an extended
period of time, so that the Langerhans islets are depleted of the
blood cells and also proliferate sufficiently in order to be
suitable for transplantation.
[0022] The present inventions are directed to a method for in vitro
culturing and proliferating isolated Langerhans islets endocrine
cells so as to be suitable for transplantation. To initiate the
proliferation viable Langerhans islets endocrine cells including
cells capable of differentiating into insulin producing cells are
collected and placed in a first culturing medium comprising a basal
medium supplemented with serum, at least one radical scavenger
selected from the group consisting of nicotinamide, mannitol or
superoxide dismutase, at least one growth factor selected from the
group consisting of: insulin transferrin selenite-complex
(ITS-complex), epidermal growth factor (EGF), platelet derived
growth factor (PDGF), thrombin, Linoleic Acid-BSA, hydrocortisone
and progesterone, and at least one antinecrosis or antiapoptosis
factor selected from the group consisting of IGF 1, IGF 2, VeGF and
culturing the Langerhans islets endocrine cells including cells
capable of differentiating into insulin producing cells for a
period of about one day in the first culturing medium to form a
first culture growth. The first culture growth is collected and
incubated at room temperature in fresh DMEM or serum free basal
medium with anti-integrin .beta.1 antibody for 45.about.120 minutes
to form a second culture growth
[0023] The second culture growth is then suspended in a matrix
material to provide a 3-dimensional culture growth environment and
a second culturing medium comprising the supplemented basal medium
and further including, at least, another growth factor is added
thereto and then culturing proceeds for 1 or 2 days to provide a
third culture growth dispersed in the matrix material.
[0024] A third culturing medium for culturing the third culture
growth in the matrix material is provided and comprises the
supplemented basal medium but without VeGF, and optionally adding
to the third culturing medium NGF and HGF if the islets of the
third culture growth appeared thick and the center of the islets
appeared dark, or optionally adding to the third culturing medium
NGF and anti-integrin .beta.1 antibody if the islets appeared too
spread out and then culturing for a period of about one or two days
to form a fourth culture growth. The islets are then collected from
the matrix material, placed in a suitable vessel and an enzyme such
as dispase is added to the collected islets and to loosen or enable
removal of any adhering gel and then incubated for about 10 minutes
to provide an incubated product. The incubated product is then
aspirated back and forth numerous times causing the gel acted on by
the dispase to be removed from the islets thereby exposing the
fibroblasts to the force created during the back and forth
aspiration which appears to cause the fibroblasts to become
separated from the surface of the islets to prepare fibroblast free
islets.
[0025] The above process takes about 7 days and can be repeated as
follows. A fourth culturing medium comprising the basal medium
supplemented with serum, insulin-transferrin-sodium selenite (ITS),
Linoleic Acid-BSA, thrombin, EGF, nicotinamide, VeGF, IGF-1, IGF-2,
superoxide dismutase and mannitol is provided and then the
fibroblast free islets are cultured for about 8-12 hours to provide
a fifth culture growth. The fifth culture growth is collected and
cultured in a fifth culturing medium comprising DMEM with
anti-integrin .beta.1 antibody for 45.about.120 minutes at room
temperature to form a sixth culture growth. The sixth culture
growth is then suspended in a matrix material to provide a
3-dimensional culture growth environment and a sixth culturing
medium comprising the supplemented basal medium and further
including, at least, another growth factor is added thereto and
then cultured for 1 or 2 days to provide a seventh culture growth
dispersed in the matrix material.
[0026] A seventh culturing medium which comprises the supplemented
basal medium without VeGF, and optionally adding to the third
culturing medium NGF and HGF if the islets of the third culture
growth appeared thick and the center of the islets appeared dark,
or optionally adding to the third culturing medium NGF and
anti-integrin .beta.1 antibody if the islets appeared too spread
out is provided for culturing the seventh culture growth dispersed
in the matrix material for a period of about one or two days to
form an eighth culture growth. The islets are then collected from
the matrix material and an enzyme, such as dispase, is added to the
collected islets and to any adhering gel and incubated for about 10
min. to provide an incubated product. The incubated product is
aspirated back and forth numerous times causing the gel acted on by
the enzyme to be removed form the islets thereby exposing the
fibroblasts, if any, to the force created during the back and forth
aspiration causing the fibroblasts to become separated from the
surface of the islets to prepare an increased number of fibroblast
free islets.
