U.S. patent application number 15/443374 was filed with the patent office on 2017-06-15 for media for culturing, preserving, and administering regenerative cells.
The applicant listed for this patent is ArthroDynamic Holdings, LLC, InGeneron, Inc.. Invention is credited to Eckhard Alt, Ivone Bruno, Michael Coleman, Frank D. Marcum, Rudy Martinez, Amir Sanchez, Paul Shealy.
Application Number | 20170166868 15/443374 |
Document ID | / |
Family ID | 50685191 |
Filed Date | 2017-06-15 |
United States Patent
Application |
20170166868 |
Kind Code |
A1 |
Coleman; Michael ; et
al. |
June 15, 2017 |
Media for Culturing, Preserving, and Administering Regenerative
Cells
Abstract
A culture media, media supplement, or soluble matrix for
cryopreservation or enhanced regenerative cell growth in culture
and maintenance of multi-lineage differentiation potentiation. The
inventive culture media, media supplement, or soluble matrix
comprises a GAG composition comprising a sulfated GAG, such as
chondroitin sulfate. A soluble matrix, a cell administration
package or kit comprising the soluble matrix and a device for cell
administration, and a method of use thereof, for administration of
regenerative cells for treating a joint disease or other weakened
or damaged tissue comprising the specified GAG compositions are
further provided.
Inventors: |
Coleman; Michael; (Houston,
TX) ; Bruno; Ivone; (Houston, TX) ; Martinez;
Rudy; (Houston, TX) ; Sanchez; Amir; (Houston,
TX) ; Alt; Eckhard; (Houston, TX) ; Marcum;
Frank D.; (Lexington, KY) ; Shealy; Paul;
(Savannah, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
InGeneron, Inc.
ArthroDynamic Holdings, LLC |
Houston
Lexington |
TX
KY |
US
US |
|
|
Family ID: |
50685191 |
Appl. No.: |
15/443374 |
Filed: |
February 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14707564 |
May 8, 2015 |
9593309 |
|
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15443374 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A01N 1/0221 20130101;
C12N 2533/80 20130101; C12N 5/0018 20130101; C12N 2501/905
20130101; C12N 2539/00 20130101; A61K 35/28 20130101; A61P 19/02
20180101; A61K 31/737 20130101; A01N 1/0284 20130101; C12N 5/0667
20130101; C12N 2501/90 20130101; A61K 35/28 20130101; A61P 19/04
20180101; A61K 2300/00 20130101; C12N 2533/70 20130101 |
International
Class: |
C12N 5/0775 20060101
C12N005/0775; A01N 1/02 20060101 A01N001/02 |
Claims
1. A method of cryopreserving a regenerative cell comprising
combining the regenerative cell with a glycosaminoglycan (GAG)
composition at a temperature above 0.degree. C., wherein at least
one GAG is a sulfated GAG, and lowering the temperature to
-18.degree. C. or lower.
2. The method of claim 1, wherein said GAG composition comprises
hyaluronic acid, CS4 and CS6 chondroitin sulfate, and N-acetyl
D-glucosamine at a concentration of 5-60% (v/v).
3. The method of claim 2, wherein said composition is at a
concentration of about 50% (v/v).
4. The method of claim 3, wherein said composition is maintained at
a temperature of -18.degree. C. or lower for at least seven
days.
5. The method of claim 1, wherein said cell is an adipose-derived
stem cell (ADSC).
6. A method of enhancing an in vitro expansion rate of cells while
maintaining multi-lineage differentiation potential, comprising
culturing the cells in a cell culture media, treating the cells
with a media supplement, or coating a surface on which the cells
are growing with a matrix, wherein said cell culture media, media
supplement, or matrix comprises a GAG composition, wherein at least
one GAG is a sulfated GAG.
7. The method of claim 6, wherein said GAG composition comprises
hyaluronic acid, CS4 and CS6 chondroitin sulfate, and N-acetyl
D-glucosamine at a concentration of 1-10% (v/v).
8. The method of claim 7, wherein said GAG composition is at a
concentration of 1-10% (v/v).
9. The method of claim 8, wherein said composition is at a
concentration of 3-7% (v/v).
10. The method of claim 9, wherein said composition is at a
concentration of 5% (v/v).
11. The method of claim 6, wherein said cells are cultured with the
culture media comprising the GAG composition.
12. The method of claim 6, wherein said cells are treated with the
media supplement comprising one or more GAG composition and then
culturing the cells.
13. The method of claim 6, wherein said cells are grown on a
surface coated with a soluble matrix comprising the GAG
composition.
14. The method of claim 6, wherein said cell is an adipose-derived
mesenchymal stem cell (Ad-MSC).
15. A cryopreservation media or media supplement comprising a GAG
composition for cryopreservation of cells and maintenance of
multi-lineage differentiation potential comprising a sulfated
GAG.
16. The cryopreservation media or media supplement of claim 15,
wherein said GAG composition comprises hyaluronic acid, CS4 and CS6
chondroitin sulfante and N-acetyl D-glucosamine at a concentration
of 5-50% (v/v).
17. The cryopreservation media or media supplement of claim 15,
wherein said media or media supplement further comprises a carrier
or soluble matrix for administration of the cells.
18. The cryopreservation media or media supplement of claim 15,
further comprising isolated regenerative cells.
19. The cryopreservation media or media supplement of claim 18,
wherein the cells are ADSCS.
20. The cryopreservation media or media supplement of claim 18 at a
temperature of -80.degree. C.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/707,564 filed May 8, 2015, issued as U.S. Pat. No.
9,593,309, which is a continuation of PCT Application No.
PCT/US2013/069206 filed Nov. 8, 2013, and which claims priority to
U.S. Provisional Application No. 61/724,285 filed Nov. 8, 2012, the
entire contents of which are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention is generally directed to compositions
and methods for preserving, suspending, cryopreserving, or
culturing cells, including regenerative stem cells, and uses for
administration to repair or enhance weakened or damaged tissues,
such as in joints, subcutaneous tissue, or organs, in animals and
humans. More particularly, the present invention provides a media,
media supplement, or soluble matrix, and method of use thereof,
comprising a composition of glycosaminoglycans for preserving,
suspending, cryopreserving, or culturing regenerative cells,
enhancing regenerative cell growth in culture, and maintaining
multi-lineage differentiation potential. The invention is also
generally directed to therapeutic administration of such cells
either isolated from the media or co-administered therewith.
BACKGROUND OF THE INVENTION
[0003] Glycosaminoglycans (GAGs) are important components of the
extracellular matrix and play important regulatory roles in cell
and tissue physiology and pathophysiology. GAGs are long unbranched
polysaccharides consisting of a repeating disaccharide unit. The
repeating unit consists of a hexose (six-carbon sugar) or a
hexuronic acid, linked to a hexosamine (six-carbon sugar containing
nitrogen). GAGs form an important component of connective tissues.
GAG chains may be covalently linked to a protein to form
proteoglycans. Proteoglycans and collagen are the chief structural
elements of all connective tissues. Their synthesis is essential
for proper maintenance and repair of connective tissues. In vitro,
the introduction of glucosamine, a key precusor for GAGs, has been
demonstrated to increase the synthesis of collagen and GAGs in
fibroblasts. In vivo, topical application of glucosamine has
enhanced wound healing. Glucosamine has also exhibited reproducible
improvement in symptoms and cartilage integrity in humans with
osteoarthritis (L. Bucci, Nutritional Supplement Advisor, July
1992).
[0004] The major proteoglycans found in connective tissue such as
cartilage are chondroitin sulfate, dermatan sulfate, keratan
sulfate and hyaluronic acid (also known as hyaluronan or HA).
