Cell culture environments for the serum-free expansion of mesenchymal stem cells

Inactive Publication Date: 2005-12-01
BECTON DICKINSON & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] Compositions and methods for promoting mesenchymal stem cell (MSC) expansion while maintaining a pluripotent phenotype are provided. The compositions include serum-free cell culture systems for MSC expansion that comprise a serum-free cell culture medium and a two-dimensional or three-dimensional cell culture surface. In the serum-free cell culture system of the present invention, at least one insoluble substrate protein is presented from the cell culture surface. In one embodiment, the cell culture surface comprises a cell culture support bound to a cell adhesion resistant (CAR) material, which in turn is bound to at least one insoluble substrate protein. Insoluble substrate proteins for use in the present invention include extra

Problems solved by technology

Typically, MSCs do very poorly in serum-free environments because they detach and die in culture.
The creation of highly defined environments for cell expansion is of great importance for quality purposes, and serum levels are typically very ill-defined (see, e.g., U.S. Pat. No. 5,90

Method used

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  • Cell culture environments for the serum-free expansion of mesenchymal stem cells
  • Cell culture environments for the serum-free expansion of mesenchymal stem cells
  • Cell culture environments for the serum-free expansion of mesenchymal stem cells

Examples

Experimental program
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Effect test

example 1

MSC Expansion in Various Serum-Free Media

[0109] MSCs along with complete growth medium were purchased from Cambrex Biosciences (Baltimore, Md.). Frozen cells were thawed and cultured following manufacturer's instruction. After reaching ˜90% confluency, MSCs were washed once with PBS, removed from the culture surface using Trypsin / EDTA and replated at a density of 1600 cells / well (for 96 well plates) or at a density of 50,000 cells / well (for 6-well plates) which corresponds to the manufacturers recommended seeding density of 5000 cells / cm2.

[0110] At day 6 or 7, cells were washed once with PBS, fixed using 4% paraformaldehyde for 15 minutes and then stained using DAPI according to manufacturer's suggestions (Molecular Probes, Eugene, Oreg.). For 96-well plate experiments, one image per well was taken at 4× magnification using Molecular Devices' Discovery-1 high content screening system. Cell nuclei enumeration was determined using Metamorph Image Analysis Software (Molecular Devices...

example 2

Differentiation Capacity and Surface Marker Characterization of G2 Expanded MSCs

[0113] MSCs expanded in serum-free media of the present invention on CAR Col 1 / FN surface for 7 days remained multipotent. Briefly, MSCs expanded in culture environments of the present invention were removed from the serum-free media and then cultured in either adipogenic (fat) or osteogenic (bone) induction media (per manufacturer's suggestions). MSCs expanded in the serum-free environments of the present invention were able to differentiate towards both adipogenic (see FIG. 2) and osteogenic (see FIG. 3) lineages. These results demonstrate that the serum-free environments of the present invention help to maintain stem cell pluripotency in expanded MSCs at least as well as the industry standard serum-containing media (Cambrex). This demonstrates that these cells are not committed towards specific lineages, and that therefore these MSCs may still be used for a variety or research or clinical application...

example 3

Comparison of Media and Cell Culture Surface Conditions

[0115] MSCs cultured in three different media (G2, base medium without G2 growth factors / cytokines, and in serum-containing complete medium) all expand better on a Col 1 / FN CAR surface as compared to TCPS surfaces (see FIG. 4). Also, MSCs cultured in G2 serum-free medium expand to equivalent levels as MSCs cultured in serum-containing medium.

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Abstract

Compositions and methods for promoting mesenchymal stem cell expansion while maintaining a pluripotent phenotype are disclosed. Serum-free cell culture systems and kits and methods of use for mesenchymal stem cell expansion are provided. Methods also comprise the use of the expanded mesenchymal stem cells to treat various disorders or diseases, particularly those of the cardiovascular system, bone, or cartilage.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Provisional Application No. 60 / 571,212, filed May 14, 2004, the content of which is herein incorporated by reference it its entirety.FIELD OF THE INVENTION [0002] The present invention relates to serum-free cell culture systems that provide for mesenchymal stem cell expansion while maintaining a pluripotent phenotype, and methods of use for the expanded mesenchymal stem cell populations. BACKGROUND OF THE INVENTION [0003] Mesenchymal stem cells (MSCs) are present in adult tissues and constitute a population of cells that can be isolated, expanded in culture, and characterized in vitro and in vivo (Pittenger and Martin (2004) Circ. Res. 95:9-20). MSCs are able to differentiate into multiple cell lineages, including osteoblasts, chondrocytes, endothelial cells, and neuronal cells, and can express phenotypic characteristics of endothelial, neural, smooth muscle, skeletal myoblast, and cardiac myo...

Claims

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Application Information

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IPC IPC(8): A61K45/00C12N5/00C12N5/0775C12N5/0789
CPCC12N5/0647C12N2533/70C12N2500/25C12N2500/36C12N2500/42C12N2500/90C12N2501/10C12N2501/105C12N2501/11C12N2501/115C12N2501/125C12N2501/13C12N2501/135C12N2501/14C12N2501/145C12N2501/15C12N2501/155C12N2501/165C12N2501/21C12N2501/22C12N2501/23C12N2501/2303C12N2501/2305C12N2501/2306C12N2501/2311C12N2501/235C12N2501/26C12N2501/32C12N2501/39C12N2501/415C12N2501/58C12N2501/60C12N2501/70C12N2501/999C12N2533/52C12N2533/54C12N5/0663A61P19/08A61P9/00
Inventor CHEN, CHANGLIEBMANN-VINSON, ANDREAXU, RUILINGROWLEY, JONATHAN ALLENHAALAND, PERRY D.CHANEY, BRYCE N.MITCHELL, MATTHEW
Owner BECTON DICKINSON & CO
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