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  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Definitions

• 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.
• Articular cartilage is composed of chondrocytes encased within the complex extracellular matrix produced by those chondrocytes.
• the unique biochemical composition of this matrix provides for the smooth, nearly frictionless motion of articulating surfaces of the joints.
• tensile properties of human articular cartilage change as a result of biochemical changes.
• the tensile strength of articular cartilage decreases markedly. Damage to cartilage produced by trauma or disease, e.g., rheumatoid and osteoarthritis, can lead to serious physical debilitation.
• 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 adopt a fibroblastic morphology and cease to produce type Il 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., In Vitro Cell. & Devel.
• chondrocytes are typically cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS).
• DMEM Dulbecco's Modified Eagle's Medium
• FBS fetal bovine serum
• 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. Freshney (1994) Serum-free media. In: Culture of Animal Cells, John Wiley & Sons, New York, 91-99.
• defined serum-free media is particularly advantageous in the ex vivo expansion of chondrocytes for treatment of cartilage defects.
• 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.
• DM biochemically defined media
• 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.
• This invention provides compositions of chemically defined culture media (DMs), methods of making such media, and methods of using such media, e.g., for culturing cells, in particular, human articular chondrocytes for repair of cartilage defects.
• DMs chemically defined culture media
• One of the distinguishing features of the DM of the invention is the presence of one or more substantially pure cytokines of the IL-6 family, such as, e.g., oncostatin M (OSM), interleukin-6 (IL-6), and leukemia inhibitory factor (LIF).
• OSM oncostatin M
• IL-6 interleukin-6
• LIF leukemia inhibitory factor
• the invention allows one to avoid the use of serum in chondrocyte cultures, enhance cell attachment and proliferation under serum-free conditions, and/or to maintain the capacity of chondrocytes to re-express cartilage-specific phenotype.
• the invention provides DM that is 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 cells such as 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.
• Another aspect of the invention provides a method of culturing a chondrocyte to a state that is suitable for treating a patient suffering from a cartilage defect. Additional 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 DM of the invention comprises a basal medium supplemented with one or more supplements, including one or more cytokines of the IL-6 family, such as, e.g., OSM, IL-6, and LIF.
• one or more supplements including one or more cytokines of the IL-6 family, such as, e.g., OSM, IL-6, and LIF.
• the basal medium may be any suitable medium.
• the basal medium is cDRF (Table 3) or cDRFm (Table 4).
• cDRF and cDRFm are made by mixing DMEM, RPMI-1640, and Ham's F-12 at a 1 :1 :1 ratio or by appropriately combining pre-mixed media and adding certain growth supplements to arrive at the basal media as defined in Tables 3 and 4 respectively.
• the basal medium is additionally supplemented with platelet-derived growth factor (PDGF) and/or one or more lipids.
• the lipids are a chemically defined lipid mixture (CDLM; Table 5) or one or more lipids from CDLM (e.g., stearic acid, myristic acid, oleic acid, linoleic acid, palmitic acid, palmitoleic acid, arachidonic acid, linolenic acid, cholesterol, and alpha-tocopherol acetate).
• a DM of the invention may include a basal medium (e.g., cDRF or cDRFm) supplemented with:
• DM of the invention may include: (a) a basal medium; (b) 0.1-100 ng/ml PDGF; (c) 0.05-5% CDLM; (d) 0.01-10 ng/ml OSM; and/or (e) 0.01-10 ng/ml IL-6.
• Figure 1 depicts a comparison in growth of primary human chondrocytes cultured in (1) DMEM + 10% FBS or (2) the E93 medium (cDRFm, as defined in Table 4, supplemented with CDLM 1 PDGF, IL-6, and OSM) over three passages.
• DMEM + 10% FBS or (2) the E93 medium (cDRFm, as defined in Table 4, supplemented with CDLM 1 PDGF, IL-6, and OSM) over three passages.