Heparin sulfate is also a proteoglycan, although it is not a
component of articular cartilage. Newer names for proteoglycans
sometime reference function of the core protein within the molecule
found in chondroitin sulfate and keratin sulfate, e.g., aggregan, a
large proteoglycan that aggregates with hyaluronin, or reference
location (e.g., decorin (dermatan sulfate), which decorates type I
collagen fibrils), or reference primary structure, biglycan which
has two glysoaminoglycan chains. Chondrocytes are active cells
within the cartilage matrix, which manufacture new collagen and
proteoglycan molecules while excreting enzymes, which aid in
removal of damaged cartilage and proteoglycans. In other tissue
with a high content of extracellular matrix such as tendons,
ligaments, subcutaneous connective tissue or bone, tissue-specific
cells provide the respective function of synthesizing the
appropriate composition of extracellular matrix.
[0005] Hyaluronan is an integral part of both synovial fluid and
articular cartilage, as exemplary tissues. Within the articular
cartilage, hyaluronan provides viscoelastic properties allowing
ease of motion between opposing surfaces and increasing compressive
resistance. Within the synovium, hyaluronan, as a component of
synovial fluid, provides an effective barrier regulating the
introduction of plasma components. Under normal conditions, the
body will synthesize sufficient amounts of base components to
maintain and grow healthy articular cartilage, while limiting the
production and release of destructive proteinases, inflammatory
mediators and catabolic enzymes.
[0006] Hyaluronan or hyaluronic acid is a natural, highly charged,
polyanionic molecule composed of alternating units of D-glucuronic
and 2-acetamido-2-deoxy-D-glucose. These unbranched, coiled,
elongated polysaccharide chains maintain a large negative
electrostatic charge that attracts water molecules and allow the
deformation of the molecular coil as ice crystallization occurs
during freezing and thawing. It is believed that hyaluronic acid
coats and protects cells and tissues by attaching to the CD44
receptor sites on cells. Hyaluronic acid and other complex GAG
formulations are frequently administered by intra-articular
injection to treat joint disease, including osteoarthritis wherein
they improve clinical symptoms and slow disease progression.
Another example is the rebuilding of subcutaneous structures with
the injection of hyaluronic acid or hyaluronic acid and other
complex GAG formulations.
[0007] Chondroitin sulfate is broken down into sulfate
disaccharides and N-acetyl galactosamine. Chondroitin sulfate, as
CS4 and CS6 sulfated forms, within the body, is thought to be an
essential glycosaminoglycan that binds water to the articular
cartilage matrix and is necessary for the formation of
proteoglycans. In particular, chondroitin sulfate is a long
hydrophilic chain of repeating sugars. This glycosaminoglycan binds
to proteoglycan molecules aiding in water and nutrient
transportation within the articular cartilage. Chondroitin in its
sulfate form includes galactosamine, a primary substrate of
hyaluronan and a disaccharide pathway for proteoglycan synthesis
secondary to the hexosamine pathways utilized for glycosaminoglycan
production. Chondroitin sulfate chains comprise the space formation
of the cartilage matrix and integral parts of the proteoglycan
molecule. Chondroitin stimulates the production of proteoglycans,
glycosaminoglycans, and collagen, which are the building blocks of
healthy cartilage. Chondroitin sulfate also inhibits the secretion
of degenerative enzymes by the chondrocytes within articular
cartilage. Chondroitin sulfates are non-toxic and work
synergistically with glucosamine to hydrate and repair articular
cartilage.
[0008] Glucosamine is an amino sugar and a precursor for
glycosaminoglycans (GAGs). Glucosamine, as glucosamine 5-phosphate,
is naturally occurring within the body and is a component in the
biosynthesis of glycosaminoglycans, proteoglycans, hyaluronan, and
collagen. Glucosamine is available in exogenous forms, glucosamine
sulfate sodium, glucosamine hydrochloride and N-acetyl
D-glucosamine. N-acetyl D-glucosamine is also a derivative of
glucose obtained by chemical hydrolysis of chitin. This
polysaccharide is readily soluble in water and extremely
bioavailable. N-acetyl D-glucosamine binds to glucuronic acid as
well as galactose making it a precursor to hyaluronic acid,
keratan-sulfate and chondroitin sulfate. This unique derivative
aids in proteoglycan, collagen and glycosaminoglycan production.
N-acetyl D-glucosamine has also been shown to aid in the healing of
soft tissue injury. D-Glucuronic acid is a key substrate comprising
one half of the hyaluronan molecule, the other being N-acetyl
D-glucosamine.
[0009] Supplemental glucosamine has the ability to influence
connective tissue such as cartilage, and so may apply to
alleviation of various dysfunctions including arthritis. In the
joint, for example, chondroitin sulfate acts to stimulate the
production of proteoglycans, glycosaminoglycans, and collagen,
inhibits degenerative enzymes excreted by the chondrocytes, and
synoviocytes, and aids in nutrient transportation within the
synovial fluid. Glucosamine, in particular N-acetyl D-glucosamine,
increases the synoviocyte and chondrocyte production and subsequent
availability of endogenous hyaluronan by the direct in situ
inclusion of its prime substrates galactosamine (through
chondroitin sulfate assimilation) and N-acetyl D-glucosamine. The
exogenous hyaluronan acts to replace depleted endogenous hyaluronan
and to lubricate and coat healthy as well as damaged articular
tissue during the reparative process.
[0010] A GAG composition marketed as a veterinary medical device as
POLYGLYCAN (ArthroDynamic Technologies) comprises chondroitin
sulfate, N-acetyl D-glucosamine, and hyaluronic acid. Such
proteoglycan compositions are described in U.S. Pat. Nos. 6,979,679
and 7,485,629, which are hereby incorporated by reference in their
entireties.
[0011] Regenerative cells found in multi-cellular organisms are
cells capable of promoting tissue repair and regeneration and
reducing inflammation. Stem cells are regenerative cells that can
differentiate into a diverse range of specialized cell types. The
two broad types of mammalian stem cells are: embryonic stem cells
that are found in blastocysts, and adult stem cells that are found
in adult tissues. The two classical properties of stem cells are
self-renewal and potency. Self-renewal refers to the ability to go
through numerous cycles of cell division while maintaining the
undifferentiated state, and potency refers to the capacity to
differentiate into specialized cell types. Potency specifies the
differentiation potential of the stem cells to differentiate into
different cell types. For instance, totipotent stem cells are cells
produced from the fusion of an egg and sperm cell, as well as the
first few divisions of the fertilized egg, they can differentiate
into embryonic and extra-embryonic cell types. Pluripotent stem
cells are the descendants of totipotent cells and can differentiate
into cells derived from any of the three germ layers. Multipotent
stem cells can produce only cells of a closely related family of
cells (e.g. hematopoietic stem cells differentiate into red blood
cells, white blood cells, platelets, etc.). Unipotent cells can
produce only one cell type, but have the property of self-renewal
that distinguishes them from non-stem cells (e.g., muscle stem
cells). In addition, a regenerative cell population frequently
comprises not only cells but also microsomes released by the
regenerative cells that are important in functions such as
immunomodulation inside the body.
[0012] Progenitor cells refer to immature or partially
undifferentiated regenerative cells, typically found in post-natal
animals. Like stem cells, progenitor cells have a capacity for
self-renewal and differentiation, although these properties may be
more limited. Embryonic stem cells are pluripotent and show
unlimited capacity for self-renewal. Thus, they are sometimes
referred to as true stem cells. In contrast, many cells termed
adult stem cells would be better defined as progenitor cells, as
their capacities for unlimited self renewal and plasticity have not
been comprehensively demonstrated. The majority of progenitor cells
are dormant or exhibit little activity in the tissue in which they
reside. They exhibit slow growth and their main role is to replace
cells lost by normal attrition. However, upon tissue damage or
injury, progenitor cells can be activated by growth factors or
cytokines, leading to increased cell division important for the
repair process. Examples of progenitor cells include satellite
cells found in muscle and the transit-amplifying neural progenitors
of the rostral migratory stream.
