CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase Application of PCT International Application Number PCT/JP2016/083014, filed on Nov. 8, 2016, designating the United States of America and published in the Japanese language, which is an International Application of and claims the benefit of priority to Japanese Patent Application No. 2015-219996, filed on Nov. 10, 2015. The disclosures of the above-referenced applications are hereby expressly incorporated by reference in their entireties.

TECHNICAL FIELD

The present invention relates to a cell culture method using a medium containing a laminin fragment and to a cell culture medium containing a laminin fragment.

BACKGROUND ART

Human pluripotent stem cells, such as human ES cells and human iPS cells, are receiving worldwide attention for their potential application to regenerative medicine. In order to realize the application of human pluripotent stem cells to regenerative medicine, there is a need to develop culture techniques for culturing and propagating such stem cells in a safe and stable manner.

The present inventors examined the type of integrins expressed on human ES cells, and reported that α6β1 integrin is a major adhesion receptor of human ES cells and that recombinant human laminins (particularly, laminin 332 composed of α3, β3 and γ2 chains, and laminin 511 composed of α5, β1 and γ1 chains) are useful as a feeder-free culture substrate for human ES cells (see Non Patent Literature 1). The present inventors also found that the use of a laminin 511E8 fragment (laminin 511E8) as a feeder-free culture substrate enables passage culture of human pluripotent stem cells in a dissociated single-cell state, which had been previously difficult (see Patent Literature 1 and Non Patent Literature 2).

Currently, a recombinant human laminin 511E8 fragment is commercially available (trade name: iMatrix-511, Nippi Inc.), and the use of human laminin 511E8 as a culture substrate for human pluripotent stem cells, such as human ES cells and human iPS cells, is rapidly spreading. However, cell culture using human laminin 511E8 as a substrate conventionally requires precoating the surface of a culture vessel with a large amount of human laminin 511E8. Such a conventional culture method is costly, and therefore, disadvantageous. In addition, the process for culture substrate coating usually involves adding a culture substrate solution prepared using phosphate buffer etc. to a culture vessel, followed by incubation at about 4° C. to about 37° C. for a few hours to overnight. Therefore, if a low-cost, coating-free protocol for cell culture can be provided, human laminin 511E8 would be more widely used as a culture substrate for pluripotent stem cells.

CITATION LIST

Patent Literature

Patent Literature 1: WO 2011/043405

Non Patent Literature

Non Patent Literature 1:Miyazaki T, Futaki S, Hasegawa K, Kawasaki M, Sanzen N, Hayashi M, Kawase E, Sekiguchi K, Nakatsuji N, Suemori H. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375:27-35, 2008.Non Patent Literature 2:Miyazaki T, Futaki S, Suemori H, Taniguchi Y, Yamada M, Kawasaki M, Hayashi M, Kumagai H, Nakatsuji N, Sekiguchi K, Kawase E. Laminin E8 fragments support efficient adhesion and expansion of dissociated human pluripotent stem cells. Nat Commun. 3, 1236. Doi:10.1038/ncomms2231, 2012.

SUMMARY OF INVENTION

Technical Problem

An object of the present invention is to provide a cell culture method that uses a smaller amount of a laminin fragment and still achieves a comparable culture efficiency as compared with the conventional cell culture method that uses a culture vessel precoated with a laminin or a laminin fragment (precoating method).

Solution to Problem

In order to achieve the above-mentioned object, the present invention includes the following.(1) A cell culture method comprising the step of culturing cells using a medium containing a laminin fragment having integrin binding activity, the method not comprising the step of coating a culture vessel with a laminin or a laminin fragment before seeding the cells in the culture vessel.(2) The cell culture method according to the above (1), wherein the cells are seeded and cultured in a culture vessel which is not precoated with a laminin or a laminin fragment.(3) The cell culture method according to the above (1) or (2), wherein the laminin fragment having integrin binding activity is a laminin E8 fragment.

(4) The cell culture method according to any one of the above (1) to (3), wherein the laminin fragment is present in the medium at such a concentration that a predetermined volume of the medium in the culture vessel contains 0.03 μg to 2 μg of the laminin fragment per cm2of culture surface area of the culture vessel.(5) The cell culture method according to the above (4), wherein the laminin fragment is present in the medium at such a concentration that a predetermined volume of the medium in the culture vessel contains 0.06 μg to 0.5 μg of the laminin fragment per cm2of culture surface area of the culture vessel.(6) The cell culture method according to any one of the above (1) to (5), wherein the integrin is integrin α6β1, integrin α6β4, integrin α3β1 and/or integrin α7β1.(7) The cell culture method according to any one of the above (1) to (6), wherein the cells are mammalian cells.(8) The cell culture method according to the above (7), wherein the cells are mammalian stem cells or cells differentiated from the stem cells. (9) The cell culture method according to the above (8), wherein the cells are pluripotent stem cells. (10) A cell culture medium containing a laminin fragment having integrin binding activity.

(11) The cell culture medium according to the above (10), wherein the laminin fragment is present at a concentration of 0.15 μg/mL to 10 μg/mL.

Advantageous Effects of Invention

The present invention provides a cell culture method that uses a smaller amount of a laminin fragment and still achieves a comparable culture efficiency as compared with the conventional precoating method. Also provided is a cell culture medium for use in such a cell culture method. The cell culture method of the present invention does not need precoating a culture vessel with a laminin fragment, and thus can shorten the time required for cell culture. In addition, the reduced amount of the laminin fragment used leads to a lower culture cost. Moreover, personal errors in the coating results can be eliminated, and thus, a simple culture method independent of individual cell culture skills can be provided.

