U.S. patent application number 11/625720 was filed with the patent office on 2007-11-29 for serum-free media for chondrocytes and methods of use thereof.
This patent application is currently assigned to Genzyme Corporation. Invention is credited to Liesbeth Maria E. Brown.
Application Number | 20070275463 11/625720 |
Document ID | / |
Family ID | 27669045 |
Filed Date | 2007-11-29 |
United States Patent
Application |
20070275463 |
Kind Code |
A1 |
Brown; Liesbeth Maria E. |
November 29, 2007 |
SERUM-FREE MEDIA FOR CHONDROCYTES AND METHODS OF USE THEREOF
Abstract
The present invention provides defined serum-free cell culture
media useful in culturing fibroblasts, especially articular
chondrocytes, that avoids problems inherent in the use of
serum-containing media. The defined media comprise platelet-derived
growth factor (PDGF), and chemically defined lipids, or
combinations of these compounds. In another aspect, the present
invention also provides tissue culture methods that comprise
incubating chondrocytes in the defined serum free media. The
methods enhance attachment and proliferative expansion of
chondrocytes seeded at low density while maintaining their
redifferentiation potential.
Inventors: |
Brown; Liesbeth Maria E.;
(West Newton, MA) |
Correspondence
Address: |
GENZYME CORPORATION;LEGAL DEPARTMENT
15 PLEASANT ST CONNECTOR
FRAMINGHAM
MA
01701-9322
US
|
Assignee: |
Genzyme Corporation
|
Family ID: |
27669045 |
Appl. No.: |
11/625720 |
Filed: |
January 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10350816 |
Jan 24, 2003 |
7169610 |
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11625720 |
Jan 22, 2007 |
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60389078 |
Jun 14, 2002 |
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60351949 |
Jan 25, 2002 |
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Current U.S.
Class: |
435/406 ;
435/325; 435/404 |
Current CPC
Class: |
C12N 2501/58 20130101;
C12N 2500/90 20130101; C12N 2501/115 20130101; C12N 2500/38
20130101; C12N 5/0655 20130101; C12N 2500/25 20130101; C12N
2501/135 20130101; C12N 2500/36 20130101; C12N 2501/39
20130101 |
Class at
Publication: |
435/406 ;
435/325; 435/404 |
International
Class: |
C12N 5/06 20060101
C12N005/06; C12N 5/02 20060101 C12N005/02 |
Claims
1. A serum-free medium comprising a basal medium, PDGF, and at
least four lipids selected from the group consisting of stearic
acid, myristic acid, oleic acid, linoleic acid, palmitic acid,
palmitoleic acid, arachidonic acid, linolenic acid, cholesterol,
and alpha-tocopherol acetate.
2. The medium of claim 1, wherein the concentration of PDGF ranges
from 0.1-100 ng/ml.
3. The medium of claim 2, wherein the concentration of PDGF ranges
from 1-25 ng/ml.
4. The medium of claim 3, wherein the concentration of PDGF is 10
ng/ml.
5. The medium of claim 1, wherein the PDGF is PDGF-BB.
6. The medium of claim 1, wherein the medium comprises 0.5% of the
chemically defined lipid mixture (CDLM) as defined in Table 4.
7. The medium of claim 6, wherein the medium comprises 10 ng/ml
PDGF.
8. The medium of claim 7, wherein the PDGF is PDGF-BB.
9. The medium of claim 7, further comprising at least one
additional serum protein.
10. The medium of claim 7, further comprising at least one
additional growth factor.
11. The medium of any of claim 1, further comprising ITS,
hydrocortisone, fibronectin, bFGF, and albumin.
12. The medium of claim 1, wherein the basal medium comprises DRF
as defined in Table 2.
13. The medium of claim 12, wherein the basal medium comprises cDRF
as defined in Table 3.
14. The medium of claim 1, further comprising one or more
supplements selected from the group consisting of BMP, TGF-.beta.,
IGF, and insulin.
15. The medium of claim 1, wherein the BMP is BMP-4 or BMP-6.
16. The medium of claim 1, comprising at least six lipids selected
from the group consisting of stearic acid, myristic acid, oleic
acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic
acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
17. The medium of claim 16, wherein the concentration of PDGF
ranges from 0.1-100 ng/mi.
18. The medium of claim 17, wherein the concentration of PDGF
ranges from 1-25 ng/ml.
19. The medium of claim 16, comprising at least eight lipids
selected from the group consisting of stearic acid, myristic acid,
oleic acid, linoleic acid, palmitic acid, palmitoleic acid,
arachidonic acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
20. The medium of claim 19, wherein the concentration of PDGF
ranges from 0.1-100 ng/ml.
21. The medium of claim 20, wherein the concentration of PDGF
ranges from 1-25 ng/ml.
22. A serum-free medium comprising a basal medium, 1-25 ng/mi PDGF,
and 0.1-2% of the chemically defined lipid mixture (CDLM) as
defined in Table 4.
23. A composition comprising a chondrocyte and a culture medium
comprising a basal medium, PDGF, and at least four lipids selected
from the group consisting of stearic acid, myristic acid, oleic
acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic
acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
24. The composition of claim 23, wherein the concentration of PDGF
ranges from 0.1-100 ng/ml.
25. The composition of claim 24, wherein the concentration of PDGF
ranges from 1-25 ng/ml.
26. The composition of claim 25, wherein the concentration of PDGF
is 10 ng/ml.
27. The composition of claim 23, wherein the PDGF is PDGF-BB.
28. The composition of claim 23, wherein the composition comprises
the chemically defined lipid mixture (CDLM) as defined in Table
4.
29. The composition of claim 28, wherein the composition comprises
0.5% CDLM and 10 ng/ml PDGF.
30. The composition of claim 28, wherein the PDGF is PDGF-BB.
31. The composition of claim 28, further comprising at least one
additional serum protein.
32. The composition of claim 28, further comprising at least one
additional growth factor.
33. The composition of claim 23, further comprising ITS,
hydrocortisone, fibronectin, bFGF, and albumin.
34. The composition of claim 23, wherein the basal medium comprises
DRF as defined in Table 2.
35. The composition of claim 34, wherein the basal medium comprises
cDRF as defined in Table 3.
36. The composition of claim 23, further comprising one or more
supplements selected from the group consisting of BMP, TGF-.beta.,
IGF, and insulin.
37. The composition of claim 36, wherein the BMP is BMP-4 or
BMP-6.
38. The composition of claim 23, comprising at least six lipids
selected from the group consisting of stearic acid, myristic acid,
oleic acid, linoleic acid, palmitic acid, palmitoleic acid,
arachidonic acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
39. The composition of claim 38, wherein the concentration of PDGF
ranges from 0.1-100 ng/ml.
40. The composition of claim 39, wherein the concentration of PDGF
ranges from 1-25 ng/ml.
41. The composition of claim 38, comprising at least eight lipids
selected from the group consisting of stearic acid, myristic acid,
oleic acid, linoleic acid, palmitic acid, palmitoleic acid,
arachidonic acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
42. The composition of claim 41, wherein the concentration of PDGF
ranges from 0.1-100 ng/ml.
43. The composition of claim 42, wherein the concentration of PDGF
ranges from 1-25 ng/mi.