[0027] In the present method it is preferred that the serum used in
the medium is obtained from the same species as that of the
Langerhans islets to be proliferated. Thus, where the Langerhans
islets are from a rat or human, the serum used is also rat, human
serum, respectively, preferably the percent of serum in the medium
is about 10%. The growth factor added to the second and sixth
culturing medium is preferably pituitary extract.
[0028] The method of the present invention also includes using
viable Langerhans islets endocrine cells including cells capable of
differentiating into insulin producing cells for proliferation
which are derived from a patient for proliferation according to the
present invention which are then used for autotransplantation back
into the same patient This results in regeneration of the islets in
the patient as described below.
[0029] The present invention also includes a method for removing
fibroblasts growing from the surface of in vitro proliferated
Langerhans islets by providing a plurality of proliferated islets
having fibroblasts growing therewith in a gel matrix. The islets
are collected from the gel matrix, along with the fibroblasts which
are growing with the islets. An enzyme, such as dispase, is added
to the collected islets and to any gel adhering to the surface of
the collected islets and then this is incubated to provide an
incubated product. The incubated product is then aspirated back and
forth numerous times causing the gel acted on by the dispase to be
removed from the islets thereby exposing the fibroblasts to the
force being created during the back and forth aspiration which
causes the fibroblasts to become separated from the surface of the
islets to prepare fibroblast free islets
[0030] The present invention also includes the culture product of
proliferated fibroblast free islets produced by the method
according to the present invention and the use of the proliferated
fibroblast free islets produced by the method according to the
present invention in treating diabetes mellitis by transplanting
the proliferated Langerhans islets endocrine cells into a patient
suffering from diabetes mellitis. In addition, the present
invention further includes the use of the proliferated fibroblast
free islets from a patient produced by the method according to the
present invention to treat diabetes mellitis and to regenerate
islets in the patient by autotransplanting the proliferated
Langerhans islets endocrine cells into the patient suffering from
diabetes mellitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The above and other objects and aspects of the invention
will become apparent from the following description of embodiments
with reference to the accompanying drawings in which:
[0032] FIG. 1 is a photograph magnified 100 times (.times.100)
showing blood cells released from islet after 24 hour incubation at
37.degree. C.,
[0033] FIG. 2 is a photograph (.times.100) showing the Langerhans
islets which are cultured in the absence of radical scavengers,
[0034] FIG. 3 is a photograph (.times.100) showing the Langerhans
islets which are cultured in the presence of a radical
scavenger;
[0035] FIG. 4 is a photograph (.times.200) showing islets which
were incubated in a supplemented medium (VeGF, IGF-1, IGF-1);
[0036] FIG. 5 is a photograph showing the islets on the second day
of proliferation;
[0037] FIG. 6 is a photograph (.times.100) showing the lateral
growth of the Langerhans islets which are proliferated in the
medium containing anti-integrin .beta.1 antibody on the 3rd or 4th
day;
[0038] FIG. 7 is a photograph (.times.100) showing an islet
cultured in the presence of migrating factors, showing horizontal
growth or spreading of the islet, for example HGF, on the 3d or 4th
day;
[0039] FIGS. 8A and 8B are photographs showing the islet growing in
all directions after proliferating for 10 days according to the
method of the present invention;
[0040] FIG. 9 is a photograph showing a human islet proliferated in
vivo for a period of 8 days in the presence of human serum;
[0041] FIG. 10 is a graph showing the change in the DNA amount of
the Langerhans islets with culturing time in days;
[0042] FIGS. 11A, 11B and 11C show the process of fibroblast
development and its depletion from the islets. Many fibroblasts
grew out from islet during islet proliferated (FIG. 11A) even
though the islets looked pure when they were collected. The
fibroblasts were almost all removed (FIG. 11B) and completely
removed (FIG. 11C) from the islets;
[0043] FIG. 12 shows the in vitro functions (glucose-response) of
fresh (square) and proliferated (circle) islets;
[0044] FIG. 13 is a graph in which the levels of glucose in the
blood of the diabetic rats which were transplanted with 1350 and
1650 islets just isolated, were measured and were plotted against
time in days;
[0045] FIG. 14 shows blood glucose level profile of STZ-induced
diabetic rat transplanted with 500 proliferated rat islets with
days;
[0046] FIG. 15 is a graph of blood glucose (mg/dl) levels against
time in days comparing 500, 600, 800, 1000, and 1200 fresh rat
islets transplanted into the spleen of STZ-induced diabetic mice,
indicating the 800 islets are the minimum islet number required to
recover and maintain normoglycaemia;
[0047] FIG. 16 is a photograph (.times.200) of a fresh islet
transplanted into the spleen of an SZT-induced diabetic nude
mouse.
