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.

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-β, 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-β, 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.