44. The composition of claim 41, comprising stearic acid, myristic
acid, oleic acid, linoleic acid, palmitic acid, palmitoleic acid,
arachidonic acid, linolenic acid, cholesterol, and alpha-tocopherol
acetate.
45. The composition of claim 44, wherein the concentration of PDGF
ranges from 0.1-100 ng/ml.
46. The composition of claim 45, wherein the concentration of PDGF
ranges from 1-25 ng/ml.
47. A composition comprising a chondrocyte and a culture medium
comprising a basal medium, 1-25 ng/ml PDGF, and 0.1-2% of the
chemically defined lipid mixture (CDLM) as defined in Table 4.
Description
[0001] This is a continuation of U.S. application Ser. No.
10/350,816, filed Jan. 24, 2003, which is incorporated herein by
reference. U.S. application Ser. No. 10/350,816 claims the benefit
of U.S. Provisional Application No. 60/389,078, filed Jun. 14,
2002, and of U.S. Provisional Application No. 60/351,949, filed
Jan. 25, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of cell and
tissue culture. More specifically, the invention relates to methods
and compositions for ex vivo propagation of cells capable of
forming cartilaginous tissue intended for treatment or repair of
cartilage defects.
BACKGROUND OF THE INVENTION
[0003] Articular cartilage is composed of chondrocytes encased
within the complex extracellular matrix produced by these cells.
The unique biochemical composition of this matrix provides for the
smooth, nearly frictionless motion of articulating surfaces of the
knee joint. With age, tensile properties of human articular
cartilage change as a result of biochemical changes. After the
third decade of life, the tensile strength of articular cartilage
decreases markedly. Damage of cartilage produced by trauma or
disease, e.g., rheumatoid and osteoarthritis, can lead to serious
physical debilitation.
[0004] The inability of cartilage to repair itself has led to the
development of several surgical strategies to alleviate clinical
symptoms associated with cartilage damage. More than 500,000
arthroplastic procedures and joint replacements are performed
annually in the United States alone. Autologous chondrocyte
implantation is a procedure that has been approved for treatment of
articular cartilage defects. The procedure involves harvesting a
piece of cartilage from a non-weight bearing part of the femoral
condyle and propagating the isolated chondrocytes ex vivo for
subsequent implantation back into the same patient (Brittberg et
al. (1994) New England J. of Medicine, 331: 889-895).
[0005] Articular chondrocytes express articular cartilage-specific
extracellular matrix components. Once articular chondrocytes are
harvested and separated from the tissue by enzymatic digestion,
they can be cultured in monolayers for proliferative expansion.
However, during tissue culture, these cells become phenotypically
unstable, adopt a fibroblastic morphology, and then cease to
produce type II collagen and proteoglycans characteristic of
hyaline-like articular cartilage. Such "dedifferentiated" cells
proliferate rapidly and produce type I collagen, which is
characteristic of fibrous tissue. Nevertheless, when placed in an
appropriate environment such as suspension culture medium in vitro
(Aulthouse et al. (1989) In Vitro Cell. & Devel. Biology, 25:
659-668) or in the environment of a cartilage defect in vivo
(Shortkroff et al. (1996) Biomaterials, 17: 147-154), the cells
redifferentiate, i.e., express articular cartilage-specific matrix
molecules again. The reversibility of dedifferentiation is key to
the successful repair of articular cartilage using cultured
autologous chondrocytes.
[0006] Human chondrocytes are typically cultured in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal
bovine serum (FBS) (Aulthouse et al. (1989) In Vitro Cell. &
Devel. Biology, 25: 659-668; Bonaventure et al. (1994) Exp. Cell
Res., 212: 97-104). However, even though serum is widely used for
mammalian cell culture, there are several problems associated with
its use (Freshney (1994) Serum-free media. In Culture of Animal
Cells, John Wiley & Sons, New York, 91-99): 1) serum contains
many unidentified or non-quantified components and therefore is not
"defined;" 2) the composition of serum varies from lot to lot,
making standardization difficult for experimentation or other uses
of cell culture; 3) many of the serum components affect cell
attachment, proliferation, and differentiation making it difficult
to control these parameters; 4) some components of serum are
inhibitory to the proliferation of specific cell types and to some
degree may counteract its proliferative effect, resulting in
sub-optimal growth; and 5) serum may contain viruses and other
pathogens which may affect the outcome of experiments or provide a
potential health hazard if the cultured cells are intended for
implantation in humans.
[0007] Thus, the use of defined serum-free media is particularly
advantageous in the ex vivo expansion of chondrocytes for treatment
of cartilage defects. However, such defined serum-free media must
be sufficient for attachment of adult human articular chondrocytes
seeded at low density, sustain proliferation until confluent
cultures are attained, and maintain the capacity of chondrocytes to
re-express the articular cartilage phenotype.
[0008] There has been some effort to develop biochemically defined
media (DM) for cell culture. DM generally includes nutrients,
growth factors, hormones, attachment factors, and lipids. The
precise composition must be tailored for the specific cell type for
which the medium is designed. Successful growth of some cell types,
including fibroblasts, keratinocytes, and epithelial cells has been
achieved in various DM (reviewed by Freshney, 1994). However,
attachment and proliferation of cells in the known media are often
not optimal.
[0009] Additionally, the amounts of starting cell material
available for autologous chondrocyte implantation are generally
limited. Therefore, it is desirable to seed articular chondrocytes
at a minimal subconfluent density. Attempts to culture articular
chondrocytes at subconfluent densities in DM have been only
partially successful. Although DM that can sustain the
proliferative capacity of the chondrocytes seeded at low density
have been developed, the use of these media still requires serum
for the initial attachment of cells to the tissue culture vessel
after seeding (Adolphe et al. (1984) Exp. Cell Res., 155: 527-536,
and United States Patent No. 6,150,163).
[0010] A need exists to optimize, standardize, and control
conditions for attachment, proliferation and maintenance of
redifferentiation-capable chondrocytes for use in medical
applications, especially, in humans.
SUMMARY OF THE INVENTION
[0011] It is an object of the invention to provide safe, effective,
and inexpensive culture medium compositions and methods for
culturing articular chondrocytes.
[0012] It is another object of the invention to provide a method
for culturing articular chondrocytes which does not involve the use
of serum.
[0013] It is yet another object of the invention to provide a
simple method for culturing articular chondrocytes in a single
defined cell culture medium.
[0014] It is yet another object of the invention to provide a
method for culturing articular chondrocytes under serum-free
conditions, wherein chondrocytes are seeded at low subconfluent
densities.
[0015] Still another object of the invention is to provide a method
for ex vivo expansion of articular chondrocytes, in which the cells
retain their redifferentiation capacity.
[0016] The invention provides a method for culturing human
articular chondrocytes and compositions of chemically defined
culture media. The DM of the invention avoid the use of serum at
any stage of chondrocyte culture and enhance cell attachment and
proliferation under serum-free conditions while maintaining the
capacity of chondrocytes to re-express cartilage-specific
phenotype.
[0017] One aspect of the invention provides defined cell culture
media that are sufficient for the initial attachment of cells to a
culture substratum, thereby eliminating a need for a
serum-containing medium in the initial stage of cell culture.