• Figure 2 depicts a comparison demonstrates a comparison in cell yield of primary human chondrocytes cultured in (1) DMEM + 10% FBS or (2) the E93 medium (cDRFm, as defined in Table 4, supplemented with CDLM 1 PDGF, IL-6, and OSM) over three passages.
• DMEM + 10% FBS or (2) the E93 medium (cDRFm, as defined in Table 4, supplemented with CDLM 1 PDGF, IL-6, and OSM) over three passages.
• Figure 3 shows an RPA of cell lysate from chondrocytes grown in E93 (lanes 2, 3, 4) or DMEM + 10% FBS (lanes 5, 6, 7).
• the cartilage markers, collagen 2 and aggrecan, are expressed in all samples.
• This invention provides compositions of chemically defined culture media (DMs), methods of making such media, and methods of using such media, e.g., for culturing cells, such as human articular chondrocytes, for repair of cartilage defects.
• the invention is based, at least in part, on the discovery that the basa! medium referred to as cDRFm supplemented with PDGF and CDLM and one or more cytokines of the IL-6 family 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.
• the cytokines of the IL-6 family of cytokines include, for example, OSM, IL-6, and LIF.
• the invention provides a culture medium comprising a basal medium supplemented with one or more supplements, which include one or more cytokines of the IL-6 family, such as, e.g., OSM, IL-6, and LIF.
• cytokines of the IL-6 family such as, e.g., OSM, IL-6, and LIF.
• the term "supplemented with” indicates that a supplement has been added to a starting material to arrive at an ending material. Unless specifically indicated, the supplement or supplements need not be added at a specific time or in a specific order. The term “supplemented with” does not preclude the starting material from being additionally supplemented with other supplements, at any point in time, before or after being supplemented with the present supplement. Unless specifically indicated, supplements are added to the medium in a “substantially pure” form. The term “substantially pure” indicates that a supplement is substantially free of components with which it naturally occurs in nature. For example, a substantially pure cytokine could be a purified cytokine or a cytokine that is recombinantly produced.
• the first step in preparing defined, serum-free media (DM) of.the invention is to obtain a basal medium.
• the basal medium may be any suitable medium.
• the basal medium is cDRF as defined in Table 3.
• cDRF can be prepared from commercially available starting components as described below.
• cDRF is a modification of the DM developed by Adolphe et al. (Exp. Cell Res. 155:527-536 (1984)) and by McPherson et al. (U.S. Patent No. 6,150,163).
• the three starting components of cDRF are DMEM, RPMM 640, and Ham's F12 (Invitrogen; Carlsbad, CA).
• the starting components are combined at a 1:1 :1 ratio. All three media can be combined at once, or any two of the media can be premixed and then combined with an appropriate amount of a third medium.
• the precise composition of starting components is set forth in Table 1.
• the resulting medium (defined in Table 2 and referred to as DRF) is then supplemented with ITS (10 ⁇ g/ml insulin, 5.5 ⁇ g/ml transferrin, 7 ng/ml selenium, and, optionally, 2.0 ⁇ g/ml ethanolamine; Invitrogen, Carlsbad, CA), human fibronectin (BD Biosciences; San Jose, CA), human serum albumin (HSA) (Grifols; Los Angeles, CA; or Baxter; Westlake Village, CA), linoleic acid (Sigma- Aldrich; St.
• ITS 10 ⁇ g/ml insulin, 5.5 ⁇ g/ml transferrin, 7 ng/ml selenium, and, optionally, 2.0 ⁇ g/ml ethanolamine; Invitrogen, Carlsbad, CA), human fibronectin (BD Biosciences; San Jose, CA), human serum albumin (HSA) (Grifols; Los Angeles, CA; or Baxter; Westlake Village,
• cDRFm modified cDRF
• the basal medium is a 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/or 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.
• 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, but are not limited to, growth factors, lipids, serum proteins, vitamins, minerals, and carbohydrates.
• growth factors or hormones that promote chondrocyte redifferentiation such as TGF- ⁇ (TGF-P1 , - ⁇ 2, - ⁇ 3), IGF, and insulin, as described in U.S. Patent No. 6,150,163.
