Swellable (meth)acrylate surfaces for culturing cells in chemically defined media
Synthetic surfaces capable of supporting culture of undifferentiated human embryonic stem cells in a chemically defined medium include a swellable (meth)acrylate layer and a peptide conjugated to the swellable (meth)acrylate layer. The swellable (meth)acrylate layer may be formed by polymerizing monomers in a composition that includes hydroxyethyl methacrylate, 2-carboxyehylacrylate, and tetra(ethylene glycol)dimethacrylate. The conjugated peptide may include an amino acid sequence of Xaa n ProGlnValThrArgGlyAspValPheThrMetPro, where n is an integer from 0 to 3 and where Xaa is any amino acid. Further, disclosed herein is a swellable (meth)acrylate synthetic surface which can be sterilized by gamma irradiation.
1. A method for culturing an isolated population of undifferentiated stem cells comprising:
providing a synthetic cell culture surface comprising a swellable (meth)acrylate layer, wherein the swellable (meth)acrylate layer is formed from a composition comprising 2-carboxyethylacrylate, tetra(ethylene glycol)dimethacrylate and one of hydroxyethyl methacrylate, hydroxypropyl methacrylate, 2-hydroxyethyl acrylate, 1-vinyl-2-pyrrolidone, di(ethylene glycol) ethyl ether methacrylate, acrylamide, ethylene glycol methyl ether methacrylate or 3-sulfopropyldimethyl-3-methacrylamidopropyl-ammonium; and a peptide comprising an amino acid sequence of
XaanProGlnValThrArgGlyAspValPheThrMetPro (SEQ ID NO:1), wherein Xaa is any amino acid and n is an integer from 0 to 3;
wherein the peptide is conjugated to the swellable (meth)acrylate layer; and,
wherein the cell culture article has been sterilized by irradiation;
providing undifferentiated stem cells on the synthetic culture surface;
culturing the undifferentiated stem cell in chemically defined media; and,
maintaining undifferentiated stem cells in culture in the chemically defined media for at least five passages.
2. The method of claim 1 wherein the stem cells comprise undifferentiated human embryonic stem cells.
3. The method of claim 2 wherein the stem cells comprise H1 or H7 cells.
4. The method of claim 1 , wherein the swellable (meth)acrylate layer is free of polypeptide crosslinkers.
5. The method of claim 1 , wherein the swellable (meth)acrylate layer is formed from a composition comprising hydroxyethyl methacrylate, 2-carboxyethyacrylate, and tetra(ethylene glycol)dimethacrylate.
6. The method of claim 1 , wherein the swellable (meth)acrylate layer is formed from a composition comprising about 60-90% hydroxyethyl methacrylate by volume, about 10-40% 2-carboxyethyacrylate by volume, and about 1-5% tetra(ethylene glycol)dimethacrylate by volume.
7. The method of claim 5 , wherein the volume ratio of hydroxyethyl methacrylate/2-carboxyethylacrylate/tetra(ethylene glycol)dimethacrylate is about 80/20/3.
8. The method of claim 5 wherein the percentage of hydroxyethyl methacrylate/2-carboxyethylacrylate/tetra(ethylene glycol)dimethacrylate in solvent is from 0.1% to 99.5%.
9. The method of claim 5 , wherein the swellable (meth)acrylate layer is formed from a composition consisting essentially of hydroxyethyl methacrylate/2-carboxyethylacrylate/tetra(ethylene glycol)dimethacrylate at a volume ratio of about 80/20/3.
10. The method of claim 1 , wherein the swellable (meth)acrylate layer is a uniform layer.
11. The method of claim 1 , wherein the peptide is conjugated to the swellable (meth)acrylate at a density of greater than about 5 picomoles per square millimeter of a surface of the swellable (meth)acrylate.
12. The method of claim 1 further comprising a peptide comprising an amino acid sequence of LysGlyGlyProQlnValThrArgGlyAspValPheThrMetPro (SEQ ID NO:6).
13. The method of claim 1 wherein the chemically defined medium comprises basic fibroblast growth factor and transforming growth factor-β1.
14. The method of claim 1 , wherein the chemically defined medium comprises about 80 ng/ml basic fibroblast growth factor and about 0.5 ng/ml transforming growth factor-β1.
15. The method of claim 1 , wherein at least one undifferentiated human embryonic stem cell is maintained in culture in the chemically defined media for at least ten passages.
16. The method of claim 1 the sterilization method is gamma irradiation.
17. The method of claim 16 wherein the gamma irradiation comprises 10-25 kGy treatment.