U.S. patent number 6,617,161 [Application Number 09/851,921] was granted by the patent office on 2003-09-09 for serum-free cell growth medium.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Ludwig Erlacher, Frank P. Luyten.
United States Patent |
6,617,161 |
Luyten , et al. |
September 9, 2003 |
**Please see images for:
( Certificate of Correction ) ** |
Serum-free cell growth medium
Abstract
A chemically defined-serum free growth medium for the in vitro
and ex vivo of cells and cell lines. The medium consists of about a
one to one ratio (v/v) of two basal growth media containing
.alpha.-ketoglutarate, insulin, transferrin, selenium, bovine serum
albumin, linoleic acid, ceruloplasmin, cholesterol,
phosphatidyl-ethanolamine, .alpha.-tocopherol acid succinate,
reduced glutathione, taurine, triiodothyronine, hydrocortisone,
parathyroid hormone, L-ascorbic acid 2-sulfate,
.beta.-glycerophosphate, PDGF, EGF and FGF. Chondrocytes, when
cultured in this medium in the presence of a cartilage derived
morphogenetic protein or bone morphogenetic protein, retain their
cartilaginous phenotype.
Inventors: |
Luyten; Frank P. (Kraainem,
BE), Erlacher; Ludwig (Vienna, AT) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
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Family
ID: |
21966803 |
Appl.
No.: |
09/851,921 |
Filed: |
May 9, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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468562 |
Dec 21, 1999 |
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PCTUS9812958 |
Jun 22, 1998 |
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Current U.S.
Class: |
435/375;
424/93.1; 424/93.7; 435/325; 435/366; 435/374; 435/383; 435/388;
435/389; 435/404; 435/405 |
Current CPC
Class: |
C12N
5/0655 (20130101); A61K 35/12 (20130101); C12N
2500/32 (20130101); C12N 2500/33 (20130101); C12N
2500/34 (20130101); C12N 2500/36 (20130101); C12N
2500/38 (20130101); C12N 2501/11 (20130101); C12N
2501/115 (20130101); C12N 2501/135 (20130101); C12N
2501/20 (20130101); C12N 2501/37 (20130101); C12N
2501/39 (20130101); C12N 2501/395 (20130101); C12N
2500/90 (20130101) |
Current International
Class: |
C12N
5/06 (20060101); A61K 35/12 (20060101); C12N
005/00 (); C12N 005/08 (); A01N 063/00 () |
Field of
Search: |
;424/93.7
;435/325,366,374,375,383,384,388,389,404,405 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 91/10726 |
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Jul 1991 |
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WO |
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WO 95/00632 |
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Jan 1995 |
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WO |
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WO 96/14335 |
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May 1996 |
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WO |
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Other References
Aulthouse et al. : Expression of the Human Chondrocyte Phenotype in
Vitro; In Vitro Cellular & Development Biology, 1989, vol. 25,
No. 7, pp. 659-668.* .
Bonaventure et al. Reexpression of Cartilage-Specific Genes by
Dedifferentiated Human Articulare Chondrocytes Cultured in Alginate
Beads; Experimental Cell Research, 1994, 212, pp. 97-104.* .
Harrison et al. Osteogenin Promotes Reexpression of Cartilage
Phenotype by Dedifferentiated Articular Chondrocytes in Serum-Free
Medium; Bone Cell Bio. (1991) 265, pp. 340-345.* .
Zenzius et al. Bone Morphogenic Protein-2 (BMP-2) Maintains the
Phenotype of Articular Chondrocytes in Long Term Monolayer Culture;
Molecular Biology of the Cell (1995), vol. 6, No. Suppl. pp. 391A.*
.
Sailor, L. Z., et all, "Recombinant Human Bone Morphogenetic
Protein-2 Maintains the Articular Chondrocyte Phenotype in
Long-Term Culture", Journal of Orthopaedic Research, 14:937-945
(1996). .
Bang, et al., Growth, Differentiation and the
.E-backward.-Adrenergic Signal System of HL-60 Cells; Biochemical
Pharmacology 38(21):3723-3729 (1989). .
Chang, et al., Cartilage-derived Morphogenetic Proteins; J. of
Biological Chemistry 269(45):28227-28234 (1994). .
Chen, et al., Osteogenic protein-1 promotes growth and maturation
of chick sternal chondrocytes in serum-free cultures; J. of Cell
Science 106:105-114(1995). .