• TGF- ⁇ TGF-P1 , - ⁇ 2, - ⁇ 3
• IGF insulin
• Such growth factors and hormones are commercially available.
• BMPs bone mo ⁇ hogeneteic proteins
• BMPs bone mo ⁇ hogeneteic proteins
• BMPs 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, MN). The concentration of various such supplements in DM of the invention can be determined with minimal 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 ⁇ g/ml.
• DM of the invention have advantages in addition to avoiding the use of serum.
• the 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.
• serum e.g., fetal calf serum
• the DM of the invention may be supplemented with 10% or less, for example, 8% or less, 6% or less, 4% or less, 2% or less, or 1% or less of serum.
• 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.
• cDRF Basal medium equivalent to cDRF
• 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 consistency and encompasses the use of media ranging from liquid to semi-solid and includes solidified media and solid compositions suitable for reconstitution.
• Vitamin Bi 2 0.68 0.005
• ITS-X supplement (insulin, 1% transferrin, selenium, ethanolamine)
• ITS-X supplement (insulin, 1% transferrin, selenium, ethanolamine)
• PDGF Platelet-Derived Growth Factor
• the basal medium is supplemented with substantially pure PDGF.
• 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.
• 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.
• PDGF-BB Human recombinant PDGF-BB (hrPDGF-BB) used in the Examples below was purchased from R&D Systems (Minneapolis, MN; 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 3 H-thymidine incorporation in quiescent NR6R-3T3 fibroblast as described by Raines et al. (Meth. Enzymol. 109:749-773 (1985)).
• the ED 50 for PDGF in this assay is typically 1-3 ng/ml.
• 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.
• cDRF is supplemented with 1-25 ng/ml, more preferably, 5 - 15 ng/ml and, most preferably, about 10 ng/ml of PDGF.
• the PDGF is PDGF-BB.
• PDGF could be of another type, e.g., PDGF-AB, PDGF-BB, or a mix of any PDGF types.
• the DM of the invention further or alternatively comprises additional supplements as described below.
• the basal medium is supplemented with CDLM (Table 5) or, alternately, one or more lipids from CDLM.
• 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. See, e.g., Weiss et al., In Vitro 26:30A (1990); Gorfien et al.. In Vitro 26.37A (1990); Fike et al., In Vitro 26:54A (1990). Thus, supplementation of serum-free media with various lipids to replace those normally supplied by serum may be desirable.
• 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.
• the basal medium is supplemented with the chemically defined lipid mixture (CDLM), shown in Table 5.
• CDLM is available from Invitrogen. As supplied by Invitrogen, in addition to the lipid components, CDLM contains ethanol (100 g/L) and emulsifiers Pluronic F68 ® (100 g/L) and Tween 80 ® (2.2 g/L).
• the concentrations of individual lipid components of CDLM shown in Table 5 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.
• the DM of the invention comprises at least one, two, four, six, eight, or all lipid components of CDLM.
• the DM comprises PDGF and CDLM as defined in Table 5.
• the DM comprises PDGF and lipid combinations as set forth in Table 6.
• CDLM chemically defined lipid mixture
• arachidonic acid linoleic acid, linolenic acid, alpha-tocopherol acetate, stearic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid
• arachidonic acid linoleic acid, linolenic acid, stearic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid
• 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%.
• the DM is additionally supplemented with 1 to 25 ng/ml, more preferably, 5 to 15 ng/ml, and, most preferably, about 10 ng/ml of PDGF.
• the DM comprises approximately 0.5% (v/v) CDLM and 10 ng/ml PDGF.
• IL-6 family of cytokines each can utilize a shared signal transducing receptor subunit, gp130, which is found in a wide range of cell types. See, e.g., Hirano et al. (2001) IL-6 Ligand and Receptor Family. In: Cytokine Reference, Academic Press, San Diego, 523-535.