Another aspect of the invention provides defined serum-free cell
culture media that promote proliferation of chondrocytes without
use of serum at any stage during cell culture. Yet another aspect
of the invention provides cell culture media that may be used to
prime chondrocytes prior to implantation into a subject or included
as a redifferentiation-sustaining medium to chondrocytes embedded
in a matrix intended for implantation into cartilage defects.
[0018] In certain embodiments, the DM of the invention comprises a
basal medium. In one embodiment, the basal medium is prepared using
commercially available culture media such as DMEM, RPMI-1640, and
Ham's F-12. In one embodiment, DMEM, RPMI-1640, and Ham's F-12 are
mixed at a 1:1:1 ratio and combined with growth supplements to
produce the basal medium defined in Table 3 (referred to
hereinafter as cDRF). In addition to a basal medium, the DM of the
invention comprises at least two of the supplements selected from
the group consisting of: platelet-derived growth factor (PDGF), and
one or more lipid components selected from the group consisting of
stearic acid, myristic acid, oleic acid, linoleic acid, palmitic
acid, palmitoleic acid, arachidonic acid, linolenic acid,
cholesterol, and alpha-tocopherol acetate. In a particular
embodiment, DM comprises PDGF and at least one lipid component. In
related embodiments, DM comprises PDGF and at least two, four, six,
eight, or all of the lipid components set forth in Table 4. In a
further embodiment, the PDGF is PDGF-BB. In certain embodiments,
the concentration of PDGF is chosen from 0.1-1 ng/ml, 1-5 ng/ml,
5-10 ng/ml, 10 ng/ml, 10-15 ng/ml, 15-50 ng/ml, and 50-100 ng/ml.
In certain other embodiments, the concentration (v/v) of lipid
components is chosen from 0.05-0.1%, 0.1-0.5%, 0.5%, 0.5-1%, 1-2%,
and 2-5%.
[0019] Additional objects and advantages of the invention will be
set forth in part in the description which follows, and in part
will be obvious from the description, or may be learned by practice
of the invention. The objects and advantages of the invention will
be realized and attained by means of the elements and combinations
particularly pointed out in the appended claims.
[0020] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed.
[0021] The accompanying figures, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a diagram of growth index for human articular
chondrocytes propagated ex vivo for four passages in DMEM/10% FBS
or cDRF (defined in Table 3), cDRF supplemented with 10 ng/ml PDGF,
and cDRF supplemented with 10 ng/ml PDGF and 5 .mu.l/ml of the
chemically defined lipid mixture (CDLM) set forth in Table 4.
[0023] FIG. 2 illustrates cell yields for human articular
chondrocytes plated at various seeding densities and propagated ex
vivo for four passages in DMEM/10% FBS or in the defined serum-free
media as follows: cDRF; cDRF supplemented with 10 ng/ml PDGF; and
cDRF supplemented with 10 ng/ml PDGF and 5 .mu.l/ml CDLM.
[0024] FIGS. 3A and 3B depict results of a TaqMan analysis of genes
expressed by chondrocytes expanded in DMEM/10% FBS or in cDRF
supplemented with 10 ng/ml PDGF and 5 .mu.l/ml CDLM.
DETAILED DESCRIPTION OF THE INVENTION
[0025] This invention provides a method for culturing chondrocytes
in a defined serum-free media and is based, at least in part, on
the discovery that the basal medium referred to as cDRF, as
described below, when supplemented with PDGF and at least on of the
lipids set forth in Table 4, is sufficient for attachment,
proliferation and maintenance of redifferentiation-capable
chondrocytes in culture and can substitute for a serum-containing
medium in all stages of cell culture.
Preparation of Basal Medium (cDRF)
[0026] The first step in preparing defined, serum media (DM) of the
invention is to prepare a basal medium. In a particular embodiment,
the basal medium defined in Table 3 (cDRF), is prepared from
commercially available starting components as described below. cDRF
is a modification of the DM developed by Adolphe et al. (1994) and
by McPherson et al. (U.S. Pat. No. 6,150,163).
[0027] The three starting components of cDRF are DMEM, RPMI-1640,
and Ham's F12 (Gibco BRL, Grand Island, N.Y.). The precise
composition of each of these starting components is set forth in
Table 1. The starting components are combined at a 1:1:1 ratio. The
resulting medium (defined in Table 2 and referred to as DRF) is
then supplemented with ITS (10 .mu.g/ml insulin, 5.5 .mu.g/ml
transferrin, 7 ng/ml selenium, and, optionally, 2.0 .mu.g/ml
ethanolamine), human fibronectin (Collaborative Biomedical
Products, Bedford, Mass.), human serum albumin (HSA), linoleic
acid, human basic fetal growth factor (bFGF) (R&D Systems,
Minneapolis, Minn.), gentamycin (BioWhittaker, Walkersville, Md.),
and hydrocortisone (Sigma, St. Louis, Mo.) to create cDRF. Freshly
prepared incomplete cDRF (cDRF without bFGF, fibronectin, linoleic
acid, and HSA) can be stored in the dark up to 2 weeks at
2-8.degree. C. bFGF, fibronectin, and HSA supplemented with
linoleic acid are diluted into the medium to create cDRF on the day
of use for cell culture. HSA utilized in the media of the invention
is either purified from human plasma (Grifols.RTM. HSA, SeraCare,
Oceanside, Calif.) or recombinant (New Century Pharmaceuticals,
Huntsville, Ala.). All materials are reconstituted, diluted, and
stored as per suppliers' recommendations.
[0028] The term "basal medium" is used interchangeably with
"defined basal medium" and refers to any medium that comprises all
essential components of cDRF listed in Table 3. A component or a
subset of components listed in Table 3 is non-essential if, when
its concentration is reduced, or the component is eliminated, the
properties of the medium related to chondrocyte attachment,
proliferation, and redifferentiation, remain substantially the
same. The stated concentrations of individual components may be
adjusted for specific cell culture conditions. Such adjustments can
easily be made by a person skilled in the art using routine
techniques. It will also be understood that additional components
may be added to the medium if such components are desirable and do
not negatively impact on chondrocytes attachment, proliferation,
and redifferentiation. Such components include growth factors,
lipids, serum proteins, vitamins, minerals, carbohydrates. For
example, it may be advantageous to supplement the medium with
growth factors or hormones that promote chondrocyte
redifferentiation such as TGF-.beta. (TGF-.beta.1, -.beta.2,
-.beta.3), IGF, and insulin, as described in U.S. Pat. No.
6,150,163. Such growth factors and hormones are commercially
available. Additional examples of supplements include, but are not
limited to, bone morphogeneteic proteins (BMP), of which there are
at least 15 structurally and functionally related proteins. BMP
have been shown to be involved in the growth, differentiation,
chemotaxis, and apoptosis of various cell types. Recombinant BMP-4
and BMP-6, for example, can be purchased from R&D Systems
(Minneapolis, Minn.; catalog #314-BP and 507-BP, respectively). The
concentration of various supplements in DM of the invention can be
determined without undue experimentation. The concentration of BMP
in DM of the invention is chosen from 0.01-0.1 ng/ml, 0.1-1 ng/ml,
1-10 ng/ml, 100 ng/ml, 10-50 ng/ml, 50-100 ng/ml, and 0.1-1
.mu.g/ml.