Harrison, et al., Osteogenin Promotes Reexpression of Cartilage
Phenotype by Dedifferentiated Articular Chondrocytes in Serum-Free
Medium; Experimental Cell Research 192:340-345 (1991). .
Hodgson, et al., Fetal Bovine Serum Revisited; Biotechnology
11:49-53 (1993). .
Inlow, et al., Insect Cell Culture and Baculovirus Propagation in
Protein-Free Medium; J. of Tissue Cluture Methods 12(1):13-16
(1989). .
Kunkel, T.A., Rapid and efficient site-specific mutagenesis without
phenotypic selection; Proc. Natl. Acad. Sci. USA 82:488-492 (1985).
.
Labarca, et al., A Simple, Rapid, and Sensitive DNA Assay
Procedure; Analytical Biochemistry 102:344-352 (1980). .
Luyten, et al., Recombinant Bone Morphogenetic Protein-4,
Transforming Growth Factor-.E-backward..sub.1, and Activin A
Enhance the Cartilage Phenotype of Articular Chondrocytes in vitro;
Experimental Cell Research 210:224-229 (1994). .
Vonen, et al., Effect of a New Synthetic Serum Replacement on
Insulin and Somotostatin Secretion From Isolated Rat Pancreatic
Islets in Long-Term Culture; J. Tiss. Cult. Meth. 14:45-50 (1992).
.
Sigma Cell Culture 1994 Catalogue and Price List , p. 219-220
(1994). .
ATCC Connection 16(2):5-6 (1996)..
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Primary Examiner: Tate; Christopher R.
Assistant Examiner: Patten; Patricia
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear,
LLP
Parent Case Text
RELATED APPLICATIONS
This application is a continuation of U.S. pat. appl. Ser. No.
09/468,562, filed Dec. 21, 1999 now abandoned, which claims the
benefit and is a continuation of priority of International Appl.
No. PCT/US98/12958, filed Jun. 22,1998, which claims the benefit of
priority of U.S. Appl. No. 60/050,691, filed Jun. 25,1997.
Claims
What is claimed is:
1. A method of maintaining a cartilaginous phenotype in
chondrocytes in vitro comprising contacting a culture comprising
chondrocytes, which have not lost and subsequently re-expressed a
cartilaginous phenotype, with a serum-free cell growth medium
comprising about a 1:1 ratio (v/v) of two basal cell culture media,
said medium containing effective cell growth-promoting
concentrations of .alpha.-ketoglutarate, insulin, transferrin,
selenium, bovine serum albumin, linoleic acid, ceruloplasmin,
cholesterol, phosphatidylethanolamine, .alpha.-tocopherol acid
succinate, reduced glutathione, taurine, triiodothyronine,
hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate,
.beta.-glycerophosphate, platelet-derived growth factor (PDGF),
epidermal growth factor (EGF) and basic fibroblast growth factor
(bEGF) and at least one morphogenetic protein selected from the
group consisting of cartilage-derived morphogenetic proteins and
bone morphogenetic proteins, whereby a cartilaginous phenotype is
maintained in said chondrocytes in vitro.
2. A method of repairing a joint surface defect in a mammal in need
thereof comprising the steps of: removing normal cartilage in the
vicinity of said surface defect; isolating chondrocytes from said
cartilage; contacting a culture comprising said chondrocytes, which
have not lost and subsequently re-expressed a cartilaginous
phenotype, with a serum-free cell growth medium comprising about a
1:1 ratio (v/v) of two basal cell culture media, said medium
containing effective cell growth-promoting concentrations of
.alpha.-ketoglutarate, insulin, transferrin, selenium, bovine serum
albumin, linoleic acid, ceruloplasmin, cholesterol,
phosphatidylethanolamine, .alpha.-tocopherol acid succinate,
reduced glutathione, taurine, triiodothyronine, hydrocortisone,
parathyroid hormone, L-ascorbic acid 2-sulfate,
.beta.-glycerophosphate, platelet-derived growth factor (PDGF),
epidermal growth factor (EGF) and basic fibroblast growth factor
(bEGF) and at least one morphogenetic protein selected from the
group consisting of cartilage-derived morphogenetic proteins and
bone morphogenetic proteins, whereby a cartilaginous phenotype is
maintained in said chondrocytes; and implanting said chondrocytes
into said surface defect.