• IL-6- family cytokines include, but are not limited to, oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), ciliary neurotrophic factor (CNTF), interleukin-11 (IL-11 ), cardiotrophin 1 (CT-1), and neurotrophin 1 / B cell- stimulating factor 3 (NNT-1/BSF-3).
• OSM oncostatin M
• IL-6 interleukin-6
• LIF leukemia inhibitory factor
• CNTF ciliary neurotrophic factor
• IL-11 interleukin-11
• CT-1 cardiotrophin 1
• NNT-1/BSF-3 neurotrophin 1 / B cell- stimulating factor 3
• Human OSM is a secreted glycoprotein that is initially translated as a 252-amino-acid polypeptide with a 25-residue hydrophobic signal sequence at the N-terminus that is removed during the secretion process.
• An additional post-translational cleavage event removes 31 C-terminal residues, leaving a 192- amino-acid disulfide-linked mature protein.
• OSM binds and signals through two different receptor complexes - the LIF receptor (LIFR) / gp130 heterodimer and the OSM receptor (OSMR) / gp130 heterodimer. Binding to either receptor complex leads to activation of the Janus kinase / signal transducers and activators of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) signaling pathways.
• LIFR LIF receptor
• OSMR OSM receptor
• OSM has been reported to inhibit the growth of some, but not all, human tumor cell lines. In contrast, OSM has also been reported to stimulate the growth of some normal fibroblasts, such as human foreskin fibroblasts or WI-38 cells. Zarling et al., Proc. Nat. Acad. Sci. USA 83:9739-9743 (1986). Thus, OSM may be useful for stimulating the growth of certain cells in vitro. A more detailed description of OSM can be found in U.S. Patent Nos. 5,202,116 and 5,814,307.
• OSM is readily available from commercial sources.
• a 196-amino-acid recombinant OSM produced in E. coli was obtained from R&D Systems (Minneapolis, MN) (catalog No.295-OM, see also Linsley et al., MoI. Cell. Biol. 10:1882-1890 (1990)).
• the biological activity of OSM may be assayed by testing in a human erythroleukemic cell line proliferation assay, as described, e.g., in Kitamura et al., J. Cell Physiol. 140:323-334 (1989).
• human OSM is used to produce the media of the invention.
• one skilled in the art would recognize that OSM from other species, naturally occurring mutants, and engineered mutants may also be effective.
• IL-6 lnterleukin-6
• IL-6 has many alternative names, including: interferon ⁇ 2; B-cell differentiation factor; B-cell stimulatory factor 2; hepatocyte stimulatory factor; hybridoma growth factor; and CTL differentiation factor.
• Human IL-6 is a 186-ami no-acid secreted glycoprotein that is synthesized as a 212-amino-acid precursor protein. Matsuda et al., (2001) IL-6. In: Cytokine Reference, Academic Press, San Diego, 538-563. In humans, IL-6 binds and signals through a complex of the IL-6 receptor (IL-6R) and a gp130 homodimer.
• IL-6R IL-6 receptor
• IL-6 Binding of IL-6 to the IL-6R receptor leads to activation of the Janus kinase / signal transducers and activators of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) signaling pathways.
• JAK/STAT Janus kinase / signal transducers and activators of transcription
• MAPK mitogen-activated protein kinase
• IL-6 has been reported to induce differentiation of PC12 neuronal cells, to induce clonogenic maturation of bone marrow progenitor cells, and to induce the growth of T cells. In contrast, IL-6 has also been shown to inhibit the growth of myeloid leukemia cells and breast cancer cells. Thus, IL-6 may be useful for stimulating the growth of certain cells in vitro. A more detailed description of IL-6 biology can be found in U.S. Patent No. 5,188,828.
• IL-6 is available from commercial sources.
• a 184-amino-acid recombinant IL-6 produced in E. coli was obtained from R&D Systems (Minneapolis, MN) (catalog No.206-IL 1 see also Hirano et al., Nature 324:73-76 (1986)).
• the biological activity of IL-6 is assayed by testing in a plasmacytoma proliferation assay as described in, e.g., Nordan et al., J. Immunol. 139:813 (1987).