[0048] FIG. 17 plots 150-200 proliferated islets transplanted into
the spleens STZ-induced diabetic mice against time in days; and
[0049] FIG. 18 is a photograph (.times.400) showing a proliferated
islet transplanted into the spleen of STZ-induced diabetic nude
mouse.
DETAILED DESCRIPTION OF THE INVENTION
[0050] To better illustrate the present invention FIGS. 1 through
18 are presented and described below. FIG. 1 shows the islet cells
with released red blood cells after being incubated for 24 hours at
37.degree. C. in a medium of rat serum. FIGS. 2 and 3 show the
difference when culturing in the absence of and the presence of
radical scavengers, respectively. FIG. 4 shows islets which were
incubated in a medium supplemented with VeGF, IGF-1 and IGF-2 to
increase islet viability. The supplements function as
anti-apoptotic and anti-necrotic factors. The islets are intact
without dead cells on the surface. FIG. 5 is a photograph showing
the islets on the second day of proliferation with the islets
having a smooth surface and high viability. The medium used in FIG.
6 included anti-integrin .beta.1 antibody and shows growing cells
appearing on the surface of the islet. FIG. 7 shows the effect of
adding a migrating factor(s), for example, hepatocyte growth factor
(HGF), to the medium which appears to cause the islet to spread
horizontally.
[0051] FIGS. 8A and 8B show that the medium used in the present
invention enables the islets to grow in all directions FIG. 9 shows
a human islet proliferated in vitro for 8 days using rat islet
proliferation methods of the present invention. FIG. 10 shows the
increase in DNA content of in vitro proliferating Sprague-Dawley
(SD) rat islets against the number of days incubated FIG. 11A shows
fibroblasts growing out from pure islets during proliferation
(.times.100 magnification), and FIGS. 11B and 11C show the
fibroblasts removed prior to transplantation (.times.200
magnification). FIG. 12 shows glucose stimulated insulin secretion
from freshly isolated (square) and proliferated (circle) islets.
FIG. 13 is a graph of blood glucose (mg/dl) plotted against time in
days for 1650 (circle) and 1350 (triangle) fresh rat islets
transplanted into the liver of a STZ induced diabetic rat via the
hepatic portal vein
[0052] FIG. 14 is a graph of blood glucose levels (mg/dl) plotted
against time in days, 0 being the day of transplant of 500
proliferated rat islets into four (4) rats. FIG. 15 is a graph of
blood glucose (mg/dl) levels plotted against the comparing 500,
600, 800, 1000 and 1200 fresh rat islets, respectively,
transplanted into the spleen of STZ-induced diabetic mice. The
numbers represent the number of islets transplanted. 800 islets are
the minimum islet number required to recover and maintain
normoglycaemia. The blood glucose levels of the diabetic rats which
were transplanted with the Langerhans islets just isolated, were
plotted against time in days.
[0053] FIG. 16 is a photograph (.times.200) of a fresh islet
transplanted into the spleen of an SZT-induced diabetic nude mouse.
The blood glucose concentration recovered and was maintained for 30
days. Then the mouse was sacrificed to collect transplanted islet.
The islet was sectioned and stained in an insulin-specific staining
method to identify the islet.
[0054] FIG. 17 is a graph of blood glucose levels (mg/dl) plotted
against time in days comparing different numbers (200, 180, 150) of
proliferated islets transplanted into spleens of STZ-induced
diabetic mice. Blood glucose levels were measured every other day
for several months. The mice used in this experiment had no working
immune system; however, in two mice one with an implanted islet
number of 150 and 180, respectively, it is believed that the immune
system functioned to the extent that it attacked the implanted
islets resulting in the sudden increase in blood glucose level at
around day 25.
[0055] FIG. 18 is a photograph (.times.400) showing a proliferated
islet transplanted into the spleen of STZ-induced diabetic nude
mouse. The mouse recovered and remained normoglycaemia for 3 months
Then the mouse was sacrificed to remove the spleen from the mouse.
The spleen was sectioned and stained in insulin-specific staining
method to identify the transplanted islet.
[0056] Before culturing, the Langerhans islets isolated from the
pancreas may be stored. Also, after culturing the proliferated
Langerhans islets must be properly stored unless they all are used
immediately To this end, they are preferably frozen in liquid
nitrogen.
