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1Department of Chemical Engineering and Materials Science, City of Hope Cancer Center
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Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.
Zhang, L., Chan, C. Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs. J. Vis. Exp. (37), e1852, doi:10.3791/1852 (2010).
1. Isolation of Rat MSCs
Mesenchymal stem cells were isolated from 6-8 week old Sprague-Dawley female rat as previously described 1, 2. Isolated MSCs can adhere to plastic surface and easily expand during in vitro culture.
2. Enrichment of Rat MSCs
A number of surface proteins have been used to enrich MSCs, including CD54, CD90, CD73, CD105 and CD271 3-5. In our study, we used CD54 and CD90 as markers to enrich MSCs by magnetic cell sorting.
3. Verification of Surface Marker Expression by Flow Cytometry
Flow cytometry analysis was performed to verify the cells we obtained express CD54 and CD90. The HSC marker CD45 was used to confirm that the MSCs were depleted of HSCs.
4. Separation of Single-colony Derived MSCs
MSCs is a heterogeneous population composed of different subpopulations with different cell shape, growth rate as well as differentiation ability 6. However, all the subpopulations express the known MSC markers and therefore it is not feasible to use markers to separate out these subpopulations. Therefore, we applied cloning cylinders to separate out the different subpopulations, which are colonies formed by single cells.
5. Representative Results
According to the protocol described in the part for rat MSCs isolation, plastic adherent MSCs should be visible the next day after plating. As cells continue to proliferate, the confluent cells should look like the cells shown in Figure 1A. When cells reach ~80% confluency (Figure 1B), subculturing can be carried out. During subculture, trypsin-EDTA was used to detach cells and lifted cells are small and round as shown in Figure 1C.
Figure 1. Phase contrast images of rat MSCs. (A) Confluent MSCs. The majority of cells are spindle-like or star-like. (B) MSCs around 80% confluency. (C) Lifted cells after trypsinization are small and round.
Once MSCs are enriched by magnetic cell sorting, flow cytometry analysis is performed to verify the surface marker expressions. If the enrichment is good, the cells should show positive staining against MSC markers CD54 and CD90 but negative against the HSC marker CD45 (Figure 2). Isotype controls IgG2a and IgG1 are used as negative controls.
Figure 2. Flow cytometry analysis of MSCs for surface markers. MSCs were labeled with antibodies against IgG2a (isotype control 1), IgG1 (isotype control 2), CD45, CD54 and CD90. MSCs expressed CD54 and CD90 but not CD45.
When cells are seeded at proper clonal density, colonies should rise from single cells. Figure 3A represents a colony formed by a single cell. Cloning cylinders can then be used to separate the colonies and cells derived from the colonies can be cultured separately. Figure 3B and 3C represents cells derived from two individual colonies. The cells derived from colony 1 are spindle like (Figure 3B) whereas the cells derived from colony 2 are round (Figure 3C).
Figure 3. Colony formation by MSCs and single-colony derived cells. MSCs cultured at clonal density form individual colonies. These colonies can be separated by cloning cylinders and cells from different colonies can be cultured separately. (A) A representative colony formed by MSCs when plated at clonal density. (B) Spindle-like cells derived from one colony. (C) Round cells derived from another colony.
This protocol describes how to isolate and enrich MSCs. A method to separate the single-colony derived cells is also incorporated. There are several steps that are important for a successful isolation, enrichment and colony separation. While doing cell isolation from rat, it is recommended to filter through a cell strainer or a sterile nylon mesh of similar size to get rid of the blood clots and bone debris. After plating the cells overnight, many dead cells will be floating in the medium and the dead cells are removed by replacing with fresh medium which should help the growth of the attached cells.
The magnetic cell sorting in this protocol describes how to perform a positive selection, and similar procedures can be used to perform a negative selection. The amount of antibodies to be added may differ and optimization is required to achieve better sorting. This is also true for labeling the cells for flow cytometry analysis. If not running flow cytometry analysis for the labeled samples immediately, samples can be fixed with 2% formaldehyde and run later. However, long-term storage is not recommended since this tends to increase the auto-fluorescence and sacrifice sample quality.
The key part for the colony separation is seeding at the right cell density (which should be optimized experimentally) and locating single clones that are not surrounded by other clones. If there are other clones nearby, the cloning cylinder may encompass the nearby clones and the cells obtained will no longer be from one clone When placing the cloning cylinder over the clone, also be careful not to slide it over the dish surface as this will cause the silicon grease at the bottom of the cloning cylinder to cover the cells and prevent the trypsin from reaching the cells to detach them.
The work was supported in part by National Institute of Health (R01GM079688, R21RR024439, and R21GM075838), National Science Foundation (CBET 0941055), and the MSU Foundation.
|Cell strainer||BD Biosciences||352350|
|BD Imagnet||BD Biosciences||552311|
|Biotin mouse IgG2a||BD Biosciences||555572|
|Biotin mouse IgG1||BD Biosciences||555747|
|Biotin anti-rat CD54||Cedarlane Labs||CL054B|
|Biotin anti-rat CD90||Cedarlane Labs||CL005B|
|Biotin anti-rat CD45||Cedarlane Labs||CL001B|
|BD Imag buffer||BD Biosciences||552362|
|Round-bottom tube||BD Biosciences||352063|
|Streptavidin particles||BD Biosciences||557812|
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