[0029] A skilled artisan will appreciate that DM of the invention
have advantages in addition to avoiding the use of serum. Thus, it
may be desirable to utilize DM of the invention in applications
where the use of undefined components is acceptable. Consequently,
DM of the invention may be supplemented with serum e.g., fetal calf
serum, or other chemically undefined components such as, for
example, animal or plant tissue extracts. In certain embodiments,
the DM of the invention may be supplemented with 10% or less than
8%, 6%, 4%, 2%, or 1% of serum.
[0030] A skilled artisan will also appreciate that equivalents of
cDRF may be prepared from a variety of known media, e.g., Basal
Medium Eagle medium (Eagle, Science, 122: 501, 1955), Minimum
Essential medium (Dulbecco et al., Virology, 8: 396, 1959), Ham's
medium (Ham, Exp. Cell Res., 29: 515,1963), L-15 medium (Leibvitz,
Amer. J. Hyg., 78:173,1963), McCoy 5A medium (McCoy et al., Proc.
Exp. Biol. Med., 100: 115,1959), RPMI medium (Moore et al., J. A.
M. A., 199: 519,1967), Williams' medium (Williams, Exp. Cell Res.,
69:106-112,1971), NCTC 135 medium (Evans et al., Exp. Cell Res.,
36: 439,1968), Waymouth's medium MB752/1 (Waymouth, Nat. Cancer
Inst., 22: 1003,1959), etc. These media may be used singularly or
as mixtures in suitable proportions to prepare a basal medium
equivalent to cDRF. Alternatively, cDRF or its equivalent can be
prepared from individual chemicals or from other media and growth
supplements. The invention is not limited to media of any
particular TABLE-US-00001 TABLE 1 Compositions of Starting Media
DMEM RPMI-1640 Ham's F-12 1.times. Liquid, 1.times. Liquid,
1.times. Liquid, mg/L mg/L mg/L Inorganic Salts CaCl.sub.2 (anhyd.)
200.00 33.22 Ca(NO.sub.3).sub.2.cndot.4H.sub.2O 100.00
CuSO.sub.4.cndot.5H.sub.2O 0.0024
Fe(NO.sub.3).sub.2.cndot.9H.sub.2O 0.10 FeSO.sub.4.cndot.7H.sub.2O
0.83 KCl 400.00 400.00 223.60 MgSO.sub.4 (anhyd.) 97.67 48.84
MgCl.sub.2 (anhyd.) 57.22 NaCl 6400.00 6000.00 7599.00 NaHCO.sub.3
3700.00 2000.00 1176.00 NaH.sub.2PO.sub.4.cndot.H.sub.2O 125.00
Na.sub.2HPO.sub.4 (anhyd.) 800.00 142.00 ZnSO.sub.4.cndot.7H.sub.2O
0.86 Other Components D-Glucose 4500.00 2000.00 1802.00 Glutathione
(reduced) 1.00 Hypoxanthine Na 4.77 Linoleic Acid 0.084 Lipoic Acid
0.21 Phenol Red 15.00 5.00 1.20 Putrescine.cndot.2HCl 0.161 Sodium
Pyruvate 110.00 Thymidine 0.70 Amino Acids L-Alanine 8.90
L-Arginine 200.00 L-Arginine.cndot.HCl 84.00 211.00
L-Asparagine.cndot.H.sub.2O 15.01 L-Asparagine (free base) 50.00
L-Aspartic Acid 20.00 13.30 L-Cystine.cndot.2HCl 63.00 65.00
L-Cysteine.cndot.HCl.cndot.H.sub.2O 35.12 L-Glutamic Acid 20.00
14.70 L-Glutamine 584.00 300.00 146.00 Glycine 30.00 10.00 7.50
L-Histidine.cndot.HCl.cndot.H.sub.2O 42.00 21.00 L-Histidine (free
base) 1.00 5.00 L-Hydroxyproline 20.00 L-Isoleucine 105.00 50.00
4.00 L-Leucine 105.00 50.00 13.10 L-Lysine.cndot.HCl 146.00 40.00
36.50 L-Methionine 30.00 15.00 4.50 L-Phenylalanine 66.00 15.00
5.00 L-Proline 20.00 34.50 L-Serine 42.00 30.00 10.50 L-Threonine
95.00 20.00 11.90 L-Tryptophan 16.00 5.00 2.00
L-Tyrosine.cndot.2Na.cndot.2H.sub.2O 104.00 29.00 7.81 L-Valine
94.00 20.00 11.70 Vitamins Biotin 0.20 0.0073 D-Ca pantothenate
4.00 0.25 0.50 Choline Chloride 4.00 3.00 14.00 Folic Acid 4.00
1.00 1.30 I-Inositol 7.20 35.00 18.00 Niacinamide 4.00 1.00 0.036
Para-aminobenzoic Acid 1.00 Pyridoxine HCl 1.00 0.06 Pyridoxal HCl
4.00 Riboflavin 0.40 0.20 0.037 Thiamine HCl 4.00 1.00 Vitamin
B.sub.12 0.005 1.40
[0031] TABLE-US-00002 TABLE 2 Composition of DRF 3.times. Liquid,
mg/L Inorganic Salts CaCl.sub.2 (anhyd.) 233.22
Ca(NO.sub.3).sub.2.cndot.4H.sub.2O 100.00
CuSO.sub.4.cndot.5H.sub.2O 0.0024
Fe(NO.sub.3).sub.2.cndot.9H.sub.2O 0.10 FeSO.sub.4.cndot.7H.sub.2O
0.83 KCl 1023.60 MgSO.sub.4 (anhyd.) 146.51 MgCl.sub.2(anhyd.)
57.22 NaCl 19999.00 NaHCO.sub.3 6876.00
NaH.sub.2PO.sub.4.cndot.H.sub.2O 125.00 Na.sub.2HPO.sub.4(anhyd.)