• human IL-6 is used to produce the media of the invention.
• IL-6 from other species, naturally occurring mutants, and engineered mutants may also be effective.
• LIF Leukemia Inhibitory Factor
• LIF has several alternative names, including: cholinergic differentiation factor; human interleukin in DA cells; differentiation stimulating factor; MLPLI; and Emfilermin.
• Human LIF is a 180-amino-acid secreted glycoprotein. Kondera-Anasz et al., Am. J. Reprod. Immunol. 52:97-105 (2004).
• LIF binds and signals through the LIF receptor (LIFR) / gp130 heterodimer. Binding of LIF to the LIF receptor leads to activation of the Janus kinase / signal transducers and activators of transcription (JAK/STAT) and mitogen-activated protein kinase (MAPK) signaling pathways. Heinrich et al., Biochem. J. 374:1-20 (2003).
• LIF has been reported to inhibit the proliferation of M1 myeloid leukemia cells. See, e.g., U.S. Patent No. 5,443,825. In contrast, LIF has also been reported to stimulate the growth of neurons as well as to promote the differentiation of neurons from an adrenal medullary phenotype to an acetylcholinergic phenotype. See, e.g., U.S. Patent No. 5,968,905. The addition of LIF to severed nerves can also enhance nerve regeneration. See, e.g., U.S. Patent No. 6,156,729. Thus, LIF may be useful for promoting the growth of certain cells in vitro.
• LIF is available from commercial sources.
• a 181-amino-acid recombinant human LIF produced in E. coli was obtained from Sigma-Aldrich (St. Louis, MO) (catalog No. L 5283, see also Gearing et al., EMBO J. 6:3995 (1987)).
• the biological activity of LIF is assayed by testing for its ability to stimulate the differentiation of M1 mouse myeloid leukemia cells as described, e.g., in Gearing et al., EMBO J. 6:3995 (1987).
• human LIF is used to produce the media of the invention.
• one skilled in the art would recognize that LIF from other species, naturally occurring mutants, and engineered mutants may also be effective.
• the DM of the invention is cDRF supplemented with PDGF 1 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, and one or more cytokines.
• DM of the invention is cDRF supplemented with PDGF, 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, and one or more of the group consisting of OSM, IL-6, and LIF.
• the concentration of cytokine is chosen from 0.01-0.1 ng/ml, 0.1-1 ng/ml, 1-5 ng/ml, 5-10 ng/ml, 10-15 ng/ml, 15-50 ng/ml, and 50-100 ng/ml.
• cDRF is supplemented with 0.01-10 ng/ml, more preferably, 0.1-2 ng/ml and, most preferably, 0.5-1 ng/ml of OSM, IL-6, and/or LIF.
• cDRF is supplemented with approximately 10 ng/ml PDGF, 0.5% CDLM, 1 ng/ml IL-6, and 0.5 ng/ml OSM.
• the DM of the invention further comprises additional supplements as described below.
• the DM of the invention comprises at least one, two, or all three of OSM, IL-6, and LIF.
• the DM comprises combinations of OSM, IL-6. and LIF as set forth in Table 7.
• the DM comprises any combination of OSM, IL-6, and LIF set forth in Table 7, PDGF 1 and CDLM as defined in Table 5.
• the DM comprises any combination of OSM, IL-6, LIF, PDGF, and lipids set forth in Table 7.
• the DM comprises OSM, IL-6, PDGF and CDLM as defined in Table 5.
• the DM is cDRFm as defined in Table 4.
• the medium may comprise cDRFm, OSM, IL-6, PDGF and CDLM.
• Table 7 Illustrative Combinations of OSM, IL-6, and LIF
• the DM of the invention may optionally be supplemented with any number of additional supplements needed to promote the growth of cells in culture.
• additional supplements may include, but are not limited to, BMP family members, TGF- ⁇ family members, IGF 1 and insulin.