[0057] Dimethyl sulfoxide (DMSO) is useful to protect the
Langerhans islets upon freezing. At a convenient or proper time,
the frozen stock of the Langerhans islets is thawed for
culturing.
[0058] In accordance with the present invention, a culture system
contains a matrix material in order to provide a three dimensional
environment for Langerhans islets. Under this circumstance, the
Langerhans islets show a high degree of proliferation. For the
matrix material, collagen, complex collagen, tail complex collagen
or other biogels (e.g., Matrigel.RTM.), or the like, may be
used.
[0059] The culture medium of the present invention (rat serum for
rat islets and human serum would be used for human islets) also
contains a radical scavenger which plays the role of protecting the
proliferated cells from radical damage. Nicotinamide, mannitol or a
superoxide dismutase is added to the culture medium as a radical
scavenger.
[0060] As mentioned above, the proliferation rate necessary for
transplantation must amount to at least 500%. For this, the
isolated Langerhans islets are cultured in the presence of growth
factors, and cell migrating/scattering factors. Suitable growth
factors are selected from the group consisting of insulin
transferrin selenite (ITS), epidermal growth factor (EGF), platelet
derived growth factor (PDGF), thrombin, progesterone, Linoleic
Acid-BSA, pituitary extract and hydrocortisone. Examples of cell
migrating/scattering factors include hepatic growth factor (HFG)
and tumor promoting activator (TPA).
[0061] During culturing, the blood cells go from the Langerhans
islets into the medium. As the culturing goes on, the blood cells
are subjected to necrosis. Since the blood cells have MHC class II,
a major factor which causes the immune rejection upon tissue
transplantation, the cultured Langerhans islets can be transplanted
in a host with little immune rejection, if any. In the present
invention, a cytoskeleton activator is added in the culture medium
to enhance islet proliferation. Preferably, anti-integrin .beta.1
antibody is used for this purpose.
[0062] A better understanding of the present invention may be
obtained in light of the following examples which are set forth to
illustrate, but should not be construed to limit the present
invention.
EXAMPLE I
Freeze Storage and Thaw of Langerhans Islets
[0063] 1 ml of 10% fetal calf (bovine) serum (FCS) medium which
contained 2,000 (rat) Langerhans islets was added to 0.5 ml of 2M
DMSO and allowed to stand at room temperature for 5 min. The
procedure of adding DMSO and standing at room temperature was
further repeated twice for the first time, with 0.5 ml of 2M DMSO
and a period of 25 min., and for the second time, with 0.5 ml of 3M
DMSO and a period of 15 min. Thereafter, the medium was allowed to
stand for 5 min on ice and then, for 15 min at -7.5.degree. C. This
medium was subjected to nucleation using previously chilled forceps
and allowed to stand for 15 min at -7.5.degree. C. The temperature
was lowered down to -35.about.40.degree. C. at a rate of 0.2-0.3
per min. Once reaching the temperature, the vial containing the
medium was stored in a liquid nitrogen tank.
[0064] For thawing, the vial stored in the liquid nitrogen tank was
transferred to a cryovial in a water bath of 37.degree. C. until
the ice crystals started to thaw. Then, the vial was placed on ice
and allowed to stand in order that the Langerhans islets may settle
on the bottom. The supernatant was drained out using a Pasteur
pipette. The Langerhans islets were added with 10% FCS supplemented
with 1 ml of 0.75 M sucrose and allowed to stand on ice for 30 min.
Then, the Langerhans islets were added with 1 ml, 2 ml, 4 ml and 8
ml of 10% FCS, successively After every addition, the Langerhans
islets were allowed to stand for 5 min at room temperature. The
resulting supernatant was removed and the remaining Langerhans
islets were re-suspended in a culture medium and cultured at
37.degree. C. This procedure may be used for rat, mouse, human, and
the like, Langerhans islets.
EXAMPLE II
Proliferation of the Langerhans Islets
[0065] In this Example, the Langerhans islets which were frozen and
thawed as in Example I were used. However, freshly isolated
Langerhans islets could also be used as is appreciated by one
skilled in the art During incubation 5% CO.sub.2 is added to
ambient air.
