Mice aged 4 weeks or 8 weeks are terminated by cervical dislocation and placed in a 100-mm cell culture dish (Becton Dickinson, Franklin Lakes, NJ, USA), where the whole body is soaked in 70% (v/v) ethanol for 2 minutes, and then the mouse is transferred to a new dish ( Fig. 1A). Therefore, an easy and effective protocol for isolation of mouse BM-MSCs is needed. Frequent medium change method is inconvenient because it is required to change the culture medium every 8 hours during the first 72 hours of the initial culture. Cells obtained using a positive selection method show higher proliferation ability compared with the negative selection method, but the method was only repeated in the C57B1/6 mice and failed to repeat in other strains of mice. Negative selection method leads to granulocyte–monocyte lineage cells reappearing after 1 week of culture. However, they all had some short falls: the standard MSCs culture method based on plastic adherence has been confirmed to have lower successful rate whereas the cell sorting approach reduced the osteogenic potentials of MSCs. Several techniques have been used to purify or enrich MSCs including antibody-based cell sorting, low and high-density culture techniques, , positive and negative selection method, frequent medium changes, and enzymatic digestion approach. BM-MSCs are usually isolated and purified through their physical adherence to the plastic cell culture plate. Two main stem cell populations and their progenies, haematopoietic stem cells and BM-MSCs, are the main residents of bone marrow. However, the isolation and purification of MSCs from mouse bone marrow is more difficult than other species due to their heterogeneity and low percentage in the bone marrow,. Mice are one of the most commonly used experimental animals in biology and medicine primarily because they are mammals, small, inexpensive, easily maintained, can reproduce quickly, and share a high degree of homology with humans. MSCs have been successfully isolated and characterised from many species including mouse, rat, rabbit, dog, sheep, pig, and human, ,. Bone marrow (BM) is the most common source of MSCs. MSCs can be isolated from various sources such as adipose tissue, tendon, peripheral blood, and cord blood. Therefore, MSCs are an attractive cell source for tissue engineering and vehicles of cell therapy. MSCs are easily accessible, expandable, immunosuppressive and they do not elicit immediate immune responses. Mesenchymal stem cells (MSCs) are multipotent stem cells that have the potential to self-renew and differentiate into a variety of specialised cell types such as osteoblasts, chondrocytes, adipocytes, and neurons. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Using our protocol, the isolated mouse BM-MSCs were strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differentiation potentials. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (2) Our culture medium is not supplemented with any additional growth factor. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. There are five main features of this protocol. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy.
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