Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

Summary

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.

Abstract

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Protocol

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.

1. The animal was put into an anaesthesia chamber and anaesthetized for around five minutes. During the anaesthesia, observe the rate of blinking, breathing and motor activity. Remove the animal from the chamber immediately after it stops motor activity and the blinking rate became infrequent.
2. Lay the animal down on an operation station and kill the animal by cervical dislocation. Cut off....

Discussion

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 b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X D-PBS	Invitrogen	14040-133
DMEM	Invitrogen	11885-084
Cell strainer	BD Biosciences	352350
FBS	Invitrogen	26140-079
Pen-Strep	Invitrogen	15140
Trypsin-EDTA	Invitrogen	25200-056
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
SA-PE	R&D Systems	F0040
Cloning cylinder	Sigma-Aldrich	C2059