3. The method of claim 1 or 2, wherein two basal cell culture media
are selected and mixed in equal proportion and wherein said basal
cell culture media are selected from the group consisting of Ham's
F-12, Dulbecco's modified Eagle's medium (DMEM), Essential modified
Eagle's medium (EMEM) and RPMI-1640.
4. The method of claim 1 or 2, wherein said morphogenetic protein
is cartilage-derived morphogenetic protein and is selected from the
group consisting of CDMP-1 and CDMP-2.
5. The method of claim 1 or 2, wherein said morphogenetic protein
is bone morphogenetic protein and is selected from the group
consisting of OP-1, BMP-2, BMP-3, BMP-4, BMP-5 and BMP-6.
6. The method of claim 1 or 2, wherein the concentration of
.alpha.-ketoglutarate is about 1.times.10.sup.-4 M.
7. The method of claim 1 or 2, wherein the concentration of
ceruloplasmin is about 0.25 U/ml.
8. The method of claim 1 or 2, wherein the concentration of
cholesterol is about 5 .mu.g/ml.
9. The method of claim 1 or 2, wherein the concentration of
phosphatidyl-ethanolamine is about 2 .mu.g/ml.
10. The method of claim 1 or 2, wherein the concentration of
.alpha.-tocopherol acid succinate is about 9.times.10.sup.-7 M.
11. The method of claim 1 or 2, wherein the concentration of
reduced glutathione is about 10 .mu.g/ml.
12. The method of claim 1 or 2, wherein the concentration of
taurine is about 1.25 .mu.g/ml.
13. The method of claim 1 or 2, wherein the concentration of
triiodothyronine is about 1.6.times.10.sup.-9 M.
14. The method of claim 1 or 2, wherein the concentration of
hydrocortisone is about 1.times.10.sup.-9 M.
15. The method of claim 1 or 2, wherein the concentration of
parathyroid hormone is about 5.times.10.sup.-10 M.
16. The method of claim 1 or 2, wherein the concentration of
L-ascorbic acid 2-sulfate is about 50 .mu.g/ml.
17. The method of claim 1 or 2, wherein the concentration of PDGF
is about 4 ng/ml.
18. The method of claim 1 or 2, wherein the concentration of EGF is
about 10 ng/ml.
19. The method of claim 1 or 2, wherein the concentration of bFGF
is about 10 ng/ml.
20. The method of claim 2, wherein said isolating chondrocytes step
comprises digestion of said cartilage with collagenase.
Description
FIELD OF THE INVENTION
The present invention relates to a cell growth medium. More
specifically, the invention relates to a chemically defined
serum-free growth medium useful for the expansion of primary cells
or cell lines in culture.
BACKGROUND OF THE INVENTION
Culturing of mammalian cells is an essential technique for research
into cellular processes, production of recombinant therapeutic
proteins, and generation of expanded cells for transplantation
purposes. Cell culture studies have led to the determination of
numerous metabolic processes and the identification of growth
factors, hormones and their receptors (Bio Techniques, 5:534-542,
1987).
The composition of media used to culture cells is of paramount
importance because of its influence on cell survival and cell
response to various effectors. Conventional cell culture media
comprise basal nutrient media supplemented with serum from various
sources, most often fetal bovine serum, horse serum or human serum.
However, the use of serum is undesirable for several reasons.
Growth media containing serum may vary in composition, hormone
content, and contaminants, thereby introducing extraneous factors
and/or infections agents into the culture system (Bio Technology,
11:49-53, 1993; Pharm. Technol., 48:56, 1987). In addition, serum
is expensive, impractical for large-scale production of
therapeutics. Further, variance between serum lots and laboratory
protocols is also a problem. Recent concerns by the FDA, the
European community, and others about serum quality, contamination
(i.e., bovine spongiform encephalopathy, bovine immunodeficiency
virus), and increased demand have generated significant interest in
the development and utility of serum-free growth media.
Significant advances have been made in the development of serum
substitutes and serum-free media that address the inadequacies of
media containing serum (J. Tissue Cult. Meth., 14:45-50, 1992;
Biochem. Pharmacol., 38:3723-3729, 1989; J. Tissue Cult. Meth.,
12:13-16, 1989). Serum-free media provide many important advantages
over serum-containing media, including lot-to-lot consistency,
biological uniformity, and freedom from adventitious agents.
Serum-free media are generally cost effective and low in protein
content. All of these factors contribute to a more controlled
examination of cellular, molecular and metabolic processes (ATCC
Connection, 16(2):5-6, 1996).