942.00 ZnSO.sub.4.cndot.H.sub.2O 0.86 Other Components D-Glucose
8302.00 Glutathione (reduced) 1.00 Hypoxanthine.cndot.Na 4.77
Linoleic Acid 0.084 Lipoic Acid 0.21 PhenolRed 21.20 Putrescine
2HCl 0.161 Sodium Pyruvate 110.00 Thymidin 0.70 Amino Acids
L-Alanine 8.90 L-Arginine 200.00 L-Arginine.cndot.HCl 295.00
L-Asparagine.cndot.H.sub.2O 15.01 L-Asparagine (free base) 50.00
L-Aspartic Acid 33.30 L-Cystine.cndot.2HCl 128.00 L-Cysteine
HCl.cndot.H.sub.2O 35.12 L-Glutamic Acid 34.70 L-Glutamine 1030.00
Glycine 47.50 L-Histidine.cndot.HCl.cndot.H.sub.2O 63.00
L-Histidine (free base) 15.00 L-Hydroxyproline 20.00 L-Isoleucine
159.00 L-Leucine 168.10 L-Lysine.cndot.HCl 222.50 L-Methionine
49.50 L-Phenylalanine 86.00 L-Proline 54.50 L-Serine 82.50
L-Threonine 126.90 L-Tryptophan 23.00 L-Tyrosine
2Na.cndot.2H.sub.2O 140.81 L-Valine 125.70 Vitamins Biotin 0.2073
D-Ca pantothenate 4.75 Choline Chloride 21.00 Folic Acid 6.30
i-Inositol 60.20 Niacinamide 5.036 Para-aminobenzoic Acid 1.00
Pyridoxine.cndot.HCl 1.06 Pyridoxal.cndot.HCl 4.00 Riboflavin 0.637
Thiamine.cndot.HCl 5.30 Vitamin B.sub.12 1.405
[0032] TABLE-US-00003 TABLE 3 Composition of cDRF 1.times. Liquid
Basal Components DRF 99% ITS 1% Supplements Linoleic Acid 5
.mu.g/ml Gentamycin 100 .mu.g/ml Hydrocortisone 40 ng/ml
Fibronectin 5 .mu.g/ml Basic FGF 10 ng/ml Human Serum Albumin 1
mg/ml
consistency and encompasses the use of media ranging from liquid to
semi-solid and includes solidified media and solid compositions
suitable for reconstitution. Supplementation of Basal Medium
[0033] Platelet-Derived Growth Factor (PDGF)
[0034] PDGF is a major mitogenic factor present in serum but not in
plasma. PDGF is a dimeric molecule consisting of two structurally
related chains designated A and B. The dimeric isoforms PDGF-AA, AB
and BB are differentially expressed in various cell types. In
general, all PDGF isoforms are potent mitogens for connective
tissue cells, including dermal fibroblasts, glial cells, arterial
smooth muscle cells, and some epithelial and endothelial cell.
[0035] Recombinantly produced PDGF is commercially available from
various sources. Human recombinant PDGF-BB (hrPDGF-BB) used in the
examples below was purchased from R&D Systems (Minneapolis,
Minn.; catalog #220-BB) and reconstituted and handled according to
the manufacturer's instructions. The E. coli expression of
hrPDGF-BB and the DNA sequence encoding the 109 amino acid residue
mature human PDGF-B chain protein (C-terminally processed from that
ends with threonine residue 190 in the precursor sequence) is
described by Johnson et al. (EMBO J., 3: 921, 1984). The
disulfide-linked homodimeric rhPDGF-BB consists of two 109 amino
acid residue B chains and has molecular weight of about 25 kDa. The
activity of PDGF is measured by its ability to stimulate
.sup.3H-thymidine incorporation in quiescent NR6R-3T3 fibroblast as
described by Raines et al. (Methods in Enzymology 109: 749-773,
1985). The ED.sub.50 for PDGF in this assay is typically 1.0-3
ng/ml.
[0036] In certain embodiments, DM of the invention is cDRF
supplemented with PDGF and BMP or one or more lipids selected from
the group consisting of stearic acid, myristic acid, oleic acid,
linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid,
linolenic acid, cholesterol, and alpha-tocopherol acetate. The
concentration of PDGF is chosen from 0.1-1 ng/ml, 1-5 ng/ml, 5-10
ng/ml, 10 ng/ml, 10-15 ng/ml, 15-50 ng/ml, and 50-100 ng/ml. In
certain embodiments, cDRF is supplemented with 1 to 25 ng/ml, more
preferably, 5 to 15 ng/ml and, most preferably, 10 ng/ml of PDGF.
In a particular embodiment, the PDGF is PDGF-BB. Alternatively,
PDGF could be of another type, e.g., PDGF-AB, PDGF-BB, or a mix of
any PDGF types. In related embodiments, the DM of the invention
further comprises additional supplements as described below.
Lipids
[0037] Lipids are important as structural components as well as
potential energy sources in living cells. In vitro, most cells can
synthesize lipids from glucose and amino acids present in the
culture medium. However, if extracellular lipid is available, lipid
biosynthesis is inhibited and the cells utilize free fatty acids,
lipid esters, and cholesterol in the medium. Serum is rich in
lipids and has been the major source of extracellular lipid for
cultured cells. Chemically undefined lipid preparations based on
marine oils have been found to be effective in promoting growth of
cells in serum free-media in several systems (Weiss et al. (1990)
In Vitro 26: 30A; Gorfien et al. (1990) In Vitro 26: 37A; Fike et
al. (1990) In Vitro 26: 54A). Thus, supplementation of serum-free
media with various lipids to replace those normally supplied by
serum may be desirable.
[0038] Suitable lipids for use in the DM of this invention include
stearic acid, myristic acid, oleic acid, linoleic acid, palmitic
acid, palmitoleic acid, arachidonic acid, linolenic acid,
cholesterol, and alpha-tocopherol acetate. In one embodiment, the
basal medium is supplemented with the chemically defined lipid
mixture (CDLM), shown in Table 4. CDLM is available from Gibco BRL
(catalog #11905-031). As supplied by Gibco BRL, in addition to the
lipid components, CDLM contains ethanol (100 g/L) and emulsifiers
Pluronic F68.RTM. (100 g/L) and Tween 80.RTM. (2.2 g/L).
[0039] In practicing the methods of the invention, the
concentrations of individual lipid components of CDLM shown in
Table 4 may be adjusted for specific cell culture conditions. Such
adjustments can easily be made by a person skilled in the art using
routine techniques. Furthermore, not all components of CDLM may be
essential. A component or a subset of components is non-essential
if, when its concentration is reduced, or the component is
eliminated, the properties of the medium related to chondrocyte
attachment, proliferation, and redifferentiation, remain
substantially the same.
[0040] In certain embodiments, the DM of the invention comprises at
least one, two, four, six, eight, or all lipid components of CDLM.
In one embodiment, the DM comprises PDGF and CDLM as defined in
Table 4. In other nonlimiting illustrative embodiments, the DM
comprises PDGF and lipid combinations as set forth in Table 5.
TABLE-US-00004 TABLE 4 Composition of CDLM Lipid components mg/L
DL-alpha-tocopherol acetate 70 Stearic acid 10 Myristic acid 10
Oleic acid 10 Linoleic acid 10 Palmitic acid 10 Palmitoleic acid 10
Arachidonic acid 2 Linolenic acid 10 Cholesterol 220
[0041] In certain embodiments, the concentration (v/v) of lipids in
the culture medium is chosen from 0.05-0.1%, 0.1-0.5%, 0.5%,
0.5-1%, 1-2%, and 2-5%. In certain other embodiments, DM is
additionally supplemented with 1 to 25 ng/ml, more preferably, 5 to
15 ng/ml, and, most preferably, 10 ng/ml of PDGF. In a particular
embodiment, DM comprises approximately 0.5% (v/v) CDLM, and 10
ng/ml PDGF.
