Tuneable cell substrates
AssigneeCambridge Enterprise Limited
InventorsChibeza Chintu Agley, Kevin Chalut, Jose Carlos Rebelo da Silva
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Abstract
This invention relates to a substrate for mammalian cell culture comprising a co-polymer hydrogel comprising monomeric units of acrylamide, bisacrylamide and a coupling compound, such as 6-acylamidohexanoic acid (6AHA). A cell adhesion molecule, such as a component of the extracellular matrix (ECM), is covalently coupled to the coupling compound monomeric units of the co-polymer. The stiffness and cell adhesion molecule density of the substrate can be independently controlled by altering the concentration of acrylamide and coupling compound in the hydrogel, respectively. Substrates and methods and kits for their production are provided, along with cell culture systems and methods of culturing mammalian cells.
Claims18
A substrate for culture of mammalian stem and progenitor cells culture comprising;
- (i) a co-polymer hydrogel formed from a co-polymer, wherein the co-polymer comprises cross-linked chains of monomeric units of acrylamide, a N,N′ methylenebisacrylamide (bisacrylamide) cross-linking agent, and a coupling compound in the hydrogel;
- and
- (ii) a cell adhesion molecule, said cell adhesion molecule being covalently coupled to coupling compound monomeric units of the co-polymer,
- wherein the coupling compound is 6-acrylamidohexanoic acid (6AHA) and
- the co-polymer hydrogel comprises 1-10% (w/v in g/100 ml) acrylamide monomeric units and 10 to 100 mM 6AHA,
- wherein the substrate constitutes a stable tunable synthetic stem cell niche for maintenance of stable mammalian stem and progenitor cell cultures;
- wherein concentration of the monomeric units of acrylamide in the co-polymer hydrogel controls stiffness of the substrate;
- and
- wherein stiffness of the hydrogel corresponds to stiffness of a biological tissue;
- wherein the cell adhesion molecule forms a molecular anchoring site for the cultured mammalian stem or progenitor cells;
- wherein concentration of the coupling compound in the copolymer hydrogel controls cell adhesion molecule density of the substrate;
- and
- wherein density of the cell adhesion molecule reflects density of the cell adhesion molecule in a biological tissue;
- wherein stiffness of the substrate and density of the cell adhesion molecules are independently tunable;
- wherein the substrate with a defined stiffness enables selection of cells from specific tissues;
- and
- wherein the substrate with a high density of cell adhesion molecules enables selection of mammalian stem or progenitor cells that originate from a high adhesion environment;
- and the substrate with a low density of cell adhesion molecules enables selection of mammalian stem or progenitor cells that originate from a low adhesion environment.
A method of producing a cell culture substrate comprising in order:
- producing a solution of acrylamide, bisacrylamide and coupling compound monomeric units,
- then polymerizing said monomers to form a co-polymer hydrogel comprising acrylamide and coupling compound monomeric units in the hydrogel;
- and
- then covalently coupling a cell adhesion molecule to the coupling compound monomeric units in the hydrogel to produce the cell culture substrate,
- wherein the coupling compound is 6-acrylamidohexanoic acid (6AHA), and
- wherein the co-polymer hydrogel comprises 1-10% (w/v in g/100 ml) acrylamide monomeric units and 10 to 100 mM 6AHA.
Dependencies are inferred from the claim text.