[0066] Serum Preparation
[0067] Rat serum was added to medium for proliferating the rat
islets. 2-5 ml. blood was obtained from a rat and transferred into
sterile 15 ml Falcon tube. The blood was left for 2-4 hours at room
temperature and then centrifuged at 3000 g for 10 minutes. The
supernatant was transferred to 1.5-2 ml tubes, which were left
overnight at 4.degree. C. and then centrifuged again in the same
way. The supernatant was stored at -20.degree. C. until use. Human
islets were proliferated in the medium containing 10% human serum
instead of rat serum that was made in the same way.
[0068] The basal medium (50 ml) is prepared by mixing together:
[0069] 1.1 mg (100 .mu.l) pyrubate, (Gibco brl); 0.25 .mu.g (100
.mu.l) hydrocortisone (Sigma); 100 units/(100 .mu.l) (1 ml)
penicillin/streptomycin; 4.456 mg (4 .mu.l) B-mercaptoethanol
(Sigma); 14.6 mg L-glutamate (Gibco); 238.3 (1 ml) Hepes Buffer
(Gibco); 100 mg (11 mM) glucose (Sigma) plus DMEM to make a volume
of 50 ml.
[0070] Experiment Example II-I:
[0071] Culturing of Langerhans Islets Under Basic Conditions.
[0072] First Day: 500 islets were cultured under basic conditions.
The freshly isolated islets were incubated overnight at 37.degree.
C. in 6 ml basal medium supplemented with 600 .mu.l rat serum
(10%), 30 .mu.g of insulin-transferrin-sodium selenite (ITS) (5
.mu.g/ml, I-1884, Sigma USA), 6 mg of Linoleic Acid-BSA (1 mg/ml,
L-8384, Sigma USA), 60 ng (platelet-derived growth factor), PDGF
(10 ngm/ml P8147 Sigma USA), 600 ng thrombin (100 ngm/ml T-4393
Sigma USA), 60 ngm (epidermal growth factor), EGF (10 ngm/ml E-1264
Sigma USA), 187.2 mg of 10.sup.-4 M superoxide dismutase (product
no. S-9636, Sigma, USA), 109.32 .mu.g of 10.sup.-4 M mannitol
(M-9546 Sigma USA), 61 mg (3 mM) nicotinamide (N-0636 Sigma USA),
12 ng (vascular endothelial growth factor) VeGF (2 ngm/ml V-7259
Sigma USA), 600 ngm (insulin-like growth factor-I and -II) IGF-1
and IGF-2 (I-3769 and I-213P, respectively, Sigma USA).
[0073] Experiment Example II-II:
[0074] Second Day:
[0075] 1. The 500 islets were incubated for 45.about.120 minutes at
room temperature with 50 ng (25 ng/ml) of anti-integrin .beta.1
antibody (Cat. No. I-41720 Transduction Labs. USA) in 2 ml DMEM
medium or serum free basal medium.
[0076] 2. The islets were then suspended in 200 .mu.l of 80 to 100%
Matrigel.RTM. (Collaborative Biomedical Product, USA) in order to
provide a three-dimensional growth environment.
[0077] 3. Added 2 ml of the above supplemented basal medium and
which was also supplemented with 100 .mu.g pituitary extract (50
.mu.g/ml P-1167 Sigma, USA) and cultured for 1-2 days at 37.degree.
C.
[0078] Experiment Example II-III:
[0079] Third or Fourth Day:
[0080] The culture medium was replaced with fresh supplemented
basal medium having the same composition as that of the first day
except that VeGF is not added and, depending on the islet shape,
other factors were added to the medium, as follows:
[0081] 1. 20 ng (nerve growth factor) NGF (10 ng/ml N-6009 Sigma,
USA), 50 ng HGF (25 ng/ml Collaborative Biomedical Product, USA Lot
902287), were added to 2 ml of the supplemented basal medium if the
islets became thick and dark in the center of the islet and then
the islets were cultured in the medium for 1 or 2 days, or
[0082] 2. 20 ng NGF and 100 ng anti-integrin .beta.1 antibody (50
ng/ml) were added to 2 ml of the supplemented basal medium if the
islets looked spread out and then cultured for 1-2 days.
[0083] Observation with a microscope at .times.200 magnification
was shown in FIG. 9.