[0042] The media can be used to seed, grow, and maintain
chondrocytes capable of redifferentiation in culture without the
use of serum. The stated ranges of concentrations of PDGF and
lipids may need to be adjusted for specific cell culture
conditions. Such adjustments can easily be made by a person skilled
in art using routine techniques. TABLE-US-00005 TABLE 5
Illustrative Lipid Combinations 1 cholesterol 2 cholesterol,
arachidonic acid 3 cholesterol, arachidonic acid, linoleic acid 4
cholesterol, arachidonic acid, linoleic acid, linolenic acid 5
cholesterol, arachidonic acid, linoleic acid, linolenic acid,
alpha-tocopherol acetate 6 cholesterol, arachidonic acid, linoleic
acid, linolenic acid, alpha-tocopherol acetate, stearic acid 7
cholesterol, arachidonic acid, linoleic acid, linolenic acid,
alpha-tocopherol acetate, stearic acid 8 cholesterol, arachidonic
acid, linoleic acid, linolenic acid, alpha-tocopherol acetate,
stearic acid, myristic acid 9 cholesterol, arachidonic acid,
linoleic acid, linolenic acid, alpha-tocopherol acetate, stearic
acid, myristic acid, oleic acid 10 cholesterol, arachidonic acid,
linoleic acid, linolenic acid, alpha-tocopherol acetate, stearic
acid, myristic acid, oleic acid, palmitic acid 11 cholesterol,
arachidonic acid, linoleic acid, linolenic acid, alpha-tocopherol
acetate, stearic acid, myristic acid, oleic acid, palmitic acid,
palmitoleic acid 12 arachidonic acid, linoleic acid, linolenic
acid, alpha- tocopherol acetate, stearic acid, myristic acid, oleic
acid, palmitic acid, palmitoleic acid 13 arachidonic acid, linoleic
acid, linolenic acid, stearic acid, myristic acid, oleic acid,
palmitic acid, palmitoleic acid 14 arachidonic acid, linoleic acid,
linolenic acid, stearic acid, myristic acid, oleic acid, palmitic
acid 15 arachidonic acid, linoleic acid, linolenic acid, stearic
acid, myristic acid, oleic acid 16 arachidonic acid, linoleic acid,
linolenic acid, stearic acid, myristic acid 17 arachidonic acid,
linoleic acid, linolenic acid, acetate, stearic acid 18 arachidonic
acid, linoleic acid, linolenic acid, stearic acid 19 arachidonic
acid, linoleic acid, linolenic acid 20 arachidonic acid, linoleic
acid 21 arachidonic acid 22 cholesterol, linoleic acid 23
cholesterol, linoleic acid, linolenic acid 24 cholesterol, linoleic
acid, linolenic acid, stearic acid 25 cholesterol, linoleic acid,
linolenic acid, stearic acid, myristic acid 26 cholesterol,
linoleic acid, linolenic acid, stearic acid, myristic acid, oleic
acid 27 cholesterol, linoleic acid, linolenic acid, stearic acid,
myristic acid, oleic acid, palmitic acid 28 cholesterol, linoleic
acid, linolenic acid, stearic acid, myristic acid, oleic acid,
palmitic acid, palmitoleic acid 29 cholesterol, linoleic acid,
linolenic acid, alpha-tocopherol acetate, stearic acid, myristic
acid, oleic acid, palmitic acid, palmitoleic acid 30 linoleic acid
31 cholesterol, linoleic acid 32 cholesterol, arachidonic acid,
linoleic acid 33 cholesterol, arachidonic acid, linoleic acid,
linolenic acid 34 cholesterol, arachidonic acid, linoleic acid,
linolenic acid, alpha-tocopherol acetate 35 cholesterol,
arachidonic acid, linoleic acid, linolenic acid, alpha-tocopherol
acetate, stearic acid 36 cholesterol, arachidonic acid, linoleic
acid, linolenic acid, alpha-tocopherol acetate, stearic acid,
myristic acid 37 cholesterol, arachidonic acid, linoleic acid,
linolenic acid, alpha-tocopherol acetate, stearic acid, myristic
acid, oleic acid 38 cholesterol, arachidonic acid, linoleic acid,
linolenic acid, alpha-tocopherol acetate, stearic acid, myristic
acid, oleic acid 39 cholesterol, arachidonic acid, linoleic acid,
linolenic acid, alpha-tocopherol acetate, stearic acid, myristic
acid, oleic acid, palmitic acid, palmitoleic acid 40 linolenic acid
41 cholesterol, linolenic acid 42 cholesterol, alpha-tocopherol
acetate, stearic acid, myristic acid, oleic acid, palmitic acid,
palmitoleic acid 43 cholesterol, alpha-tocopherol acetate 44
cholesterol, stearic acid, myristic acid, oleic acid, palmitic
acid, palmitoleic acid 45 stearic acid, myristic acid, oleic acid,
palmitic acid, palmitoleic acid 46 cholesterol, myristic acid,
oleic acid, palmitic acid, palmitoleic acid 47 cholesterol, oleic
acid, palmitic acid, palmitoleic acid 48 cholesterol, stearic acid,
myristic acid, oleic acid, palmitic acid, palmitoleic acid 49
cholesterol, myristic acid, oleic acid, palmitic acid 50
cholesterol, arachidonic acid, linoleic acid, linolenic acid,
palmitic acid, palmitoleic acid
[0043] Chondrocytes and Other Suitable Cells
[0044] The present invention is generally suitable for ex vivo
proliferation of cells capable of producing cartilaginous tissue.
Chondrocytes are cells found in various types of cartilage, e.g.,
hyaline cartilage, elastic cartilage, and fibrocartilage.
Chondrocytes are mesenchymal cells that have a characteristic
phenotype based primarily on the type of extracellular matrix they
produce. Precursor cells produce type I collagen, but when they
become committed to the chondrocyte lineage, they stop producing
type I collagen and start synthesizing type II collagen, which
constitutes a substantial portion of the extracellular matrix. In
addition, committed chondrocytes produce proteoglycan aggregate,
called aggrecan, which has glycosaminoglycans that are highly
sulfated.
[0045] Chondrocytes can be isolated from any mammal, including,
without limitation, human, orangutan, monkey, chimpanzee, dog, cat,
rat, rabbit, mouse, horse, cow, pig, elephant, etc. Chondrocytes
used in the present invention can be isolated by any suitable
method. Various starting materials and methods for chondrocyte
isolation are well known in the art (Freshney (1987) Culture of
Animal Cells: A Manual of Basic Techniques, 2d ed. A. R. Liss,
Inc., New York, pp. 137-168; Klagsburn (1979) Methods Enzymol. 58:
560-564). By way of example, articular cartilage can be harvested
from femoral condyles of human donors, and chondrocytes can be
released from the cartilage by overnight digestion in 0.1%
collagenase/DMEM. The released cells are expanded as primary cells
in a suitable medium such as the DM of this invention or DMEM
containing 10% FBS. Cells can be passaged at 80-90% confluence
using 0.05% trypsin-EDTA, diluted for subculture, and reseeded for
second and subsequent passages to allow for further expansion. At
any time, trypsinized cells can be frozen in DMEM containing 10%
DMSO and 40% HSA or in other compositions known in the art, e.g.,
as described in U.S. Pat. No. 6,365,405.
[0046] It may be desirable in certain circumstances to utilize
chondrocyte progenitor stem cells such as mesenchymal stem cells
rather than cells from cartilage biopsies that are already
differentiated into chondrocytes. Examples of tissues from which
such stem cells can be isolated include placenta, umbilical cord,
bone marrow, skin, muscle, periosteum, or perichondrium.
Chondrocytes can be obtained by inducing differentiation of such
cells into chondrocytes in vitro.