Harrison et al. (Exp. Cell Res., 192:340-345, 1991) describe a
serum-free medium used for chondrocytes in suspension/agarose
cultures. Addition of a cocktail of growth factors to this medium
was sufficient to promote colony formation at levels comparable to
medium containing 10% fetal bovine serum (FBS).
Serum-free technology is not entirely problem-free. Many serum-free
media are highly specific to a particular cell type, are for
short-term culturing only, or contain components extracted from
serum. Very few available products can support primary cell
cultures. Ideally, serum-free media should be compatible with
various basal media and be useful for primary culturing and
long-term maintenance of established cell lines. The present
invention provides such a medium.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a cell growth medium
consisting essentially of about a 1:1 ratio (v/v) of two basal cell
culture media containing effective cell growth-promoting amounts of
.alpha.-ketoglutarate, ceruloplasmin, cholesterol,
phosphatidylethanolamine, .alpha.-tocopherol acid succinate,
reduced glutathione, taurine, triiodothyronine, hydrocortisone,
parathyroid hormone, L-ascorbic acid 2-sulfate,
.beta.-glycerophosphate, platelet-derived growth factor (PDGF),
epidermal growth factor (EGF) and basic fibroblast growth factor
(bFGF). Preferably, the medium contains about: 1.times.10.sup.-4 M
.alpha.-ketoglutarate, 0.25 U/ml ceruloplasmin, 5 .mu.g/ml
cholesterol, 2 .mu.g/ml phosphatidylethanolamine, 9.times.10.sup.-7
M .alpha.-tocopherol acid succinate, 10 .mu.g/ml reduced
glutathione, 1.25 mg/ml taurine 1.6.times.10.sup.-9 M
triiodothyronine, 1.times.10.sup.-9 M hydrocortisone,
5.times.10.sup.-10 M parathyroid hormone, 50 .mu.g/ml L-ascorbic
acid 2-sulfate, 10 mM .beta.-glycerophosphate,4 ng/ml PDGF, 10
ng/ml EGF and 10 ng/ml bFGF. Advantageously, the two basal cell
culture media are selected from Dulbecco's modified Eagle's medium
(DMEM), Ham's F-12, Essential modified Eagle's medium (EMEM) and
RPMI-1640. The medium described above may further contain at least
one cartilage derived morphogenetic protein. Preferably, the
cartilage derived morphogenetic protein is CDMP-1 or CDMP-2.
Advantageously, the medium further contains at least one bone
morphogenetic protein. Preferably, the bone morphogenetic protein
is OP-1.
The present invention also provides a method of growing or
expanding cells or cell lines, comprising contacting the cells with
a cell growth medium consisting essentially of about a 1:1 ratio
(v/v) of two basal cell culture media containing effective cell
growth-promoting amounts of .alpha.-ketoglutarate, ceruloplasmin,
cholesterol, phosphatidylethanolamine, .alpha.-tocopherol acid
succinate, reduced glutathione, taurine, triiodothyronine,
hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate,
.beta.-glycerophosphate, platelet-derived growth factor, epidermal
growth factor and basic fibroblast growth factor. Preferably, the
cells are primary cells. In one aspect of this preferred
embodiment, the cells are chondrocytes or marrow stromal
fibroblasts. Advantageously, the cells are grown in vitro.
Alternatively, the cells are grown ex vivo.
Another embodiment of the present invention is a method of
maintaining a cartilaginous phenotype in chondrocytes in vitro,
comprising culturing the chondrocytes in serum-free medium
comprising a cartilage-derived morphogenetic protein and/or bone
morphogenetic protein.
The present invention also provides a method of repairing a joint
surface defect in a mammal in need thereof, comprising the steps
of: isolating normal cartilage in the vicinity of the surface
defect; isolating chondrocytes from the cartilage; culturing the
chondrocytes in serum-free medium comprising a cartilage-derived
morphogenetic and/or bone morphogenetic protein whereby the
chondrocytes are expanded; and implanting the expanded chondrocytes
into the surface defect.
Preferably, the serum-free medium is the medium described above.
Advantageously, the step of isolating chondrocytes comprises
digestion of cartilage with collagenase.