[0084] After the Sixth or Seventh day, the islets culturing
sequence was performed as follows:
[0085] Seventh Day
[0086] The islets were collected from the 3-dimensional gel. The
fibroblasts were removed using dispase by the process described
below The collected islets were cultured overnight in a floating
culture, i.e., the islets were not fixed in a medium, such as a gel
medium. The culturing medium was 6 ml of the basal supplemented
medium, as described above, supplemented with 600 .mu.l of rat
serum (10%), 30 .mu.g of insulin-transferrin-sodium selenite (ITS),
6 mg of Linoleic Acid-BSA (1 mg/ml), 600 ng thrombin (100 ngm/ml
T-4393 Sigma USA), 60 ng EGF, 101 ng nicotinamide, 12 ng VeGF, 600
ng IGF-1, 600 ng IGF-2, 187.2 mg of 10.sup.-4 M superoxide
dismutase (Sigma), 109.32 .mu.g of 10.sup.-4 M mannitol.
[0087] Eighth Day
[0088] 1. The islets are incubated in fresh DMEM supplemented with
50 ng of anti-integrin .beta.1 antibody for 45.about.120 minutes at
room temperature.
[0089] 2 The islets were suspended in Matrigel.RTM. to grow in a
3-dimensional environment.
[0090] 3. 2 ml of the basal supplemented medium was added plus 100
.mu.g pituitary extract and were cultured for 1-2 days at
37.degree. C.
[0091] Ninth or Tenth Day
[0092] 1. Basal supplemented medium added as first prepared, except
that VeGF is not used.
[0093] 2. Depending on the islet shape, the islets were cultured
for 2-3 days in the above medium plus the added factors as
described in the Third or Fourth Day culture medium.
[0094] Fourteenth Day
[0095] 1. The islets were collected from the gel by using dispase
as described below and as done at around the Seventh Day This step
is mainly to release the islets from the gel However, if any
fibroblasts were to remain, they also would be removed from the
surface of the islet.
[0096] 2. The islets were incubated at 37.degree. C. in a floating
culture (islets not fixed as in a matrix) in a medium having the
same constitutional amounts and components as in the first day.
These islets may be transplanted but are preferably subjected to
removal as described below.
[0097] Experiment Example III:
[0098] During culturing the amounts of DNA of the Langerhans islets
were measured every other day and their relative amounts were
plotted for culture times on the basis of the day on which the
Langerhans islets were isolated As seen in FIG. 10, the Langerhans
islets proliferated up to about 1,000% on the 14th day after
culturing.
EXAMPLE IV
[0099] Test for in vivo function of the Langerhans Islets
[0100] Experiment Example IV-I:
[0101] Blood glucose level of diabetic rats transplanted with
freshly isolated Langerhans islets.
[0102] For this, streptozotocin (STZ) was intraperitoneally
injected to the rats at a dosage of 53-55 mg per kg of body weight
and the level of glucose in the blood was checked every day for two
weeks. If the blood glucose level reached 250 ng/mg, the
Sprague-Dawley (SD) rats were judged to be diabetic.
[0103] 5.5 collagenase XI (0.7 mg/ml type XI Sigma, USA) was
introduced to the pancreases of healthy rats by injection via the
common bile duct to distend the pancreases and to digest them for
17 min. at 37.degree. C.
[0104] After the digested pancreas was washed three times with
Dulbecco modified Eagle medium (DMEM), the Langerhans islets were
isolated therefrom and collected in a discontinuous bovine serum
albumin (BSA) concentration gradient method. The collected
Langerhans islets were added with DMEM to a final volume of 100-150
.mu.l.
[0105] With the aid of 1 ml syringes, the resulting solution was
injected into the hepatic portal vein of the diabetic rats
anesthetized by intraperitoneal injection of Entobal (Hanlim
Pharmacy Co., Ltd., Korea) at a dosage of 60 mg per kg body
weight.
[0106] 1650 (islet equivalents (IEQ) 2750) and 1350 (IEQ 2400)
fresh Wistarkyoto (WK) rat islets were transplanted into the livers
of 175 g streptozotocin-induced diabetic rats via hepatic portal
vein 1650 fresh islets are enough mass to recover and maintain
normoglycaemia but 1350 fresh islets are not enough mass to recover
and maintain normoglycaemia, see FIG. 13. Therefore, 1650 fresh
islets are minimum required islet number
[0107] Experiment Example IV-II:
[0108] Blood glucose level after transplantation of in vitro
proliferated rat islets.
[0109] The procedure of Experiment Example III-I was repeated using
the same Langerhans islets as those of Example II, which were
proliferated in a medium supplemented with collagen, radical
scavengers, cell migrating/scattering factors, growth factors and
anti-integrin .beta.1 antibody
[0110] The blood glucose levels of the rat hosts transplanted with
500 proliferated rat islets were measured and plotted with times.