• the medium of the invention can be used to seed, grow, and maintain chondrocytes capable of redifferentiation in culture without the use of serum.
• concentrations of PDGF, lipids, OSM, IL-6, and LIF 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.
• the culture medium of the invention is not supplemented with substantially pure jagged 1 (JAG1) and/or substantially pure interleukin-13 (IL-13).
• the culture medium of the invention is not supplemented with any of the specific combinations of supplements set forth in U.S. Patent Application Publication Nos. US 2005/0265980 A1 (e.g., at paragraphs 59 to 68) and US 2005/0090002 A1 (e.g., at paragraphs 10 to 14), although it may be supplemented with a subset of any combination disclosed therein as long as the medium excludes at least one or more of the supplements from that combination.
• the culture medium of the invention is not supplemented with any specific one, two, three, four or more supplements selected from the group consisting of substantially pure epidermal growth factor (EGF), substantially pure stem cell factor (SCF), substantially pure insulin-like growth factor 1 (IGF-1 ), substantially pure brain- derived neurotrophic factor (BDNF), substantially pure erythropoietin (EPO), substantially pure FMS-related tyrosine kinase-3 (Flt-3/Flk-2) ligand, and/or a substantially pure member of the wingless-type MMTV integration site (WNT) family.
• the medium of the invention does not contain dexamethasone.
• the methods of the invention can be used with any suitable cells.
• the methods are particularly suitable for ex vivo propagation of cells capable of producing cartilaginous tissue, such as chondrocytes.
• 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 Il 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.
• aggrecan proteoglycan aggregate
• chondrocyte refers to a differentiated cell obtained from the cartilage, including a de-differentiated chondrocyte as grown in culture which retains the capacity to differentiate into a chondrocyte.
• chondrocyte refers to a chondrocyte regardless of whether it is primary or passaged, autologous, heterologous, allogeneic, xenologous, 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, Culture of Animal Cells: A Manual of Basic Techniques, 2d ed. A. R. Liss, Inc., New York, pp. 137-168 (1987); Klagsburn, Methods Enzymol. 58:560-564 (1979); R. Tubo and L. Brown, Articular Cartilage. In: Human Cell Culture; Volume V 1 Koller et al. (eds.) (2001 ); and Kandel et al., Art. Cells, Blood Subs., and Immob. Biotech. 25(5), 565-577 (1995).
• 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.
• chondrocyte progenitor stem cells such as mesenchymal stem cells rather than cells from cartilage biopsies that are already differentiated into chondrocytes. Chondrocytes can be obtained upon differentiation of such cells into chondrocytes. Examples of tissues from which such stem cells can be isolated include synovium, placenta, umbilical cord, bone marrow, adipose, skin, muscle, periosteum, or perichondrium. _
• chondrocytes and chondrocyte progenitor stem cells it may be desirable in certain circumstances to utilize other cells with chondrocyte potential, such as cells of mesenchymal lineage that can be trans-differentiated into chondrocytes.
• Chondrocytes can be obtained by inducing differentiation of such cells into chondrocytes in vitro.
• examples of such other cells with chondrocyte potential include osteoblasts, myocytes, adipocytes, fibroblasts, epithelial cells, keratinocytes, and neuronal cells.
• Chondrocytes, chondrocyte progenitor cells, and other cells with chondrocyte potential may be cultured to a state that is suitable for treating a patient suffering from a cartilage defect.
• Such therapeutically useful chondrocytes should express articular cartilage-specific extracellular matrix components, including, but not limited to, type Il collagen and proteoglycans characteristic of hyaline-like articular cartilage.
• Assays to determine the differentiation state of chodrocytes are known in the art and described in, e.g., R. Tubo and L. Brown, Articular Cartilage. In: Human Cell Culture; Volume V, Koller et al., eds. (2001) and the Examples.
• Other cells for which the DM of the present invention may be used include any primary or passaged cells, or cells as part of cultured tissues, that are capable of growing in the DM. Examples of other cells include hepatocytes, beta cells, and islet cells.