BRIEF DESCRIPTION OF THE FIGURE
FIG. 1 is a graph showing human fetal chondrocyte expansion as
monolayer cultures in the serum-free medium of the invention (basal
medium+EGF+PDGF+FGF), the serum-free medium+cartilage-derived
morphogenetic protein 1 (CDMP-1), or basal medium containing 10%
fetal bovine serum.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a chemically defined serum-free
medium which can be used to maintain and expand cells, primary
cells and cell lines both in vitro and ex vivo. The medium can be
used for any desired cell type, and can be used for both short-term
and long-term culturing of cells. The medium consists of two basal
cell culture media (about 1:1, v/v) (Collaborative Biomedical
Products, Bedford, Mass.; GIBCO/BRL, Gaithersburg, Md.) containing
effective cell growth-promoting amounts of insulin, transferrin,
selenium, bovine serum albumin, linoleic acid,
.alpha.-ketoglutarate, ceruloplasmin, cholesterol,
phosphatidylethanolamine, .alpha.-tocopherol acid succinate,
reduced glutathione, taurine, triiodothyronine, hydrocortisone,
parathyroid hormone, L-ascorbic acid 2-sulfate and
.beta.-glycerophosphate. The medium also contains platelet-derived
growth factor (PDGF)-AB or BB, epidermal growth factor (EGF) and
basic fibroblast growth factor (bFGF). All supplements are
available from Sigma Chemical Co., St. Louis, Mo. Growth factors
are available from, for example, Collaborative Medical
Products.
Although Ham's F-12/Dulbecco's modified Eagle's medium (DMEM)
(about 1:1) are the two preferred basal media, the use of other
basal media combinations is also contemplated, including Essential
modified Eagle's medium (EMEM) and RPMI-1640. In a preferred
embodiment, the concentrations of the compounds listed above are
about:
TABLE 1 Compound Concentration .alpha.-ketoglutarate 1 .times.
10.sup.-4 M insulin 6.25 .mu.g/ml transferrin 6.25 .mu.g/ml
selenium 6.25 ng/ml bovine serum albumin 1.25 mg/ml linoleic acid
5.35 .mu.g/ml ceruloplasmin 0.25 U/ml cholesterol 5 .mu.g/ml
phosphatidylethanolamine 2 .mu.g/ml .alpha.-tocopherol acid
succinate 9 .times. 10.sup.-7 M reduced glutathione 10 .mu.g/ml
taurine 1.25 .mu.g/ml triiodothyronine 1.6 .times. 10.sup.-9 M
hydrocortisone 1 .times. 10.sup.-9 M parathyroid hormone 5 .times.
10.sup.-10 M L-ascorbic acid 2-sulfate 50 .mu.g/ml
.beta.-glycerophosphate 10 mM PDGF-AB or -BB 4 ng/ml EGF 10 ng/ml
bFGF 10 ng/ml
Although the preferred concentration of each component is as listed
in Table 1, these concentrations can be adjusted to suit any
desired cell or cell line. This serum-free medium comprises basal
medium (BM) plus various components and growth factors. The medium
is suitable for anchorage-dependent growth of cells for laboratory
experimentation, for large-scale production of recombinant proteins
and for expansion of cells for transplantation or implantation. For
example, fetal or adult pancreatic islet cells can be expanded in
vitro in the serum-free medium of the invention using standard cell
culture techniques, then implanted into a diabetic patient. The
medium can also be used to effectively culture marrow stromal
fibroblasts which differentiate into bone cells.
The medium can also be used to expand chondrocytes as discussed in
Example 1. This will allow in vivo cartilage repair. In this
embodiment, cartilage is removed from a non-damaged cartilage area
around a damage site and digested with collagenase. The resulting
chondrocytes are expanded ex vivo in the serum-free medium of the
invention, then injected or implanted into the cartilage defect
site. Similarly, marrow stromal fibroblasts can be isolated from a
normal area adjacent a bone defect, expanded ex vivo in serum-free
medium and administered at the site of a bone defect.
The expansion of chondrocytes (articular or fetal limb) in the
serum-free medium of the invention containing PDGF, EGF and FGF
results in the loss of two main cartilage-specific phenotypic
markers, type II collagen and proteoglycan aggrecan, which
contribute to the maintenance of the cartilaginous phenotype. These
molecules are critical to extracellular matrix synthesis.