The measurements of the blood glucose level are shown in FIG. 14.
As seen, 500 proliferated Langerhans islets completely functioned
to recover and maintain normoglycaemia, so that enough insulin was
secreted inproper response to the blood glucose levels of the
hosts.
[0111] Experiment Example IV-III:
[0112] Blood glucose concentration profile of STZ-induced diabetic
mice transplanted with different number of fresh rat islets.
[0113] Different numbers of fresh rat islets were transplanted into
spleen of STZ-induced diabetic nude mice to determine minimum
number of islets required to recover and maintain normoglycaemia.
The blood glucose levels of the most mice were measured and plotted
with respect to time. The results are given in FIG. 15. The figure
shows the profile of blood glucose levels against time in days. At
least 800 fresh islets are required to recover and maintain
normoglycaemia.
[0114] The blood glucose level profile of STZ-induced diabetic mice
of FIG. 15 is based on STZ-induced diabetic mice transplanted with
proliferated rat islets.
[0115] The Langerhans islets isolated from Sprague-Dawley (SD) rats
were proliferated in the same medium as in Example II.
[0116] About 150-200 proliferated rat islets were transplanted into
the spleen of STZ-induced diabetic nude mice The 150-200
proliferated rat islets are sufficient in number to recover and
maintain normoglycaemia.
[0117] The islets were transplanted into the spleen of STZ-induced
diabetic nude mice. The blood glucose levels of the host mice were
measured and plotted against time in days. The results are given in
FIG. 17 As compared with FIG. 15, in vitro proliferated (for 5-6
days) islets showed 3-5 fold higher in vivo function than fresh
islets.
[0118] As described hereinabove, the Langerhans islets, whether
they are just isolated from pancreas or thawed from a frozen state,
can be proliferated in volume upon culturing in a species similar
serum (i.e., rat serum or human serum for rat or human Langerhans
islets, respectively) medium further supplemented with radical
scavenger, collagen, growth factors and cell migrating/scattering
factors. Further, the Langerhans islets which are cultured for a
long time in the supplemented medium, are depleted of blood cells,
so that they can be transplanted and function well enough to
recover and maintain normoglycaemia
[0119] Experiment Example V:
[0120] In vitro function (glucose response) of proliferated
islets.
[0121] Fresh and proliferated rat islets were incubated with 2.7
and 16.7 mM glucose for 1 hour and secreted insulin concentration
were measured by using 125I-insulin KIT.
[0122] Proliferated islets respond nearly null to low glucose
concentration and very strongly to high glucose concentration;
whereas, fresh islets respond higher to low glucose, lower to high
glucose concentration than proliferated islets (as see FIG. 12).
The results indicated that in vitro proliferated islets have more
desirable functional properties than fresh islets, therefore
proliferated islets can be used as autotransplantation
material.
[0123] Experiment Example IV:
[0124] Fibroblast Removal
[0125] Fresh islets are likely to be contaminated with fibroblasts
even though they are collected in a pure state as can be seen by
the fact that many fibroblasts grow out from the islets during the
proliferation. The fibroblasts are removed completely prior to
islet transplantation, as seen in FIG. 11C Islets are dispersed in
a gel (e.g., Matrigel.RTM.) during proliferation At about the
seventh or fourteenth day, the islets are collected from the gel
and 400 .mu.l of dispase (Collaborative Biomedical Products, USA,
Cat. 40235) is added and incubated for about 10 minutes at
37.degree. C. Then the islets are aspirated back and forth several
times causing the gel acted on by the dispase to be removed from
the islets which exposes the fibroblasts to the force created
during the back and forth aspiration causing the fibroblasts to
become separated from the surface of the islets to prepare
fibroblast-free islets.
[0126] Islets proliferated up to 5- and 10-fold for the first and
second week during in vitro culturing, respectively. These
proliferated islets showed a more desirable in vitro
glucose-response pattern than fresh islets. Their transplantation
results showed that those islets have a 3.about.4 fold higher
capability to recover and maintain normoglycaemia than fresh
islets. These data suggest that in vitro proliferated islets are a
better source than fresh islets for transplantation to treat
diabetes. Consequently, the present invention can produce a large
quantity of the Langerhans islets and, thus, transplantation using
the proliferated islets according to the present invention is a
promising therapeutic means for the treatment of diabetes.
[0127] Experiment Example VII:
[0128] Islet Autotransplantation-Stimulated Islet Regeneration.
