Method for enhancing activity of laminin fragments as cell culture matrix

Patent metadata

Field	Value
Publication number	US-10428311-B2
Application number	US-201515325743-A
Country	US
Kind code	B2
Filing date	Jul 15, 2015
Priority date	Jul 16, 2014
Publication date	Oct 1, 2019
Grant date	Oct 1, 2019

Abstract

The present invention provides a novel technique in a cell culture method using a cell culture vessel coated with a laminin fragment, which novel technique achieves cell culture as in the case of using a recommended coating concentration even when the coating concentration is lower than the recommended coating concentration. The present invention relates to a method for enhancing an activity for mammalian cultured cells of a laminin fragment or a variant thereof each having integrin binding activity.

First claim

The invention claimed is:

1. A method for enhancing an activity for mammalian cultured cells of a laminin fragment or a variant thereof each having integrin binding activity,
the method comprising bringing a culture surface of a cell culture vessel into contact with a coating solution containing the laminin fragment or a variant thereof and a protein that is neither a laminin nor a laminin fragment, thereby achieving coating of the culture surface with the laminin fragment or a variant thereof,
the coating solution containing the laminin fragment or a variant thereof at a concentration of 5 μg/mL or lower,
the protein that is neither a laminin nor a laminin fragment being present at a concentration which is 100-fold or more that of the laminin fragment or a variant thereof and is 500 μg/mL or higher in the coating solution,
the protein that is neither a laminin nor a laminin fragment is gelatin or serum albumin,
wherein the laminin fragment is a laminin E8 fragment,
wherein the variant is a chimeric molecule in which the laminin E8 fragment is conjugated with a cell adhesion molecule or a growth factor binding molecule, and
the activity for mammalian cultured cells being at least one kind selected from activity for binding to cell-surface adhesion receptors, cell-adhesive activity, cell growth-supporting activity and colony formation-promoting activity.

2. The method according to claim 1 , wherein the laminin fragment is derived from at least one kind selected from laminin α5β1γ1, laminin α5β2γ1, laminin α4β1γ1 and laminin α2β1γ1.

3. A method for culturing mammalian cells using a cell culture vessel coated with a laminin fragment or a variant thereof each having integrin binding activity,
the cell culture vessel being prepared by bringing a culture surface of the cell culture vessel into contact with a coating solution containing the laminin fragment or a variant thereof and a protein that is neither a laminin nor a laminin fragment,
the coating solution containing the laminin fragment or a variant thereof at a concentration of 5 μg/mL or lower,
the protein that is neither a laminin nor a laminin fragment being present at a concentration which is 100-fold or more that of the laminin fragment or a variant thereof and is 500 μg/mL or higher in the coating solution,
the protein that is neither a laminin nor a laminin fragment is gelatin or serum albumin,
wherein the laminin fragment is a laminin E8 fragment, and
wherein the variant is a chimeric molecule in which the laminin E8 fragment is conjugated with a cell adhesion molecule or a growth factor binding molecule.

4. The method according to claim 3 , wherein the laminin fragment is derived from at least one kind selected from laminin α5β1γ1, laminin α5β2γ1, laminin α4β1γ1 and laminin α2β1γ1.

5. A solution for coating a culture surface of a cell culture vessel with a laminin fragment or a variant thereof,
the solution containing a laminin fragment or a variant thereof each having an integrin binding activity and a protein that is neither a laminin nor a laminin fragment,
the laminin fragment or a variant thereof being present at a concentration of 5 μg/mL or lower,
the protein that is neither a laminin nor a laminin fragment being present at a concentration which is 100-fold or more that of the laminin fragment or a variant thereof and is 500 μg/mL or higher,
the protein that is neither a laminin nor a laminin fragment is gelatin or serum albumin,
wherein the laminin fragment is a laminin E8 fragment, and
wherein the variant is a chimeric molecule in which the laminin E8 fragment is conjugated with a cell adhesion molecule or a growth factor binding molecule.

6. The solution according to claim 5 , wherein the laminin fragment or a variant thereof is present in an amount adjusted so that the culture surface of the cell culture vessel is coated with the laminin fragment or a variant thereof at a concentration lower than 0.5 μg/cm 2 .

Assignees

• Univ Osaka

Inventors

• Sekiguchi Kiyotoshi
• Tsutsui Ko

Classifications

• C12N15/09

  Recombinant DNA-technology · CPC title

• C12N5/0696 Primary

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• C12N15/00

  Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor (mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00) · CPC title

• C12N2533/52

  Fibronectin; Laminin · CPC title

• C07K2319/43

  containing a FLAG-tag · CPC title