[0013] Mesenchymal stem cells ("MSCs") (i.e., stromal cells) are
pluripotent regenerative cells that can differentiate into a
variety of cell types. MSCs can be derived from many tissues
including bone marrow, adipose, umbilical cord, and dental pulp.
MSCs adhere to plastic tissue culture and are commonly selected
from more diverse regerative cell populations based on this
property of plastic adherence. For example, bone marrow MSCs
(BM-MSCs) can be selected from the cell population present in a
bone marrow aspirate by culturing the cells that adhere to the
plastic tissue culture surface, and adipose MSC (Ad-MSC) can be
selected from adipose stromal vascular fraction (SVF) cells in the
same manner. Adipose SVF is the non lipid-filled cell population
from adipose tissue and contains a high proportion of regenerative
cells. Among the cell types that MSCs have been shown to
differentiate into in vitro or in vivo are osteoblasts,
chondrocytes, myocytes, adipocytes, neuronal cells, and
beta-pancreatic islets cells. MSCs provide the supportive structure
in which the functional cells of the tissue reside. In addition,
MSCs play roles in tissue healing and repair.
[0014] Because in adult organisms, MSCs act as a repair system for
the body replenishing specialized cells but also maintaining tissue
homeostasis and they are capable of differentiating into different
types of tissues, they have been utilized in the treatment of, for
example, skeletal and connective tissue disorders.
[0015] There is increasing evidence that regenerative cells derived
from other tissue, such as adipose-derived regenerative cells, are
equally or even more capable than bone marrow derived regenerative
cells in repairing or alleviating connective tissue dysfunctions.
(See, Toghraie et al., "Treatment of Osteoarthritis with
Infra-Patellar Fat Pad Derived Mesenchymal Stem Cells in Rabbit,"
Knee, 2011, 18 (2):71-75; and Frisbie et al., "Evaluation of
Adipose Derived Stromal Vascular Fraction or Bone Marrow Derived
Mesenchymal Stem Cells for Treatment of Osteoarthritis," J. Orthop.
Res., 2009, 27 (12):1675-1680.)
[0016] Intra-articular administration of stem and regenerative
cells, particularly MSCs from adipose tissue and bone marrow, is
increasingly utilized in clinical practice. Current practice is to
administer cells suspended in an inactive carrier such as saline or
platelet rich plasma, or in a single component non-sulfated GAG,
such as hyaluronan. To date, minimal data have been reported as to
the effects of GAGs on stem and regenerative cells and on the
optimal composition of GAG formulations for combination with stem
and regenerative cells for in vitro and in vivo applications.
SUMMARY OF THE INVENTION
[0017] Use of cells suspended in a GAG composition that includes
hyaluronic acid in combination with at least one sulfated GAG,
either as an acceptable admixture or in concurrent or sequential
administration, is one aspect of the present invention.
Administration of cells in such a GAG formulation provides benefits
not only for intra-articular injection but also for other modes of
administration such as subdermal, subcutaneous, topical,
intra-muscular, intravenous, intra-capillary such as the cavernous
body, intra-arterial, intra-thecal, directly into organs, into the
urinary bladder, into tendons and the peri-tendineum, in the
periosteum, and into cavities such as bone cysts.
[0018] In addition, culturing, preserving, or cryopreserving of
cells in such a GAG composition provides benefits for cell
proliferation, viability, and regenerative potential. The invention
provides compositions and methods for the use of a GAG formulation
that includes hyaluronic acid in combination with at least one
sulfated GAG for culturing, suspending, preserving, cryopreserving,
or administering or any combination thereof of cells derived from
animals or humans.
[0019] In one aspect, the present invention provides a media
supplement for enhanced effectiveness in preservation, suspension,
storage, cryopreservation, culturing, growing, and proliferating
cells, such as adipose-derived regenerative cells (ADRCs), while
maintaining differentiation potential in vitro and in vivo. In
certain embodiments, the media or media supplement of the present
invention comprises a glycosaminoglycan (GAG) formulation
comprising one or more GAGs in a defined concentration. In certain
embodiments, particularly adapted for culturing, growing, and
proliferating cells, while maintaining pluripotency, the specified
GAG composition is diluted to a concentration of 1-10% (v/v),
2.5-7.5% (v/v), 3-7% (v/v), or 5% (v/v) based on the final volume
of the media. In other embodiments, particularly adapted for
preservation, suspension, storage and cryopreservation, the
specified GAG formulation is diluted to a concentration of 5%
(v/v), 10% (v/v), 20% (v/v), 30% (v/v), 40% (v/v), 50% (v/v), 60%
(v/v), or 70% (v/v), or more based on the total volume of the
media.
[0020] In an embodiment of the present invention, a media or media
supplement comprising a GAG composition comprises proportions of
about 1:20:20 (mg/ml) of hyaluronic acid (HA):chondroitin sulfate
(CS):N-acetyl D-glucosamine (NaDg). Such a composition can, for
example, comprise hyaluronic acid sodium salt (5 mg/ml), sodium
chondroitin sulfate (100 mg/ml) (either or both CS4 and CS6 forms),
and N-acetyl D-glucosamine (100 mg/ml) such as in the commercially
available POLYGLYCAN composition. The proportions of the GAG
composition of the present invention can also vary from 0.1-10
HA:2-200 CS:2-200 NaDg (mg/ml).
[0021] In certain embodiments, the present invention provides that
adipose derived regenerative cells (ADRCs) proliferate more
adherent cells, and are blocked from chondrogenic and osteogenic
differentiation in the presence of a GAG combination of hyaluronic
acid, chondroitin sulfate and N-acetyl D-glucosamine in the cell
media at 1%-10% (v/v) or about 5% (v/v) concentration. In other
embodiments, the present invention provides that a formulation
containing only hyaluronic acid (HA) is pro-mitotic, but to a
lesser magnitude.
[0022] One aspect of the present invention provides a method of
enhancing an in vitro expansion rate of cells while maintaining
differentiation potential. In this aspect, the invention method
comprises culturing the cells in the culture media of the present
invention, or treating the cells with the media supplement of the
present invention, or coating the surface on which the cells are
growing with a soluble matrix of the present invention, wherein the
culture media, media supplement, cryopreservant, or soluble matrix
of the present invention comprises a specified GAG composition,
which comprises one or more GAGs at a defined concentration. In
certain embodiments, the specified GAG formulation comprises a
POLYGLYCAN composition of hyaluronic acid, chondroitin sulfate and
N-acetyl D-glucosamine at a concentration of up to 50% (v/v), 1-10%
(v/v), 3-7% (v/v), or about 5% (v/v). In other embodiments, the
specified GAG formulation comprises hyaluronic acid (HA), and
optionally a sulfated chondroitin, at a concentration of up to 50%
(v/v), 1-10% (v/v), 2.5-7.5% (v/v) or 5% (v/v). In certain
embodiments, the cells are cultured in such culture media of the
present invention for hours and days, in some cases for 12-20
hours, before being harvested for further use. In other
embodiments, the cultured cells remain in the media, or are exposed
to supplemental media, before further use.
