Serum-Free Chemically Defined Cell Culture Medium

Abstract

Embodiments of chemically defined cell culture media containing nutrients and growth factors free of any serum for culturing cells such as mesenchymal stem cells and methods of using embodiments of the cell culture medium for expanding cell populations such as mesenchymal stem cells while maintaining a pluripotent phenotype and methods of inducing chondrogenesis and osteogenesis of mesenchymal stem cells by admixing differentiation factors into embodiments of the cell culture medium.

1 . A serum-free chemically defined cell culture medium, comprising:
 a) a base medium, wherein said base medium is serum-free; and   b) a base medium supplement, including:
 i) insulin; 
 ii) one or more phospholipid growth factors; and 
 iii) one or more WNT signaling pathway activators. 
   
     
     
         2 . The serum-free chemically defined cell culture medium of  claim 1 , wherein said base medium comprises an amount of DMEM and an amount of MCDB having a ratio of DMEM to MCDB in the range of about 0.75:1.25 v/v to about 1.25:0.75 v/v. 
     
     
         3 . (canceled) 
     
     
         4 . The serum-free chemically defined cell culture medium of  claim 2 , further including one or more cell growth factors selected from the group consisting of: bFGF, PDGF-bb, EGF, TGF-beta1 and IGF-1. 
     
     
         5 . The serum-free chemically defined cell culture medium of  claim 1 , wherein said insulin has a concentration in said serum-free chemically defined cell culture medium in a range of about 4 mg/L and about 6 mg/L. 
     
     
         6 . The serum-free chemically defined cell culture medium of  claim 4 , wherein each of said bFGF, PDGF-bb, EGF, TGF-beta1 and IGF-1 has a concentration in said serum-free chemically defined cell culture medium in a range of about 1 ng/mL to about 20 ng/mL. 
     
     
         7 . (canceled) 
     
     
         8 . The serum-free chemically defined cell culture medium of  claim 2 , wherein said one or more lipid growth factors is selected from the group consisting of: LPA, and S1P. 
     
     
         9 . The serum-free chemically defined cell culture medium of  claim 8 , wherein said one or more lipid growth factors have a concentration in said base medium in the range of about 100 nM and about 200 nM. 
     
     
         10 . (canceled) 
     
     
         11 . The serum-free chemically defined cell culture medium of  claim 2 , wherein said one or more WNT signaling pathway activators is selected from the group consisting of: a human WNT3A and RSPO 1 . 
     
     
         12 . The serum-free chemically defined cell culture medium of  claim 11 , wherein said one or more WNT signaling pathway activators has a concentration in said base medium in the range of about 20 ng/mL and about 200 ng/mL. 
     
     
         13 . The serum-free chemically defined cell culture medium of  claim 12 , wherein said one or more WNT signaling pathway activators comprises a combination of a human WNT3A having a concentration of about 20 ng/mL and RSPO 1  having a concentration of about 40 ng/mL. 
     
     
         14 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising one or more steroids. 
     
     
         15 . The serum-free chemically defined cell culture medium of  claim 14 , wherein said one or more steroids is selected from the group consisting of: dexamethasone and hydrocortisone. 
     
     
         16 . The serum-free chemically defined cell culture medium of  claim 14 , wherein said one or more steroids comprises a combination of said dexamethasone having concentration of about 2.0 μg/mL and about 5.0 μg/mL and said hydrocortisone having a concentration of about 0.5 mg/mL and about 1.5 mg/mL. 
     
     
         17 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising a regulated iron source. 
     
     
         18 . The serum-free chemically defined cell culture medium of  claim 17 , wherein said regulated iron source comprises an amount of transferrin having a concentration in said base medium in the range of about 2 mg/L and about 10 mg/L. 
     
     
         19 . (canceled) 
     
     
         20 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising an electron transport activator. 
     
     
         21 . The serum-free chemically defined cell culture medium of  claim 20 , wherein said electron transport activator is selected from the group consisting of: an amount of selenium, and an amount of selenous acid. 
     
     
         22 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising an amount of HSA. 
     
     
         23 . The serum-free chemically defined cell culture medium of  claim 22 , wherein said amount of HSA has concentration in said base medium in a range of about 100 μg/mL and about 350 μg/mL. 
     
     
         24 . The serum-free chemically defined cell culture medium of  claim 23 , wherein said amount of HSA comprises and amount of recombinant HSA. 
     
     
         25 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising one or more antioxidants. 
     
     
         26 . The serum-free chemically defined cell culture medium of  claim 25 , wherein said antioxidants are selected from the group consisting of: α-tocopherol acetate and ascorbic acid-2 phosphate. 
     
     
         27 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising an amount of 5-hydroxytryptamine in the range of about 0.001 g/L and about 0.003 g/L. 
     
     
         28 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising a buffer. 
     
     
         29 . The serum-free chemically defined cell culture medium of  claim 25 , wherein said buffer comprises an amount of sodium bicarbonate in the range of about 3.2 g/L and about 4.2 g/L. 
     
     
         30 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising an amount of TGFB1 sufficient to derive chondrocytes from mesenchymal stem cells cultured in said serum-free chemically defined cell culture medium. 
     
     
         31 . The serum-free chemically defined cell culture medium of  claim 2 , further comprising an amount of BMP2 sufficient to derive chondrocytes from mesenchymal stem cells cultured in said serum-free chemically defined cell culture medium. 
     
     
         32 - 73 . (canceled)