Chondrocytes cultured in this serum free medium are fibroblastic in
appearance and longitudinal in shape, compared to the their typical
round morphology. The presence of these two phenotypic markers is
important, because the Food and Drug Administration (FDA)/Center
for Biologics Evaluation and Research (CBER) requires that
protocols relating to ex vivo expansion of articular chondrocytes
for repair of joint surfaces prove that the expanded cell
populations are very similar to the native chondrocytes. This can
be assessed by evaluating the expression of proteoglycan aggrecan
and type II collagen at the end of the expansion protocol by
Northern blotting, immunoblotting or fluorescence-activated cell
sorting (FACS).
In vitro studies were performed to investigate the role of Bone
Morphogenetic Proteins/Cartilage-Derived Morphogenetic Proteins
(BMPs/CDMPs) in maintaining the cartilaginous phenotype of human
articular and fetal limb chondrocytes under serum-free conditions
using Ham's F-12/DMEM (1:1) containing the compounds listed in
Table 1. One such BMP is osteogenic protein-1 (OP-1; also called
BMP-7) (Creative Biomolecules). The inclusion of CDMP-1 and/or
OP-1, both at 50 ng/ml in the serum-free medium contributed to the
maintenance and re-expression of proteoglycan aggrecan and type II
collagen in cultured human fetal chondrocytes as determined by
northern blotting of mRNA using labeled cDNA probes to type II
collagen and proteoglycan aggrecan. It is contemplated that the
inclusion of one or more CDMPs (CDMP-1, CDMP-2) and/or one or more
BMPs (i.e. OP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6) ) in any
serum-free medium capable of supporting cell growth will contribute
to the maintenance of the cartilaginous phenotype and the
proteoglycan aggrecan and type II collagen markers.
In addition, other cell types and cell lines in monolayer cultures
survived in the serum-free medium containing growth factor cocktail
(PDGF, EGF, FGF) with or without CDMPs (i.e., mouse ATDC5 embryonic
teratinocarcinoma cells; mouse calvarial osteoblastic clonal cell
line MC3T3-E1; the mouse myoblast cell line C2C12; R mutant Mv1 Lu
cells; and the rat osteoprogenitor-like cell line ROB-C26), and
some continued to proliferate.
The efficacy of cell expansion was compared using Ham's F-12/DMEM
(1:1) containing the compounds shown in Table 1 with 10% fetal
bovine serum versus the same medium with growth factors (PDGF, FGF
and EGF) replacing the serum. Surprisingly, every primary cell or
cell line tested with the serum-free medium expanded in culture
almost as quickly or equally fast than in the serum-containing
medium. This medium will contribute to the reproducibility of cell
expansion protocols, provides lot-to-lot consistency, biological
uniformity and freedom from adventitious agents. The medium also
performs well regardless of the cell type used.
Various CDMP assays performed in cultured cells, including DNA
determination, alkaline phosphatase activity and proteoglycan
biosynthesis could only be performed in the serum-free medium of
the invention; the presence of serum significantly interfered with
these assays. Thus, the serum-free medium is essential to the
success of these assays.
The growth of human fetal chondrocytes in the serum-free medium of
the invention is discussed in the following example.
EXAMPLE 1
Growth of Human Fetal Chondrocytes in Serum-Free Medium
Human fetal limbs from 52 to 79 day old fetuses were provided by
the Laboratory for Human Embryology, Department of Pediatrics,
University of Washington, Seattle, Wash. This was approved by the
Office of Human Subjects Research, National Institutes of Health.
The cartilaginous cores were carefully dissected from the
surrounding fetal tissue and the chondrocytes were released by a
six hour digestion in 0.2% collagenase B (Boehringer Mannheim,
Indianapolis, Ind.) in basal medium lacking growth factors (Table
1) at 37.degree. C. Chondrocytes were cultured in either basal
medium containing EGF, PDGF and FGF (Table 1); basal medium
containing EGF, PDGF, FGF and 50 ng/ml CDMP-1; or basal medium plus
10% fetal bovine serum for two to six days. Cell expansion at days
2, 3, 4, 5 and 6 was assessed by determination of cellular DNA
content using bisbenzimide (Hoechst 33258, Sigma, St. Louis, Mo.)
(Labarca et al., Anal. Biochem., 102:344-352, 1980). As shown in
FIG. 1, by day 6 in culture, human chondrocytes exhibited the same
degree of expansion in either serum-free or serum-containing
medium.
EXAMPLE 2
Expression and Purification of CDMPs
The cDNAs encoding CDMP-1 and CDMP-2 were isolated by Chang et al.
