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US 9,068,182 B2Granted
Synthetic polysaccharide microcarriers for culturing cells
AssigneeCorning Incorporated
InventorsSophie Deshayes, David Henry, Martial Hervy
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Abstract
A cell culture polysaccharide microcarrier includes (1) a cross-linked polysaccharide microcarrier base having a neutral or negative charge at pH 7, and (ii) a polypeptide conjugated to the base. The polypeptide may contain a cell adhesive sequence, such as RGD. Cells cultured with such microcarriers exhibit peptide-specific binding to the microcarriers.
Claims20
A microcarrier for cell culture, comprising:
- a cross-linked polysaccharide hydrogel microcarrier base having a net neutral or negative charge in medium at pH 6-8, wherein the cross-linked polysaccharide hydrogel microcarrier base comprises carboxylic acid functional groups, and wherein the cross-linked polysaccharide hydrogel microcarrier base is free of positively charged functional groups in the medium at pH 6-8;
- and
- a plurality of synthetic, cell attachment polypeptides conjugated to the base, wherein each of the plurality of synthetic, cell attachment polypeptides is conjugated to the base via one of the carboxylic acid functional groups, wherein each of the plurality of synthetic, cell attachment polypeptides comprises an amino acid sequence of RGD, and wherein at least some of the carboxylic acid functional groups are unconjugated to the plurality of synthetic, cell attachment polypeptides and are blocked with a monoamine, such that the cross-linked polysaccharide hydrogel microcarrier base does not support non-specific attachment of cells thereto during cell culture.
A method for forming a cell culture microcarrier, comprising:
- providing a net negatively or neutral charged polysaccharide hydrogel microcarrier base having pendant carboxylic acid functional groups, wherein the polysaccharide hydrogel microcarrier base is free of positively charged functional groups;
- and
- conjugating a plurality of synthetic polypeptides to the microcarrier base, wherein each of the plurality of synthetic polypeptides is conjugated to one of the pendant carboxylic acid functional groups of the microcarrier base to form the microcarrer, wherein each of the plurality of synthetic polypeptides comprises an amino acid sequence of RGD, and
- blocking at least some of the carboxylic acid functional groups which are unconjugated to
- the plurality of synthetic polypeptides with a monoamine to form an amide, wherein the blocking alters an equilibrium water content of the microcarrier, such that the polysaccharide hydrogel microcarrier base does not support non-specific attachment of cells thereto during cell culture.
2 more elements
A method for culturing stem cells, comprising:
- contacting the stem cells with a cell culture medium having microcarriers, wherein the medium is a chemically defined medium, and wherein the microcarrier comprises:
- (i) a net neutral or negatively charged polysaccharide hydrogel microcarrier base, wherein the net neutral or negatively charged polysaccharide hydrogel microcarrier base comprises carboxylic acid functional groups, and wherein the net neutral or negatively charged polysaccharide hydrogel microcarrier base is free of positively charged functional groups, and
- (ii) a plurality of polypeptides conjugated to the base, wherein each of the plurality of polypeptides is conjugated to the base via one of the carboxylic acid functional groups, wherein each of the plurality of polypeptides comprises an RGD amino acid sequence, and wherein at least some of the carboxylic acid functional groups are unconjugated to the plurality of synthetic, cell attachment polypeptides and are blocked with a monoamine;
- and
- culturing the stem cells in the chemically defined medium, wherein non-specific attachment of the stem cells to the microcarrier base is not supported.
2 more elements
Dependencies are inferred from the claim text.