• Chondrocytes and other cells can be isolated from any mammal, including, without limitation, human, orangutan, monkey, chimpanzee, dog, cat, rat, rabbit, mouse, horse, cow, pig, elephant, etc.
• Cells for which the DM of the present invention may be used include any primary or passaged cells, or cells as part of cultured tissues, that are capable of growing in the DM.
• the cell can be cultured using any suitable cell culture methods appropriate for a particular cell type and application. Methods cell culture are well known in the art and described in, e.g., J. M. Davis, Basic Cell Culture, 2d ed. Oxford U. Press, 2002.
• chondrocytes 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.
• Trypsin and EDTA are both readily available from Invitrogen (Carlsbad, CA).
• cells may be passaged by incubation with a solution containing a chelating agent such as EDTA.
• a chelating agent such as EDTA.
• EDTA a chelating agent for the non-enzymatic detachment of cells is well known in the art.
• cells grown in the DM of the invention are passaged using 0.1 mM to 1 mM EDTA.
• cells grown in the DM of the invention are passaged using less than 0.0025% (or 325 units/ml), preferably 0.00025% (or 32.5 units/ml), recombinant trypsin in 0.1 mM to 1 mM EDTA.
• cells can be collected and frozen in DMEM containing 10% DMSO and 40% HSA or in other compositions known in the art, e.g., as described in U.S. Patent No. 6,365,405.
• cells can be initially cultured at low density.
• low density refers to seeding densities less than 20,000 cells/cm 2 .
• 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.
• a medium of the invention can be tested for the capacity to maintain cells in a differentiation-competent state, and in particular, for differentiation/redifferentiation into chondrocytes when the cells are placed in a permissive environment.
• Proteoglycan, aggrecan and collagen Il are examples of components of the extracellular matrix normally secreted by chondrocytes in vivo and may serve as markers of chondrocyte function.
• the capacity of medium to maintain chondrocyte differentiation potential may be determined by agarose and/or alginate assays.
• the agarose assay identifies the formation of proteoglycan by cells grown in a three-dimensional agarose matrix and is described in, e.g., Benya et al., Cell 30:215-224 (1982).
• the alginate assay measures expression of aggrecan and collagen Il genes in cells cultured in an alginate suspension and is described in, e.g., Yaeger et al., Exp. Cell. Res. 237(2):318-25 (1997); and Gagne et al., J. Orthop Res. 18(6):882-890 (2000). Vl. Methods of Using Cells
• the invention further provides cells cultured using the methods of the invention and methods of using such cells, e.g., in therapy, e.g., for treating a subject by administering to the subject such cells.
• the methods include repair of cartilage defects (e.g., due to trauma or osteoarthritis) by administering chondrocytes (e.g., autologous chondrocytes) cultured in accordance with the methods of the invention.
• Example 1 IL-6 increases cell yield and proliferation of primary human chondrocytes
• the growth index for cells grown in cDRF/P/L + IL-6 was roughly equal to the growth index for cells grown in DMEM + 10% FBS and exceeded that of cells grown in cDRF/P/L alone (Table 10). These results indicate that cDRF/P/L supplemented with IL-6 is an effective replacement for serum-containing media.
• ITS-X supplement (insulin, transferrin, selenium, 1% ethanolamine)
• Example 2 OSM increases cell yield and proliferation of primary human chondrocytes
• the growth index for cells in cDRF/P/L + OSM was roughly equal to the growth index for cells in DMEM + 10% FBS and exceeded that of cells in cDRF/P/L alone (Table 12). These results indicate that cDRF/P/L supplemented with OSM is an effective replacement for serum-containing media.
• Example 3 LIF increases cell yield and proliferation of primary human chondrocytes
• Example 4 IL-6 and OSM together increase cell yield of primary human chondrocytes
• Example 5 JAG-1 inhibits growth of chondrocytes in serum-free medium
• Example 6 IL-13 inhibits growth of chondrocytes in serum-free medium
• Example 7 The E93 medium increases cell yield and proliferation of chondrocytes
• E93 (referred herein as "E93").
