Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Method of regulating cell metabolism or physiology
Reexamination Certificate
2001-05-09
2003-09-09
Tate, Christopher R. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Method of regulating cell metabolism or physiology
C435S325000, C435S366000, C435S374000, C435S383000, C435S388000, C435S389000, C435S404000, C435S405000, C424S093700, C424S093100
Reexamination Certificate
active
06617161
ABSTRACT:
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 &agr;-ketoglutarate, ceruloplasmin, cholesterol, phosphatidylethanolamine, &agr;-tocopherol acid succinate, reduced glutathione, taurine, triiodothyronine, hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate, &bgr;-glycerophosphate, platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). Preferably, the medium contains about: 1×10
−4
M &agr;-ketoglutarate, 0.25 U/ml ceruloplasmin, 5 &mgr;g/ml cholesterol, 2 &mgr;g/ml phosphatidylethanolamine, 9×10
−7
M &agr;-tocopherol acid succinate, 10 &mgr;g/ml reduced glutathione, 1.25 mg/ml taurine 1.6×10
−9
M triiodothyronine, 1×10
−9
M hydrocortisone, 5×10
−10
M parathyroid hormone, 50 &mgr;g/ml L-ascorbic acid 2-sulfate, 10 mM &bgr;-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 &agr;-ketoglutarate, ceruloplasmin, cholesterol, phosphatidylethanolamine, &agr;-tocopherol acid succinate, reduced glutathione, taurine, triiodothyronine, hydrocortisone, parathyroid hormone, L-ascorbic acid 2-sulfate, &bgr;-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.
REFERENCES:
patent: 5723331 (1998-03-01), Tubo et al.
patent: WO 91/10726 (1991-07-01), None
patent: WO 95/00632 (1995-01-01), None
patent: WO 96/14335 (1996-05-01), None
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 ∃-Adrenergic Signal System of HL-60 Cells; Biochemical Pharmacology38(21):3723-3729 (1989).
Chang, et al.,Cartilage-derived Morphogenetic Proteins; J. of Biological Chemistry269(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 Science106:105-114(1995).
Harrison, et al.,Osteogenin Promotes Reexpression
Erlacher Ludwig
Luyten Frank P.
Patten Patricia
Tate Christopher R.
The United States of America as represented by the Department of
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