[0023] An aspect of the present invention further provides a
soluble matrix comprising a specified GAG formulation for
administration of cells for the treatment of diseases such as joint
or other connective tissue damage. In certain embodiments, the
specified GAG formulation in the soluble matrix comprises a
POLYGLYCAN composition at a concentration of 1-20% (v/v). In other
embodiments, the specified GAG formulation in the soluble matrix
comprising hyaluronic acid, chondroitin sulfate, or other GAGs in
the same ratio as those in the POLYGLYCAN composition. A cell
administration package comprising the soluble matrix of an aspect
of the present invention and a device for cell administration is
also provided.
[0024] An aspect of the present invention further provides a
method, and composition thereof, for preventing and treating a
joint disease or other tissue damage, or specifically in the repair
of cartilage and connective tissue, in an affected site in animals
or humans by administering the cells cultured and proliferated in
the culture media, or treated, stored or cryopreserved in the media
or with the media supplement of the present invention. In certain
embodiments, the cells are mixed with the soluble matrix of an
aspect of the present invention comprising a specified GAG
formulation, such as the POLYGLYCAN composition, for administration
of the cells. The pharmaceutical preparation described herein
comprises a POLYGLYCAN and other proteoglycan or GAG compositions
in combination with cells. Methods of treatment are provided herein
using the POLYGLYCAN composition and other proteoglycan or GAG
compositions in combination with regenerative cells, either as a
pharmaceutically acceptable admixture or in concurrent or
sequential administration. A cell administration package or kit
comprising the soluble matrix comprising a specified GAG
formulation, and a device for cell administration are also
provided.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIGS. 1A and 1B describe an effect of GAG concentration on
proliferation on Ad-MSCs. Fresh canine adipose SVF cells were
plated at equal nucleated cell density and grown for 7 days in
culture on plastic tisse culture surface in the presence of
complete growth media (.alpha.-MEM with 20% (v/v) FBS) and the
respective concentrations (v/v) of GAG formulation (FIG. 1A).
Nucleated cell counts were performed by Syto13 staining followed by
hemacytometer counting under fluorescence microscopy (FIG. 1B).
[0026] FIG. 2 shows that Ad-MSCs proliferate faster in POLYGLYCAN
formulation at 5% concentration (v/v). Fresh canine adipose-derived
stromal cells were plated at equal nucleated cell density and grown
for 7 days in culture on plastic tisse culture surface in the
presence of complete growth media and the respective concentrations
(v/v) of GAG formulation. Nucleated cell counts were performed by
Syto13 staining followed by hemacytometer counting under
fluorescence microscopy *P<0.05 .
[0027] FIGS. 3A and 3B show that Ad-MSCs proliferate faster and
express higher levels of Sox2 when cultured in GAG formulations.
Canine Ad-MSCs were grown for 6 days in culture in the presence of
complete growth media and the respective 5% (v/v) GAG formulation.
Magnification=100.times.. Sox2 levels, a marker of stem cell
proliferation, were measured in total RNA samples and normalized to
.beta.-Actin.
[0028] FIGS. 4A and 4B show that colony formation is enhanced by
POLYGLYCAN. A total of 0.25.times.10.sup. 6 nucleated canine
adipose SVF cells were plated and grown for 14 days in culture on
tissue culture plastic surface. Results shown depict colony
formation in Growth Media with 5% (v/v) GAG formulation,
*P<0.05.
[0029] FIG. 5 shows Ad-MSC cell surface marker expression after
exposure to HA formulations in vitro for six days. Human Ad-MSC CD
marker profiling after 6 days culture in GAG formulation. FACS
analysis was performed using the GALLIOS.TM. Flow Cytometer.
[0030] FIG. 6 shows that Ad-MSC proliferation is maintained while
osteogenic differentiation is inhibited by GAG formulations
containing sulfated GAG in vitro. Cultures were maintained in
osteogenic differentiation induction medium for 14 days, in 5%
(v/v) formulation. Cultures were fixed with 4% formalin and stained
with Alzarin red that stains calcium.
[0031] FIG. 7 shows that Ad-MSC proliferation is maintained while
chondrogenic differentiation is inhibited by GAG formulations
containing sulfated GAG in vitro. Cultures were maintained in
chondrogenic differentiation induction medium for 14 days, in 5%
(v/v) formulation. Cultures were fixed with 4% formalin and stained
with Alcian Blue that contains chondrocytes.
[0032] FIG. 8 shows that osteogenic differentiation is highly
efficient in Ad-MSC after in vitro exposure and later removal of
GAG-containing formulations. Cultures were maintained in osteogenic
differentiation medium for 14 days after being cultured in GAG
formulation for 6 days. Cultures were fixed with 4% formalin and
stained with Alzarin red that stains calcium.
[0033] FIG. 9 shows that chondrogenic differentiation is highly
efficient in Ad-MSC after in vitro exposure and later removal of
HA-containing formulations. Cultures were maintained in
chondrogenic differentiation induction medium for 14 days after
being cultured in GAG formulation for 6 days. Cultures were fixed
with 4% formalin and stained with Alcian Blue.
[0034] FIG. 10 shows cell counts of nucleated viable canine stromal
vascular cells after cryopreservation in the presence of
GAG-containing formulations compared to cells cryopreserved in 5%
DMSO and 85% fetal bovine serum (FBS).
[0035] FIG. 11 shows flow activated cell sorting (FACS) analysis of
cell surface marker expression of canine stromal vascular cells
after cryopreservation in the presence of GAG-containing
formulations compared to cells cryopreserved in 5% DMSO and 85%
fetal bovine serum (FBS).
[0036] FIG. 12 shows expression of stem cell markers in Canine
Ad-MSC growing in GAG formulations.
[0037] FIG. 13 shows POLYGLYCAN induced changes in gene expression
in hADSC after culturing for 6 days.
[0038] FIG. 14 shows cryopreservation recovery after 1 month in
-80.degree. C.
[0039] FIG. 15 shows cultured hADSC stability analysis after
shipment.
[0040] FIG. 16 shows fresh hADSC stability analysis after
shipment.
DETAILED DESCRIPTION OF THE INVENTION
[0041] Additional objects, advantages and other novel features of
the invention will be set forth in part in the description that
follows and in part will become apparent to those skilled in the
art upon examination of the foregoing or may be learned with the
practice of the invention. Additionally, throughout this document,
various publications and patents have been cited, the contents of
which are incorporated herein by reference in their entirety.
[0042] In one aspect, the present invention provides a media or
media supplement for enhanced cell growth and maintenance of
differentiation potential. In one embodiment, the invention media
or media supplement comprises a specified proteoglycan or GAG
formulation, in certain embodiments comprising a POLYGLYCAN
composition, or hyaluronic acid (HA) and other GAGs in a defined
concentration. In certain embodiments, the invention media or media
supplement comprises the POLYGLYCAN composition consisting of
hyaluronic acid sodium salt (5 mg/ml), sodium chondroitin sulfate
(100 mg/ml), and N-acetyl D-glucosamine (100 mg/ml). In certain
embodiments, the media or media supplement comprises a composition
in mg/ml proportions of about 1:20:20 of hyaluronic
acid:chondroitin sulfate:N-acetyl D-glucosamine. Supplement
compositions can comprise sodium hyaluronic acid and other sulfated
glycosaminoglycans (GAGs). In another embodiment of the present
invention, a media or media supplement comprising a composition in
mg/ml proportions of about 1:10:10 of hyaluronic acid:chondroitin
sulfate:N-acetyl D-glucosamine can be applied. As can be
appreciated, the final dilution of the composition can be adjusted
according to the intended use.