(J. Biol. Chem., 269:28227-28234, 1994) and are also described in
International Publication WO96/14335. A cDNA encoding the mature
cdmp-1 was modified for insertion into an E. coli expression vector
by site directed mutagenesis using the method of Kunkel (Proc.
Natl. Acad. Sci. U.S.A., 82:488-492, 1985). Following the
pro-domain and in close proximity to the RXXR processing site, a
leucine residue was converted to a methionine translational
initiation codon with a corresponding NcoI restriction site, while
a 3' XhoI site was similarly introduced immediately after the
translational stop codon. The NcoI to XhoI fragment containing the
open reading frame for mature cdmp-1 was ligated to a tetracycline
resistant pBR322-derived expression vector. Fermentation was done
in shaker flasks using 2YT medium with addition of indolacrylic
acid at the appropriate time for induction of the tryptophan
promoter (Nichols et al., J. Mol. Biol., 146:45-54, 1981). Large
exclusion bodies containing CDMP-1 accumulated. A cDNA for cdmp-2
was similarly tailored for expression. Since the CDMP-2 expression
level was quite low, part of the N-terminal region of the highly
expressed cdmp-1 was spliced with the 7-cysteine domain of cdmp-2
at the first cysteine, where both genes share a PstI site. Induced
cell cultures (250 ml) were centrifuged (11,000.times.g, 10 min.,
4.degree. C.), followed by resuspension of the cell pellets in 50
ml of 25 mM Tris-HCl, 10 mM EDTA, pH 8.0 (1.times.TE) containing
100 .mu.g/ml lysozy cell suspensions were incubated overnight at
37.degree. C., chilled on ice and sonicated. Inclusion bodies were
isolated by centrifugation (11,000.times.g, 20 min., 4.degree. C.)
and resuspended in 1.times.TE. The final washed inclusion body
pellets were resuspended in 40 ml 1.times.TE, 15% glycerol and
stored at -20.degree. C.
Reduced and denatured inclusion body protein solutions were
prepared by dissolving aliquots of pelleted inclusion bodies in 100
mM Tris-HCl, 10 mM EDTA, 6 M guanidine HCl, 10 mM dithiothreitol
(DTT), pH 8.0 (final protein concentration of 4-6 mg/ml). The
inclusion body protein solutions were incubated at 37.degree. C.
for 30 min., then diluted 40-fold with refolding buffer (100 mM
Tris, 10 mM EDTA, 1 M NaCl, 2% 3-[3-cholamidopropyl)
dimethylammonio]-2-hydroxy-1-propanesulfonic acid (CHAPS), 5 mM
reduced glutathione, 2.5 mM oxidized glutathione, pH 8.7). The
refolding reactions were incubated for 72 hours at 4.degree. C. The
folding reactions were dialyzed extensively against 10 mM HCl, then
clarified by centrifugation (11,000.times.g, 20 min., 4.degree.
C.). The solutions were concentrated using a stirred cell
concentrator and YM10 MWCO membranes (Amicon). The concentrated
proteins were then lyophilized. The lyophilized proteins were
resuspended in 0.8 ml 0.1% trifluoroacetic acid (final acetonitrile
concentration--30%). The protein solutions were fractionated by
semi-preparative C4 Reverse Phase High Performance Liquid
Chromatography (HPLC) using a linear acetonitrile gradient (30-70%
in 0.1% TFA). Aliquots of each fraction were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using
15% gels under non-reducing conditions after reduction and
alkylation. Peak dimer fractions were pooled and concentrations
were estimated by UV absorbance spectra obtained at 280 nm. Protein
pools were stored at -20.degree. C.
EXAMPLE 3
Repair of Cartilage Defects
An individual with a joint surface (i.e. knee) defect is identified
and healthy cartilage surrounding the defect is surgically removed.
The cartilage is then digested with collagenase using standard
methods to isolate individual chondrocytes. Chondrocytes are
cultured in basal medium containing the components listed in Table
1 and containing CDMP-1 and/or CDMP-2. The expanded chondrocytes
are tested for the presence of proteoglycan aggrecan and type II
collagen markers, then surgically implanted into the joint defect
site. Significant improvement in both clinical signs and symptoms
as well as repair of the defect of the joint surface occurs in
response to this procedure.
It should be noted that the present invention is not limited to
only those embodiments described in the Detailed Description. Any
embodiment which retains the spirit of the present invention should
be considered to be within its scope. However, the invention is
only limited by the scope of the following claims.
* * * * *