• E93 Cells were passaged upon reaching 50% to 80% confluence.
• Cells grown in DMEM + 10% FBS were rinsed with PBS 1 harvested by exposure to 325 units/ml trypsin in EDTA, counted, and reseeded.
• Cells grown in the E93 medium were rinsed with PBS, harvested by exposure to 0.00025% Trypzean TM in 0.5 mM EDTA, counted and reseeded. Cell yield was determined and population doublings calculated at the end of each passage.
• the growth index for cells in E93 was equal to or greater than the growth index for cells in DMEM + 10% FBS (Table 18, Figure 1).
• Example 8 Medium supplemented with IL-6 and OSM maintains re differentiation capacity of chondrocytes in three-dimensional culture
• Cells were passaged upon reaching 50% to 80% confluence.
• Cells grown in DMEM + 10% FBS were rinsed with PBS, harvested by exposure to 325 units/ml trypsin in EDTA, counted, and reseeded.
• Cells grown in serum- free medium were rinsed with PBS, harvested by exposure to 0.00025% Trypzean TM in 0.5 mM EDTA, counted and reseeded.
• ⁇ . agarose were cultured in DMEM + 10% FBS at 37 0 C, and refed 24 hours after plating, and every 2 to 3 days thereafter. After 21 days in culture, the plates were fixed with 10% formalin, rinsed, stained with 0.2% safranin, and rinsed extensively to remove background stain. The number of colonies that stained positive for proteoglycan, and were equal to or greater than 50 microns in size, was determined. Plates on which more than 6.8% of the cells formed proteoglycan- positive colonies and met the minimum size criteria were scored as "pass". All strains were tested in triplicate. Cell strains from six biopsies were examined.
• Example 9 Mean cell yield for ten strains of chondrocytes is greater in medium supplemented with IL6 and OSM than in DMEM supplemented with serum
• Example 10 Medium supplemented with IL6 and OSM maintains capacity of chondrocytes to re-express type 2 collagen and aggrecan in alginate suspension culture
• chondrocytes were prepared as describe in Example 9. Cells grown in DMEM + 10% FBS or E93 were harvested in third passage for alginate culture. Alginate cultures were set up by seeding 1 x 10 6 cells into a 1.2% alginate solution. Alginate cultures were fed every 3-5 days with EGHIC (DMEM, 20 ng/mL rhlGF-1, 25 ⁇ g/mL ascorbic acid, and 1 mM sodium pyruvate). After 21 days of culture, the chondrocytes were extracted from the alginate beads and mRNA for type I collagen, type Il collagen and aggrecan were detected using a ribonuclease protection assay (RPA).
• RPA ribonuclease protection assay
• type Il collagen is detected as a 310 base pair (bp) band on a gel
• type I collagen is a 260 bp band
• aggrecan is a 210 bp band.
• Figure 3 shows that increasing amounts of cell lysate from cells grown in E93 (lanes 2, 3 and 4) or DMEM supplemented with 10% serum (lanes 5, 6 and 7) contain mRNA for type Il collagen and aggrecan. This indicates that human chondrocytes grown in E93 media are capable of re-expression these important cartilage markers.
• Example 11 Karyotype and senescence of chondrocytes grown in medium supplemented with IL6 and OSM
• Example 12 Low levels of cytokines stimulate growth of chondrocytes
• Primary human chondrocytes were isolated from biopsies of articular cartilage by mincing of the sample followed by enzymatic digestion with 0.25% protease type XIV (Streptomyces griseus) for one hour and then 0.1% collagenase overnight at 37 °C. Cells were recovered by centrifugation for five minutes at 1 ,000 x g and resuspended in the appropriate test medium. Cells grown in DMEM + 10% FBS were plated at a density of 3,000 cells per cm 2 . Cells grown in serum-free medium were plated at either 5,000 cells per cm 2 . T75 flasks were used for all experiments. The following media were tested:

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