[0043] As used herein, the terms "proteoglycan composition" or "GAG
formulation" or "GAG composition" described herein are used
interchangeably, which refer to a composition or formulation
comprising one or more glycosaminoglycans (GAGs), including but not
limited to, hyaluronic acid, chondroitin sulfate and N-acetyl
D-glucosamine, and is formulated into any acceptable formulations
suitable for storage, cryopreservation, culture media, media
supplement, or matrix, or for therapeutic administration. The GAG
composition of the present invention includes, but is not limited
to, a sterile solution or suspension, or matrix or gel that can be
mixed with cells, or any cell culture, cryopreservation, or
suspension media or media supplement, known or later developed in
the art in culturing, administering, suspending, or cryopreserving
regenerative cells, or can be used to coat the surface on which the
regenerative cells are growing in any suitable media known or later
developed in the art. Methods of treatment are provided herein
using the GAG composition in combination with regenerative cells,
either as an acceptable admixture or in concurrent or sequential
administration. The GAG composition of the present invention can
also be formulated for direct application or intra-articular,
intramuscular, intravenous, subcutaneous, or other parenteral or
systemic administration to a subject in need for treating a joint
or other connective tissue damage or weakness, along with the
regenerative cells.
[0044] Depending on the embodiments, various GAGs can be included
in the specified GAG composition described herein. In certain
embodiments, the GAG composition comprises, or consists essentially
of, chondroitin sulfate, glucosamine, and hyaluronan. In certain
embodiments, the GAG composition comprises, or consists essentially
of, glucosamine and hyaluronan. In certain embodiments, the GAG
composition comprises a mixture of chondroitin sulfate,
poly-sulfated GAGs, glucosamine and hyaluronan, and can be stored
in a single container at room temperature, in a refrigerator or a
freezer. In other embodiments, the glucosamine, such as N-acetyl
D-glucosamine, is stored in a separate container at room
temperature, in a refrigerator or freezer, and can be mixed with
the chondroitin sulfate and hyaluronan mixture before
administration. In yet other embodiments, the GAG composition
comprises chondroitin sulfate, (both CS4 and CS6 forms) hyaluronan,
and glucosamine, such as N-acetyl D-glucosamine, all mixed together
and stored in a single container ready for mixing with media, or
coating the surface for cells to grow, or direct co-administration
with the cells to a subject in need.
[0045] In certain embodiments, the GAG composition is the
POLYGLYCAN composition (Arthrodynamic Technologies, Lexington, Ky.)
consisting essentially of an effective amount of chondroitin
sulfate, N-acetyl D-glucosamine, and hyaluronan (hyaluronic acid).
In certain embodiments, the chondroitin sulfate in the proteoglycan
composition is preferably chondroitin 4-sulfate (CS4), chondroitin
6-sulfate (CS6), or a mixture of both CS4 and CS6. An effective
amount of chondroitin sulfate and N-acetyl D-glucosamine is
preferably from between about 0.5 grams to about 1.5 grams of per
unit dose, respectively, and an effective amount of hyaluronan is
preferably from about 10 mg to about 50 mg per unit dose. The
detailed descriptions of POLYGLYCAN composition, or its
equivalents, and method of preparation and use of such composition
are described in U.S. Pat. Nos. 6,979,679 and 7,485,629, and the
entire contents of these patents are incorporated by reference
herewith.
[0046] As used herein, the term "stem cells" or "regenerative
cells" are used interchangeably and are cells capable of retaining
the ability to reinvigorate themselves through mitotic cell
division and which can differentiate into more than one specialized
cell types. In one embodiment, the regenerative cells described
herein are mesenchyme and/or stromal cells including, but not
limited to, osteoblasts, chondrocytes, chondrocyte progenitor cells
including mesenchymal stem cells or MSCs, fibroblasts,
fibroblast-like cells, and SVF cells or other stromal cells capable
of producing collagen types and proteoglycans which are typically
produced in cartilaginous tissues. In yet another embodiment, the
regenerative cells described herein are stromal cells capable of
producing osteoblasts, adipocytes, and chondroblasts. In yet
another embodiment, the regenerative cells described are able to
differentiate into mesodermal, endodermal, or ectodermal
lineages.
[0047] In yet another embodiment, the regenerative cells are
chondrogenic stem and/or progenitor cells including mesenchymal
stem cells or MCSs. In further embodiments, the mesenchymal stem
cells or MCSs are animal mesenchymal stem cells isolated from an
animal tissue specimen. In yet other embodiments, the progenitor
cells can be obtained from a patient in an autologous or allogenic
manner. The progenitor cells, fibroblast-like cells and other cells
and/or elements that comprise the stroma may be fetal or adult in
origin, and may be derived from convenient sources such as adipose,
cartilage, bone, skin, ligaments, tendons, muscles, placenta,
umbilical cord, etc. For example, stromal cells such as
chondrocytes may be derived from any type of cartilage, including
but not limited to, hyaline cartilage, costal cartilage, fibrous
cartilage, etc., which can be obtained by biopsy (where
appropriate) or upon autopsy.
[0048] Regenerative cells are typically used in the present
invention in an isolated state, in that they are provided in
concentrated numbers or a cellular culture free from at least some
of the other constituents with which they are found in nature.
Regenerative cells may be derived from various sources including
adipose tissue, bone marrow, umbilical cord, placenta, dental pulp,
tendons, muscle, or skin. Regenerative cells from a variety of
sources may be used in the present invention.
[0049] The regenerative cells used in the present invention may be
readily isolated by disaggregating an appropriate tissue.
Furthermore, once cells have been isolated, their population can be
expanded mitotically, and if preferred, enriched in certain cell
types, in order to obtain the cell preparation for the combination
with the glycosaminoglycans in the composition disclosed in the
present invention.
[0050] In certain embodiments, the present invention provides that
regenerative cell viability and proliferation of colony forming
cells is enhanced while differentiation potential is preserved. In
certain embodiments, the present invention facilitates plastic
adherence of stromal cells and reduces anoikis-induced
apoptosis.
[0051] In certain embodiments, the regenerative cells are preserved
with one or more appropriate well-known additional cryoprotectants
in the compositions described herein. In one embodiment, the
regenerative cells provided herein, either uncultured or cultured,
or previously preserved in the presence of one or more suitable
cryoprotectants, can be combined with the compositions described
herein. In addition, regenerative cells can be properly protected
during the cryopreservation process when combined with the
compositions described herein, yielding improved viability.
[0052] In certain embodiments, the effectiveness of the present
invention is demonstrated by viable cell count and flow cytometry
analysis of canine and human adipose SVF cells cryopreserved in
various formulations containing different GAGs at different
concentrations.
[0053] The present invention further provides a method, and
composition thereof, for administering into a human or animal body
regenerative cells cultured and proliferated in the culture media
or treated with the media supplement or grown on the surface coated
with the media supplement or matrix of the present invention. In
certain embodiments, the regenerative cells are cultured,
preserved, or cryopreserved in the media or media supplement of the
present invention and then mixed with the matrix of the present
invention comprising a specified GAG formulation, such as the
POLYGLYCAN composition, or any other suitable GAG formulations, for
administration of the regenerative cells. The preparation described
herein comprises a POLYGLYCAN or any other GAG formulations in
combination with regenerative cells. Methods of treatment are
provided herein using the POLYGLYCAN or any other GAG formulation
in combination with regenerative cells, either as an acceptable
admixture or in concurrent or sequential administration. However,
the dilution of the POLYGLYCAN or any other GAG formulation used in
culturing, preservation, and cryopreservation may differ from the
dilution used for the administration of regenerative cells.
Dilution of GAG formulations used for administration of
regenerative cells may vary depending on specifics of the condition
of the patient and the anatomical site of administration.
[0054] As used herein, the preparation of the present invention is
formulated in a suitable form, including, but not limited to, a
form of sterile solution, suspension, a scaffold such as a stent,
sponge, suture, or matrix, or a gel- or paste-like formulation.
Molecular weights of the GAG in the GAG formulation can be selected
to determine the physical properties of the suitable form.
