Soleimani, M. & Nadri, S. A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow. Nat. Protoc. 4, 102-106

Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, PO Box, 14115-111, Tehran, Iran.
Nature Protocol (Impact Factor: 9.67). 02/2009; 4(1):102-6. DOI: 10.1038/nprot.2008.221
Source: PubMed


We explain a protocol for straightforward isolation and culture of mesenchymal stem cells (MSCs) from mouse bone marrow (BM) to supply researchers with a method that can be applied in cell biology and tissue engineering with minimal requirements. Our protocol is mainly on the basis of the frequent medium change in primary culture and diminishing the trypsinization time. Mouse mesenchymal stem cells are generally isolated from an aspirate of BM harvested from the tibia and femoral marrow compartments, then cultured in a medium with Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) for 3 h in a 37 degrees C-5% CO(2) incubator. Nonadherent cells are removed carefully after 3 h and fresh medium is replaced. When primary cultures become almost confluent, the culture is treated with 0.5 ml of 0.25% trypsin containing 0.02% ethylenediaminetetraacetic acid for 2 min at room temperature (25 degrees C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

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    • "Culture was maintained at 37 °C in a humidified atmosphere containing 95% air and 5% CO 2 . The isolation of MSCs was carried out on the basis of the frequent medium change in primary culture and diminishing the trypsinization time [21]. Medium was changed twice during initial 72 h to remove non-adherent RBCs and macrophages, thereafter once every 3–4 days. "
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    ABSTRACT: For non-invasive stem cells tracking through MRI, it is important to understand the efficiency of in vitro labeling of stem cells with iron oxide with regard to its relaxation behavior. In this study, we have carried out a pilot study of labeling mice mesenchymal stem cells (mMSCs) with ultrasmall superparamagnetic iron oxide (USPIO) entrapped with poly-l-lysine (PLL) in different ratios and incubated with different times. Our results demonstrated that 50:1.5µg/ml of iron oxide and PLL at an incubation time of 6h with 10% serum concentration are sufficient enough for effective labeling. Optimized labeling showed that>98% of viability and<3% toxicity were observed at a total iron content of 11.8pg/cell. In vitro relaxometry study showed that almost a 6.6 fold reduction in transverse relaxation time (T2) was observed after labeling as compared to unlabeled. IO-PLL complex was more effective than iron oxide alone in labeling and a detectable lower limit found to be hundred with optimized concentration. Significant increase in Oct-4 expression on day-3 after labeling was observed, whereas CD146 expression remains unchanged in real time RT-PCR. This optimized labeling method of MSCs may be very useful for cellular MRI and stem cells tracking studies.
    Experimental Cell Research 11/2015; DOI:10.1016/j.yexcr.2015.11.010 · 3.25 Impact Factor
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    • "MSCs were isolated from the AT or BM of C57/BL6 mice according to three adapted protocols (Soleimani and Nadri, 2009; Sung et al., 2008; Yamamoto et al., 2007). Cells (2 3 10 5 ) at passage 3–5 were injected into the hindlimb of syngeneic mice the day following resection of the femoral artery. "
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    ABSTRACT: Mesenchymal stromal cells (MSCs) are defined as multipotent, self-renewing cells residing in several tissues, including the bone marrow, adipose tissue, umbilical cord blood, and placenta (Pittenger et al., 1999). These cells are defined as multipotent, as they are capable of generating different mesenchymal cell types, traditionally adipocytes, chondrocytes, and osteocytes, but also smooth muscle cells and cardiomyocytes (Makino et al., 1999 and Pittenger et al., 1999). MSCs have been at the forefront of clinical research for the therapy of cardiovascular disorders for many years. In particular, cardiac and peripheral ischemia is a leading cause of morbidity and mortality in our aging society and suffers from a lack of curative therapies (Tendera et al., 2011). In this setting, MSC transplantation has been proposed as an innovative therapy for no-option ischemic patients. Originally, the therapeutic potential of these cells was thought to arise through their putative capacity to transdifferentiate, thereby directly contributing to vasculogenesis and tissue regeneration (Quevedo et al., 2009). This attractive hypothesis led to the prompt, perhaps immature transition of the results obtained in animal models to the clinics, with the ambitious goal to regenerate ischemic tissues (Hare et al., 2009 and Tateishi-Yuyama et al., 2002). However, MSC plasticity has been later harshly questioned (Noiseux et al., 2006), and the therapeutic potential of these cells is currently considered to derive from the secretion of a variety of growth factors and cytokines exerting a paracrine, protective effect on ischemic cells (Gnecchi et al., 2012).
    Stem Cell Reports 02/2015; 110(3). DOI:10.1016/j.stemcr.2015.01.001 · 5.37 Impact Factor
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    • "The Ethical Review Board of Shanghai Sixth People's Hospital, Shanghai Jiaotong University School of Medicine approved the experimental procedures. Primary human bone marrow-derived mesenchymal stem cells (hBMSCs) were isolated and sub-cultured using a method described previously [38]. First, cell aggregates and bone debris were removed by filtering the bone marrow suspension through a 70 mm cell strainer. "
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    ABSTRACT: There is growing interest in the use of synthetic biomaterials to deliver inorganic ions that are known to stimulate angiogenesis and osteogenesis in vivo. In the present study, we investigated the effects of varying amounts of copper (Cu) in a bioactive glass on the response of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro and on blood vessel formation and bone regeneration in rat calvarial defects in vivo. Porous scaffolds of a borosilicate bioactive glass (composition 6Na2O, 8K2O, 8MgO, 22CaO, 36B2O3, 18SiO2, 2P2O5; mol%) doped with 0.5, 1.0 and 3.0 wt% CuO were created using a foam replication method. When immersed in simulated body fluid, the scaffolds released Cu ions into the medium and converted to hydroxyapatite. At the concentrations used, the Cu in the glass was not toxic to the hBMSCs cultured on the scaffolds in vitro. The alkaline phosphatase activity of the hBMSCs and the expression levels of angiogenic-related genes (vascular endothelial growth factor and basic fibroblast growth factor) and osteogenic-related genes (runt-related transcription factor 2, bone morphogenetic protein-2 and osteopontin) increased significantly with increasing amount of Cu in the glass. When implanted in rat calvarial defects in vivo, the scaffolds (3 wt% CuO) significantly enhanced both blood vessel formation and bone regeneration in the defects at 8 weeks post-implantation. These results show that doping bioactive glass implants with Cu is a promising approach for enhancing angiogenesis and osteogenesis in the healing of osseous defects. Copyright © 2014. Published by Elsevier Ltd.
    Acta Biomaterialia 12/2014; 14. DOI:10.1016/j.actbio.2014.12.010 · 6.03 Impact Factor
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