[0129] Islet autotransplantation recovered and maintained
normoglycaemia. In the diabetic rats, islet neogenesis did appear
Exogenous insulin injection also did not stimulate islet
neogenesis. However, islet autotransplantation stimulates islet
neogenesis.
[0130] The following table shows insulin content, number, size of
islets collected from rats and rice which were either
pancreatectomized (columns 2 and 3) or transplanted into diabetic
mice/rats after transplantation (columns 4, 5 and 6). The islets
used were either fresh (columns 4 and 5) or cultured/proliferated
for 6 days (column 6) according to the process of the present
invention. The transplantation (TX) was islet auto-transplantation
The results in columns 5 and 6 indicate that the transplanted
islets, either fresh or proliferated according to the present
invention result in islet regeneration in the pancreas.
1 /fresh /fresh /autotransplantation 25 day stable 13 month
normogly- normogly- Pancreatectomy caemia caemia after 80-90% 2
months 25 day unstable controlled 500 pancrea- after 90% blood
glucose by 1650 proliferated tectomy on pancrea- level after 1350
fresh after islets TX in day 0 tectomy islet TX in liver TX in
liver liver Collected 113 (EQ 235 (EQ 70 (IEQ 100) 226 (IEQ 164
(IEQ islet number 223) 505) from panc 168) from 264) from panc.
panc. islet size no data Very large very small to small islet Very
large collected very large (90 .about. 119) since normal immature
insulin High High High Low High present
[0131] The following experimental results prove this (see Table,
above)
[0132] 1 1350 fresh rat islets were transplanted into the liver of
a STZ-induced diabetic rat via hepatic portal vein. Blood glucose
concentration declined gradually to be normoglycaemia 35 days
later. The values lingered in the 200's for 20 days and finally
normoglycaemia for 1-2 days, as seen in FIG. 13. Islets were
collected from the pancreas and stained deeply with dithizone.
[0133] 2. 1650 fresh rat islets were transplanted syngeneically
into the liver of a STZ-induced diabetic rat. The rat recovered
normoglycaemia and maintained it for 25 days, as seen in FIG. 13
After sacrificing, 226 (EQ 168) small islets were collected and
were not stained with dithizone. The pancreas islets were immature
and could be produced by neogenesis. In conclusion, on comparing
both cases, islet autotransplantation-recovered normoglycaemia
seems to provide a favorable environment for islet neogenesis and
replication by secreting various natural substances.
[0134] 3. The rats transplanted syngeneically with 500 proliferated
islets maintained normoglycaemia for 13 months 164 large islets
(IEQ 264) were collected and stained deeply with dithizone The
results indicates that neogenic (pancreas) islets grew up to large
mature islets over a long period of time and islet
autotransplantation enhances islet regeneration and stimulates
islet maturation.
[0135] 4 90% pancreatectomy rat maintained normoglycaemia for 2
months. 235 large islets were collected from the remaining
pancreas. The islets were stained deeply with dithizone. They
seemed to originate from islet regeneration and grew for 2
months.
[0136] 5 90% pancreatectomy rat maintained normoglycaemia for 2
days. Islets were collected from the remaining pancreas.
[0137] 6 In 90% pancreatectomy rat, 113 (IEQ 223) islets are
collected from the remaining head part of the pancreas on the day
of the pancreatectomy On the basis of the pancreatectomy results,
islet regeneration rate and size depends on the magnitude of islet
deficiency.
[0138] Islet proliferation research is performed for the ultimate
purpose of treating a diabetic patient by islet autotransplantation
by providing a sufficient number of islets for transplant. The
present invention provides the required number of islets since it
enables successful islet proliferation in vitro That is, one of the
major reasons for the failure of transplantation is an insufficient
number of islets are available for transplant into a diabetic
patient. This problem is overcome by the present invention.
[0139] While the present invention was developed using rat and mice
islets, the application of the present invention to proliferate
human islets is within the scope of the teachings of the present
invention, as can be appreciated by those skilled in the art.
[0140] The present invention has been described in an illustrative
manner so as to be easily understood by one skilled in the art.
This description, examples and illustrations are intended to be in
the nature of the description rather than of a limitation, as
appreciated by those skilled in this art. Many modifications and
variations of the present invention are possible in light of the
above teachings. Therefore, it is to be understood that within the
scope of the appended claims, the invention may be practiced
otherwise than as specifically described.
* * * * *