Administration of regenerative cells in such a GAG formulation
provides benefits not only for intra-articular injection but also
for other modes of administration such as subdermal, subcutaneous,
topical, intra-muscular, intravenous, intra-capillary such as the
cavernous body, intra-arterial, intra-thecal, directly into organs,
into the urinary bladder, into tendons and the peri-tendineum, in
the periosteum, and into cavities such as bone cysts. Systemic
administrations can include, but are not limited to, intramuscular,
intravenous or subcutaneous injection or via direct adsorption into
the bloodstream via non-gastrointestinal transmucosal, e.g.,
sublingual administration.
[0055] It is contemplated by certain embodiments of the invention
that the trasnsmucosal delivery can include any mucosal tissue that
provides a mucosal surface area for direct adsorption into the
blood stream and that does not subject the compositions of the
invention to digestion and/or other alteration via gastric or
intestinal enzymes. The compositions can be provided as liquids or
semi-solids for direct application to the desired mucosal tissue.
The compositions can be formulated into any of a variety of
presentations designed to enhance and/or prolong contact with the
desired mucosal tissue to promote adsorption into the bloodstream.
For example, the compositions can be incorporated into a
dissolvable or biodegradable film for placement e.g., under the
tongue or as an oral or nasal spray or other presentation designed
to enhance and/or prolong contact with the mucosa of the oropharnyx
or other target tissue.
[0056] In yet another preferred embodiment, the compositions
provided herewith are attached to a sheet of material adapted for
implantation onto or between tissues of a human or animal body.
Preferably, the compositions are impregnated into a polymeric
gauze-like material or coated onto a gauze-like material or joined
to the material by adhesion and/or capillary action. The material
onto which the composition is attached may be either a permanent
implant or it may be biodegradable. In yet another preferred
embodiment, the composition provided herewith is attached to a
bandage or other surgical materials, including, but not limited to,
surgical suture material, surgical staple, or a device such as a
buckle.
[0057] The pharmaceutical preparation described herein further may
optionally comprise one or more other therapeutic agents,
including, but not limited to, synthetic and non-synthetic
corticosteroid agents, nonsteroidal anti-inflammatory drugs,
analgesics, antirheumatics, immunoregulators, immunosuppressant,
articular function augmenters, interleukin production inhibitors,
or growth factor, all of which have therapeutic effects. Any drugs,
agents, compounds, known and/or to be developed, showing any
desired therapeutic effects are within the scope of this invention.
In other embodiments, the invention may specifically exclude one or
more of the above therapeutic agents. In yet another embodiment,
the stem cells, or the pharmaceutically acceptable formulation
comprising the regenerative cells, may further comprise other cells
that aid in the production of one or more tissues including, for
example, muscle, cardiac, neural, and connective tissues.
[0058] The composition of the present invention can be used in the
prevention or treatment of connective tissue damage, which includes
any primary or secondary diseases or injuries to the connective
tissues in humans or animals. Such diseases or injuries include,
but are not limited to, arthritic diseases, osteoarthritis (OA),
rheumatoid arthritis (RA), osteochondrosis dessicans (OCD),
cartilage damage, joint injuries, joint inflammation, joint
synovitis, degenerative joint disease (DJD), post surgical DJD,
traumatic injuries, fractures, tendon damage, ligament damage,
skeletal damage, musculoskeletal damage, bone damage, fiber damage,
adipose tissue damage, blood cell damage, and plasma damage.
[0059] Having discussed the media or media supplement comprising a
specified GAG formulation, and the method of use thereof, providing
an enhanced effectiveness of culturing, growing, preserving,
cryopreserving, administering, and proliferating regenerative
cells, while maintaining differentiation potentials, and for the
treatment of animals and humans. It will be more clearly perceived
and better understood from the following specific examples that are
intended to provide examples of certain preferred embodiments and
not limit the scope of the present invention.
EXAMPLES
Materials
[0060] POLYGLYCAN is a commercially available patented formulation
of hyaluronic acid, sodium chondroitin sulfate and N-acetyl
D-glucosamine used for post-surgical lavage of synovial
compartments because it contains naturally occurring components of
synovia that play a central role in maintaining the homeostatic
environment of the joint. POLYGLYCAN is also designed to replace
synovial fluid lost during surgery. Commercially available
POLYGLYCAN (Arthrodynamic Technologies) is a highly viscous aqueous
solution of defined fractions of purified hyaluronic acid,
chondroitin sulfates C4 & C6 in a 10% solution of N-acetyl
D-glucosamine. Each 10 mL vial contains 50.0 mg hyaluronic acid
sodium salt, 1000 mg sodium chondroitin sulfate, and 1000 mg
N-acetyl D-glucosamine.
[0061] Hyaluronic acid is a natural, highly charged, polyanionic
molecule composed of alternating units of D-glucuronic and
2-acetamido-2-deoxy-D-glucose. These unbranched, coiled, elongated
polysaccharide chains maintain a large negative electrostatic
charge that attracts water molecules and allow the deformation of
the molecular coil as ice crystallization occurs during freezing
and thawing. One commercially available source of hyaluronic acid
is MAP5 (Bioniche). ADEQUAN Canine (Luitpold Animal Health) is a
prescription, water-based, intramuscular, poly sulfated
glycosaminoglycan (PSGAG).
Methods
[0062] Adipose SVF cells were obtained from canine adipose tissue
taken at spay procedures and from human lipoaspirate samples
procured under IRB protocol and with informed consent from patients
undergoing elective lipoplasty. Primary cell preparations were
obtained using a point-of-care tissue processing system and
associated disposables and reagent (ARC.TM. System and MATRASE.TM.
Reagent, InGeneron, Inc. Houston, Tex.). Ad-MSC were obtained by
culture of primary cells in Alpha DMEM, 20% (v/v) FBS, with
antibiotic (pen/strep). Commercially available formulations of
polysulfated glycosaminoglycans (ADEQUAN, Luitpold Animal Health,
Shirley, N.Y.), a formulation of hyaluronic acid, N-acetyl
D-glucosamine, and chondroitin sulfate (POLYGLYCAN, Arthrodynamic
Technologies, Lexington, Ky.), and a formulation of hyaluronic acid
(HA) (MAP-5, Bioniche, Athens, Ga.), and individual GAGs were
tested at concentrations up to 50% (v/v).
[0063] Assays for cell proliferation, colony forming units (CFU),
gene expression analyses, cell surface markers, and chondrogenic
and osteogenic differentiation were performed. Cell growth and
Colony Forming Units (CFU) were grown in monolayer on tissue
culture grade plastic in alpha MEM, 20% FBS (v/v), pen/strep.
Nucleated cell counts were performed by staining with Syto13
(Invitrogen) followed by hemacytometer counting under fluorescence
microscopy. Gene expression analysis was performed by quantitative
RT-PCR profiling, BioRad iQ. Flow Activated Cell Storing (FACS)
analyses were performed at M.D. Anderson Cancer Center FACS Core
Facilities, Houston, Tex. by GALLIOS Flow Cytometry Instruments
(Beckman Coulter). For osteogenic and chondrogenic differentiation,
cells were cultured in STEMPRO induction media (Invitrogen,
Carlsbad, Calif.) for 14 days, fixed in 4% formalin, and stained
with Alzarin red and Alcian Blue, respectively. Statistical
analyses were performed by ANOVA.