[0047] The term "chondrocytes," as used herein, refers not only to
mesenchymal stem cells, but also to cells that can be
trans-differentiated into chondrocytes, for example, adipocytes,
osteocytes, fibroblasts, and myocytes. The term "chondrocytes" also
refers to chondrocytes that are passaged or dedifferentiated.
[0048] The term "low density" refers to seeding densities less than
20,000 cells/cm.sup.2.
[0049] The methods of this invention are suitable for cells growing
in cultures under various conditions including, but not limited to,
monolayers, multilayers, on solid support, in suspension, and in 3D
cultures.
[0050] Other embodiments of the invention will be apparent to those
skilled in the art from consideration of the specification and
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with a
true scope and spirit of the invention being indicated by the
following claims.
EXAMPLES
[0051] Various aspects of the invention are further described and
illustrated in the examples presented below.
Example 1
[0052] Human articular cartilage biopsy samples from donors of
16-51 years of age were trimmed of extraneous material, minced and
subjected to enzymatic digestion using 0.25% protease from Bascilus
Thermopropolipycus for 1-2 hrs followed by an overnight digestion
in 0.1% collagenase/DMEM at 37.degree. C. Isolated articular
chondrocytes were washed twice in DMEM containing 10% human serum
albumin (DMEM/10% HSA). The isolated primary human articular
chondrocytes (HAC) were seeded at 5,000-6,000 cells/cm.sup.2 in T75
flasks using the following separate media conditions: [0053] 1)
DMEM/10% FBS (DMEM supplemented with 10% fetal bovine serum and 100
.mu.g/ml gentamycin); [0054] 2) cDRF (as defined in Table 3);
[0055] 3) cDRF/P (cDRF supplemented with 10 ng/ml PDGF); [0056] 4)
cDRF/L (cDRF supplemented with 5 .mu.l/ml CDLM (as defined in Table
4)); and [0057] 5) cDRF/P/L (cDRF supplemented with 10 ng/ml PDGF
and 5 .mu.l/ml CDLM)
[0058] Two flasks were used per each condition. At the end of each
passage, nearly confluent cells were harvested by trypsinization,
counted, washed in DMEM/10% HSA and reseeded at 5,000-6,000
cells/cm.sup.2 in the corresponding media. Cell yield was
calculated as the average of duplicate samples for each condition.
A comparison of cell yields at the end of each passage for
chondrocytes propagated under media condition defined above is
represented in Table 6. The results of this experiment demonstrate
that regardless of the passage number, cell yields were higher for
chondrocytes passaged in cDRF/P/L, as compared to either DMEM/10%
FBS or cDRF. The effect was more pronounced for higher passage
numbers. TABLE-US-00006 TABLE 6 Cell Yield per T75, .times.10.sup.7
Passage Medium 1 2 3 4 5 DMEM/10% FBS 0.95 0.6 0.25 0.24 0.3 cDRF
0.59 0.75 0.90 1.05 0.8 cDRF/P/L 1.9 2.8 1.2 2.25 2.05
Example 2
[0059] Hyaline cartilage biopsy samples collected from multiple
donors were used to compare cell yields as a function of the
passage number for chondrocytes cultured in DMEM/10% FBS or in a
completely defined serum-free medium according to this invention.
Samples were collected and treated as described in Example 1.
Isolated chondrocytes were washed twice in DMEM containing 10%
human serum albumin (DMEM/10% HSA). The isolated primary human
articular chodrocytes (HAC) were seeded at 6,000 cells/cm.sup.2 in
T75 flasks using the following media conditions: [0060] 1) DMEM/10%
FBS (DMEM supplemented with 10% fetal bovine serum and 100 .mu.g/ml
gentamycin); and [0061] 2) cDRF/P/L (cDRF supplemented with 10
ng/ml PDGF and 5 .mu.l/ml CDLM)
[0062] At the end of each passage nearly confluent cells were
harvested by trypsinization, counted, washed in DMEM/10% HSA and
reseeded at 6,000 cells/cm.sup.2 in respective media. A comparison
of cell yields at the end of each passage for chondrocytes
propagated in DMEM/10% FBS or cDRF/P/L is shown in Table 7. Cell
yields were higher in cDRF/P/L as compared to DMEM/10% FBS for
cells in passages 1-3, and significantly higher (p=0.05) for cells
in passage 4. TABLE-US-00007 TABLE 7 Passage 1 2 3 4 DMEM/10% FBS
Cell Yield (.times.10.sup.4/cm.sup.2) 7.5 .+-. 2.3 8.5 .+-. 2.4
.sup. 5 .+-. 2.7 4 .+-. 2.0 Number of Samples 9 8 5 3 cDRF/P/L Cell
Yield (.times.10.sup.4/cm.sup.2) 9.6 .+-. 7.0 12.5 .+-. 4.5 9.0
.+-. 5.0 14.0 .+-. 6.3 Number of Samples 8 8 3 3 T-test p-value
0.43 0.07 0.10 0.05*
Example 3
[0063] In this experiment, human articular chondrocytes from three
donors, ages 16, 22, and 55, were isolated and treated as described
in Example 1. Chondrocytes were seeded at 6,000 cells/cm.sup.2 in
T75 flasks and grown in DMEM/10% FBS until near confluence. The
cells were then harvested by trypsinization, washed in seeding
media, and immediately frozen in 10% DMSO/40% HSA/50% DMEM. For the
second passage, ampules of frozen cells were thawed out, rinsed in
DMEM/10% HSA and reseeded at 3,000-4,000 cells/cm.sup.2 in the
following media: 1) DMEM/10% FBS; 2) cDRF; 3) cDRF/P; and 4)
cDRF/P/L (see Example 1 for the description of the media). Two
flasks were used per each set of media conditions. At the end of
each passage nearly confluent cells were harvested by
trypsinization, washed in DMEM/10% HSA and reseeded in the
corresponding media. At the end of the third passage, cells were
harvested and counted. Growth index expressed as a number of
doublings per day at the end of a seven-day period was calculated
as the mean value for the three donor samples, with each sample
being represented by the average of duplicates derived from the
donor. A comparison of growth index for chondrocytes propagated in
DMEM/10% FBS and in completely defined serum-free media is
illustrated in FIG. 1. Growth index for chondrocytes propagated in
DMEM/10% FBS and those propagated in cDRF/P/L were comparable
whereas cells propagated in cDRF/P or cDRF had slightly lower
growth index. Chondrocytes grown as monolayer in DM of the
invention did not have the typical fibroblastic morphology when
compared with chondrocytes grown in DMEM/10% FBS, but had a very
distinct cell shape with well defined borders.
Example 4
[0064] In this experiment, the dependence on the seeding density
was investigated. Human hyaline articular chondrocytes were
obtained from the same donors and treated as described Example 3,
except each sample was split into three to be seeded at 6,000;
4,000, and 2,000 cells/cm.sup.2. At the end of each passage, cells
were reseeded at the original seeding density of that set. At the
end of the third passage, cells were harvested, counted and cell
yield was calculated for chondrocytes propagated in DMEM/10% FBS or
in DM of this invention. The results of the experiment are
presented in FIG. 2. Chondrocytes grown in cDRF/P/L had at least
comparable or higher yields than cells grown in DMEM/10% FBS or
cDRF/P, whereas cells propagated in cDRF had slightly lower yields
as compared to DMEM/10% FBS. The difference was more pronounced for
cells passaged at seeding density of 6,000 cells/cm.sup.2.