[0064] The SVF cells are obtained from subcutaneous adipose tissue
by known methods, preferably by processing with the help of
collagenase 1, collagenase 2, and the neutral protease in the
MATRASE.TM. Reagent formulation. The typical cell count obtainable
is between 600,000 and 1 million nucleated cells per gram of human
subcutaneous adipose tissue, 1 million to 1.5 million per gram of,
and 1.2 to 3 million per gram of equine adipose tissue. A higher
number of cells can be obtained in those with a lower body mass
index. This indicates that the relative percentage of adipose cells
compared to the stromal vascular fraction is lower. Also, meaning
that 1 gram of tissue contains relatively more interstitial stromal
vascular component compared to the adipocytes, while in obese
individuals the number of cells obtained per gram tissue is lower
due to the higher relative percentage of adipocytes.
[0065] After recovering the cells, they were subjected to a 50%
POLYGLYCAN dilution with serum (preferably autologous) and frozen
by known methods whereby the temperature gradient by minute change
was controlled in order to prevent cell rupture. The 50% POLYGLYCAN
dilution--due to its composition and physical properties--prevented
cell death. After storing in -18 degrees Celsius to -20 degrees
Celsius, the rate of apoptosis after thawing compared to the rate
of apoptosis at time of freezing was negligible. For prolong
periods of preservation such as months and years, storage at -70
degrees Celsius or even lower degrees centigrade prevents cell
death.
[0066] After thawing, the cells can be used for injection. However,
if they are injected into a defined compartment such as a joint, a
dilution at the final site of up to a 5 percent content of
POLYGLYCAN may be beneficial. Such a dilution can be obtained by
diluting the initial solution with regenerative cells from 50% to,
for example, 10%. As an example: if the cells were frozen in 50%
POLYGLYCAN in a glass vial of 1.8 ml, a further dilution to 9 ml
would yield a 10% POLYGLYCAN content in which the regenerative
cells are dispersed. Upon injection into, for example, a knee that
typically has about 18 ml of synovial fluid, 9 ml of this synovial
fluid is removed by puncture and 9 ml of regenerative cells
solution will be injected making the final solution of the
POLYGLYCAN in the knee joint a 5% solution which has shown a
beneficial effect on cell growth.
[0067] Shipping of regenerative cells typically results in an
increased rate of apoptosis even if the cells are kept in optimal
culture conditions with temperature, media, and CO.sub.2 adjusted
to optimum levels. This is due to anoikis, which results from a
lack of adherence to extracellular matrix. The GAG formulation of
the present invention binds to certain cell surface receptors, such
as the CD44 receptor and provides a natural matrix for preventing
anoikis and is, therefore, in a formulation, such as that of the
present invention, represents a preferable shipping medium.
RESULTS
[0068] FIGS. 1A and 1B show the effect of GAG concentration on
proliferation on Ad-MSCs was demonstrated in FIGS. 1A-B. Fresh
canine adipose SVF cells were plated at equal nucleated cell
density and grown for 7 days in culture on tissue culture plastic
in the presence of complete growth media (.alpha.-MEM containing
20% (v/v) FBS) and the respective concentrations (v/v) of GAG
formulations (FIG. 1A). Media was changed at days 3 and 6.
Nucleated cell counts were performed by Syto13 staining followed by
hemacytometer counting under fluorescence microscopy (FIG. 1B).
[0069] The invention demonstrates that Ad-MSCs proliferate faster
in POLYGLYCAN formulation at 5% concentration (v/v) (see FIG. 2).
Fresh canine adipose SVF cells were plated at equal nucleated cell
density and grown for 7 days in culture on tissue culture plastic
in the presence of complete growth media and the respective
concentrations (v/v) of GAG formulation. Nucleated cell counts were
performed by Syto13 staining followed by hemacytometer counting
under fluorescence microscopy. Data represent average +/-SD for
triplicate determinations of cell number at day 7*P<0.05 .
[0070] The invention further demonstrates that Ad-MSCs proliferate
faster and express higher levels of Sox2 when cultured in GAG
formulations (see FIGS. 3A and 3B). Canine Ad-MSCs were grown for 6
days in culture in the presence of complete growth media and the
respective 5% (v/v) GAG formulations. Magnification=100.times..
Sox2 levels, a marker of stem cell proliferation, were measured in
total RNA samples and normalized to .beta.-Actin mRNA. Here "GAG"
represents poly-sulfated glycosaminoglycan (ADEQUAN). FIG. 3B
clearly shows the combination of HA, CS and NaDg in POLYGLYCAN
resulted in a substantially greater increase in Sox2 levels.
[0071] Colony formation is also significantly enhanced by the GAG
composition of POLYGLYCAN (see FIGS. 4A, 4B) over poly-sulfated GAG
(ADEQUAN) or hyaluronan (MAP5) alone. A total of 0.25.times.10.sup.
6 nucleated canine adipose-SVF cells were grown for 14 days in
culture on tissue culture plastic. Results shown depict colony
formation in Growth Media with 5% (v/v) GAG formulation,
*P<0.05.
[0072] FIG. 5 describes Ad-MSC cell surface marker expression after
exposure to GAG formulations in vitro for six days. Human Ad-MSC CD
marker profiling after 6 days culture in GAG formulation. FACS
analysis done in GALLIOS.TM. Flow Cytometer.
[0073] FIG. 6 describes that Ad-MSC proliferation is maintained
while osteogenic differentiation is inhibited by GAG formulations
containing sulfated GAG in vitro (ADEQUAN and POLYGLYCAN). Cultures
were maintained in osteogenic differentiation induction medium for
14 days, in 5% (v/v) formulation. Cultures were fixed with 4%
formalin and stained with Alzarin red that stains calcium.
[0074] FIG. 7 describes that Ad-MSC proliferation is maintained
while chondrogenic differentiation is inhibited by GAG formulations
containing sulfated GAG in vitro (ADEQUAN and POLYGLYCAN). Cultures
were maintained in chondrogenic differentiation induction medium
for 14 days, in 5% (v/v) formulation. Cultures were fixed with 4%
formalin and stained with Alcian Blue that contains
chondrocytes.
[0075] FIG. 8 describes that osteogenic differentiation is highly
efficient in Ad-MSC after in vitro exposure and later removal of
GAG-containing formulations. Cultures were maintained in osteogenic
differentiation medium for 14 days after being cultured in GAG
formulation for 6 days. Cultures were fixed with 4% formalin and
stained with Alzarin red that stains calcium.
[0076] FIG. 9 describes that chondrogenic differentiation is highly
efficient in Ad-MSC after in vitro exposure and later removal of
HA-containing formulations. Cultures were maintained in
chondrogenic differentiation induction medium for 14 days after
being cultured in GAG formulation for 6 days. Cultures were fixed
with 4% formalin and stained with Alcian Blue.
[0077] FIG. 10 shows recovery of nucleated, viable canine adipose
SVF after cryopreservation in the presence of GAG-containing
formulations compared to cells cryopreserved in control media (5%
DMSO, 10% .alpha.-MEM, 85% fetal bovine serum (FBS)).
Cryopreservation in 50% POLYGLYCAN resulted in significantly higher
ADSC recovery than any other GAG combination. 5.5.times.10.sup.6
fresh canine stromal vascular cells were aliquotted in duplicate to
15 ml sterile conical tubes for each condition and concentrated to
a pellet by centrifugation at 600.times.g for 10 minutes.
Supernatants were removed by aspiration, and pellets were
resuspended in 1.5 control or test cryopreservation media and
transferred to individual 2.0 ml sterile cryovials. Cryovials were
then cooled to -80 C in a Mr. Frosty freezing container (Nalgene,
Rochester, N.Y.). After freezing, cryovials were maintained at -80
C for 20 days. Cryopreserved cells were rapidly thawed in a 37 C
water bath. Total cell count was determined with the aid of Syto 13
nuclear stain and nonviable cell count was determined with the aid
of Trypan blue dye. Percent viability was immediately determined as
((total cells-nonviable cells/total cells).times.100).