Additionally, cells grown in cDRF or cDRF/P had higher yields at
higher seeding densities.
Example 5
[0065] To assess the redifferentiation potential of chondrocytes
after their expansion in monolayer culture in DM of this invention,
the chondrocytes' capacity to form cartilaginous tissue was
examined. Chondrocytes were isolated and treated as described in
Example 2. At the end of the second passage chondrocytes were
trypsinized, rinsed in DMEM/10% FBS and seeded as described below
on Millicell-CM.RTM. filter inserts (12 mm diameter, 0.4 .mu.m pore
size, Millipore Corp., Bedford, Mass.). The filters were pre-coated
with type II collagen (0.5 mg/ml 0.012N HCl) [Sigma St. Louis,
Mo.]. Chondrocytes were seeded at 2.times.10.sup.6 cells/cm.sup.2
on top of the filter in DMEM/20% FBS or in a DMEM-based
differentiation medium (DMEM supplemented with 2 mg/ml HSA, 5
.mu.g/ml linoleic acid, 2% ITS). The cultures were maintained at
37.degree. C. in a humidified atmosphere supplemented with 5%
CO.sub.2. After three days in culture 100 .mu.g/ml ascorbic acid, 5
ng/ml TGF-.beta.2 and 10 ng/ml PDGF-BB were added to the media. The
media were changed every two days. After 1 week, the chondrocyte
cultures on the filter inserts were harvested at selected intervals
and fixed in 10% formalin, embedded in paraffin and cut in 5 .mu.m
sections that were then stained with toluidine blue or
safranin-.largecircle.. These reagents stain sulphated
proteoglycans. Sulphated glycosaminoglycans were quantified using a
modified dimethylmethylene blue assay according to the procedure
described by Farndale et al. (Biochimica et Biophyca Acta
883:173-177, 1986).
[0066] Immunohistochemical analysis on the paraffin-embedded
sections was performed to analyze expression of type II collagen.
Primary antibodies for type II collagen (Biodesign International,
Kennebunkport, Me.) were used at 1:50 dilution. The reaction was
carried out in a humid atmosphere at 37.degree. C. for one hour.
The tissue sections were then washed 3 times in Phosphate Buffered
Saline (PBS) and incubated with a 1:200 dilution of
rhodamine-conjugated goat anti-rabbit IgG in PBS as a secondary
antibody, under the same conditions as described for the primary
antibodies. Hoechst dye at 1 .mu.g/ml was included in some
experiments with the secondary antibody for nuclear staining. The
sections were washed three times in PBS and examined under a
fluorescence microscope.
[0067] Histological examination of the cultures showed that the
chondrocytes passaged in cDRF/P/L accumulated an extracellular
matrix, which contained proteoglycans and collagen, and formed a
continuous layer of cartilaginous tissue. These chondrocytes showed
an increase in the amount of tissue produced and the matrical
staining of proteoglycans when compared with chondrocyted
propagated with DMEM/10% FBS. The cells propagated in cDRF/P/L
readily underwent differentiation from a monolayer to round cells
with lacunae chondrogenic morphology and expressed more type 11
collagen as compared to DMEM/10% FBS. The results of this
experiment demonstrate that chondrocytes propagated in DM of the
invention, are capable of re-expressing their chondrocyte
phenotype, i.e., they retain their redifferentiation potential. The
results also demonstrate the feasibility of producing preformed
cartilage grafts from propagated chondrocytes isolated and expanded
in DM of the invention.
Example 6
[0068] Normal adult human articular chondrocytes dedifferentiate as
a consequence of expansion in monolayer in vitro. To confirm that
chondrocytes cultured in DM of the invention have retained their
capacity to redifferentiate, a TaqMan analysis of gene expression
of cartilage-specific markers was performed. The analysis of gene
expression begins with the isolation of quality total RNA from ex
vivo formed cartilagenous tissue and normal articular
cartilage.
[0069] Initially, chondrocytes were isolated and treated and
expanded in DMEM/10% FBS or cDRF/P/L described in Example 2. These
cultures were then harvested at 1, 2, 3, and 4 weeks from seeding
on the filter inserts. Subsequently, chondrocytes cultures were
grown on Millicell-CM.RTM. filter inserts as described in Example
5. Gene expression was analyzed for the following proteins:
aggrecan, type I collagen, type II collagen, type X collagen,
osteocalcin, osteopontin, and versican.
[0070] Total RNA was isolated using a modification of a published
protocol (Reno et al. (1997) Biotechniques 22: 1082-1086). First
total RNA is isolated from the tissue using the TRIzol reagent
(catalog #15596-026, Invitrogen Life Technologies, Carlsbad,
Calif.) along with mechanical homogenization using a handheld
tissue homogenizer. The isolated RNA was resuspended in 10 .mu.l of
nuclease-free water and purified over a RNeasy Mini spin column
(calalog #74104, QIAGEN, Valencia, Calif.) following a protocol as
supplied by the manufacturer. Contaminating genomic DNA is removed
using DNA-free kit (catalog #1906, Ambion, Austin, Tex.). An equal
amount of total RNA was taken from each sample and
reverse-transcribed (RT) using beads with an oligo-dT primer
(catalog #27-9264-01, Amersham Biosciences, Piscataway, N.J.). A
Picogreen assay ( catalog # P-7589, Molecular Probes, Eugene,
Oreg.) was performed to measure the efficiency of the RT reaction.
Next, a TaqMan assay was performed to quantify of the absolute copy
number of each gene using 25 ng of starting material from each
sample. The number of gene copies was determined for each gene
using a standard curve created with commercially available plasmid
standards. The final results were adjusted in accordance with
results of the PicoGreen assay.
[0071] The results of a TaqMan assays for samples from two subjects
are presented in FIGS. 3A and 3B. These results demonstrate a
sustained (2-4 weeks) elevated expression of the type II collagen
gene, a major marker for articular cartilage, in cells propagated
in DMEM/P/L. The results of this experiment confirm that that
chondrocytes propagated in DM of the present invention retain their
capacity to re-express the chondrocyte phenotype. The results also
demonstrate the feasibility of producing preformed cartilage grafts
from propagated chondrocytes isolated and expanded in DM of the
invention.
[0072] The specification is most thoroughly understood in light of
the teachings of the references cited within the specification, all
of which are hereby incorporated by reference in their entirety.
The embodiments within the specification provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention.
[0073] Unless otherwise indicated, all numbers expressing
quantities of ingredients, cell culture conditions, and so forth
used in the specification, including claims, are to be understood
as being modified in all instances by the term "about."
Accordingly, unless otherwise indicated to the contrary, the
numerical parameters are approximations and may very depending upon
the desired properties sought to be obtained by the present
invention. A skilled artisan will recognize that many other
embodiments are encompassed by the claimed invention and that it is
intended that the specification and examples be considered as
exemplary only, with a true scope and spirit of the invention being
indicated by the following claims.
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