[0078] FIG. 11 shows Flow Cytometry analysis of cell surface marker
expression by human adipose SVF cells after cryopreservation in the
presence of POLYGLYCAN (PO) GAG-containing formulations (5-50%
(v/v)) compared to cells cryopreserved in control media (5% DMSO,
10% .alpha.-MEM, 85% fetal bovine serum (FBS)). Cryopreservation
was performed as described for FIG. 10. After storage at -80 C for
14 days, cells were thawed and placed in culture for 24 h in 75
cm.sup.2 plastic tissue culture dishes with media (.alpha.-MEM
containing 20% (v/v) FBS). Surface marker expression for CD34,
CD44, CD45, CD73, CD90, CD105, and CD117 was assessed on plastic
adherent cells by Flow Cytometery using a GALLIOS.TM. Flow
Cytometer (Beckman Coulter, Brea, Calif.) and compared to cells
from the same sample cultured for 24 h under the same conditions
but not cryopreserved.
[0079] FIG. 12 shows changes in gene expression in canine Ad-MSC
after culturing in media containing GAG formulation for 6 days.
Fresh canine adipose SVF cells were plated at a density of
5.times.10.sup.5 cells per well in 6 well plates and cultured for 6
days on tissue culture plastic in .alpha.-MEM containing 20% (v/v)
FBS and GAG formulations ranging from 2.5-10% (v/v). Media was
changed at day 3. At day 6 total RNA was prepared and gene
expression for selected genes was determined by quantitative
rt-PCR.
[0080] FIG. 13 shows changes in gene expression in human Ad-MSC
after culturing in media containing GAG formulation for 6 days.
Fresh human adipose SVF cells were plated at a density of
7.5.times.10.sup.5 cells per well in 6 well plates and cultured for
6 days on tissue culture plastic in control media (.alpha.-MEM
containing 20% (v/v) FBS) or control media supplemented with 5%
(v/v) POLYGLYCAN or MAP5 media was changed at day 3. At day 6 total
RNA was prepared and gene expression for selected genes was
determined by quantitative rt-PCR. Bars depict relative changes in
gene expression for cells cultured in media containing GAG
formulation to cells cultured in control media.
[0081] FIG. 14 shows recovery of nucleated, viable canine stromal
vascular cells after cryopreservation in the presence of POLYGLYCAN
GAG-containing formulations compared to cells cryopreserved in
control media (5% DMSO, 10% .alpha.-MEM, 85% fetal bovine serum
(FBS)) or commercial cryopreservation media (CRYOSTOR, BioLife
Solutions, Inc., Bothell, Wash.). 5.5.times.10.sup.6 fresh canine
adipose SVF cells were aliquotted in duplicate to 15 ml sterile
conical tubes for each condition and concentrated to a pellet by
centrifugation at 600.times.g for 10 minutes. Supernatants were
removed by aspiration, and pellets were resuspended in 1.5 control
or test cryopreservation media and transferred to individual 2.0 ml
sterile cryovials. Cryovials were then cooled to -80 C in a Mr.
Frosty freezing container (Nalgene, Rochester, NY). After freezing,
cryovials were maintained at -80 C for 20 days. Cryopreserved cells
were rapidly thawed in a 37 C water bath. Total cell count was
determined with the aid of Syto 13 nuclear stain and nonviable cell
count was determined with the aid of Trypan blue dye. Percent
viability was immediately determined as ((total cells-nonviable
cells/total cells).times.100).
[0082] FIG. 15 shows the effect of storage solution composition on
viability of cultured hAd-MSC subjected to overnight shipment.
1.5.times.10.sup.6 viable human Ad-MSC were suspended in 1.0 ml
test solution and transferred to sterile cryovials. Cryovials were
placed into a styrofoam shipping container with a cold pack to
maintain temperature at 2-8 C. The shipping container was sealed,
transported to a local shipping center, and shipped by overnight
courier to the laboratory location. 24 h after placement of
cryovials in the container, the container was opened to recover
cells from cryovials and assess cell viability as described for
FIG. 10. Bars depict the mean of triplicate determinations for each
test solution.
[0083] FIG. 16 shows the effect of storage solution compositions on
viability of fresh adipose SVF cells subjected to overnight
shipment. Immediately after isolation from human lipoaspirate
1.5.times.10.sup.6 viable human stromal vascular cells were
suspended in 1.0 ml test solution and transferred to sterile
cryovials. Cryovials were placed into a styrofoam shipping
container with a cold pack to maintain temperature at 2-8 C. The
shipping container was sealed, transported to a local shipping
center, and shipped by overnight courier to the laboratory
location. 24 h after placement of cryovials in the container the
container was opened to recover cells from cryovials and assess
cell viability as described for FIG. 10. Bars depict the mean of
triplicate determinations for each test solution.
[0084] In view of the studies and results provided above,
regenerative cells exposed to low concentrations of GAG
formulations of the invention such as a POLYGLYCAN composition
increase cell proliferation and colony forming potential
(P<0.05), and enrich the proportion of cultured cells expressing
key regenerative cell markers such as CD44, CD90, CD146, CD117,
CD73, and CD105. In addition, these formulations significantly
increased Sox2 levels, (a marker of a cell's stemness or potency)
(P<0.05), and influence cellular pathways such as the Wnt
pathways, which are known to be key in the regulation of
regenerative cell proliferation. Noted effects were markedly dose
dependent. The presence of these GAGs in vitro promoted
proliferation and self-renewal, even in the presence of
differentiation cues in the culture media. These effects were
reversible as efficient differentiation was observed in the absence
of GAG formulations.
[0085] In view of the effects of POLYGLYCAN on gene expression in
cultured canine Ad-MSC, the data indicate a siginificant increase
in CD44 levels (receptor for hyaluronic acid (HA)), a decrease in
the level of differentiation markers, a significant decrease in the
levels of apoptosis related genes, a significant decrease in
expression of inflammatory cytokines such as IL1b and IL6, an
increase in the leval of Col.1A, and increase in the levels of
(FGFs) growth factor.
[0086] Thus, the Examples provided above demonstrate that effects
of GAG formulations were strongly dose-dependent, and profound
differences were observed between GAG formulations. A combination
of hyaluronic acid, chondroitin sulfate, and glucosamine in
POLYGLYCAN composition at dilutions of 1-10% (v/v) in growth media
was found to be optimal for promoting proliferation and
self-renewal. Addition of HA alone to culture media elicited
qualitatively similar, but of lesser magnitude effects on the
marker of stemness, Sox2, correlated with effects on cell
proliferation. Removal of HA containing formulations enabled
lineage specific differentiation in appropriate culture media. In
contrast, addition of the commercial GAG formulation ADEQUAN (only
poly-sulfated GAG) inhibited proliferation and dramatically reduced
chondrogenic potential of regenerative cells. These results
indicate that an optimal GAG formulation for culturing and
administration of stem and regenerative cells is based on GAG
ratios in the POLYGLYCAN composition.
[0087] The use of the GAG formulation for preservation, shipping,
cryopreservation, or suspension is also evidenced at the GAG
dilutions described herein. The invention provides that MSCs
proliferate more rapidly in culture media supplemented with a
formulation containing POLYGLYCAN at 5% (v/v), and MSCs are capable
of efficient differentiation in vitro once GAG formulation is
removed. POLYGLYCAN at low concentration (e.g., 5%) in culture
media induces expression of markers of MSCs. The commercial GAG
formulation MAP5 containing only HA appears to be pro-mitotic, but
less potent than POLYGLYCAN. Further, cryopreservation media
containing 10% POLYGLYCAN, 5% DMSO, 85% Ringer's yield performs at
least as well as standard control media containing FBS and
DMSO.
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