PROTOCOL

A protocol for isolation and culture of mesenchymal

stem cells from mouse bone marrow
Masoud Soleimani1,4 & Samad Nadri2–4
1Department of Hematology, Faculty of Medical Science, Tarbiat Modares University, P.O. Box, 14115-111, Tehran, Iran. 2Department of Stem Cells and Tissue
Engineering, Stem Cell Technology Institute, P.O. Box 14155-3174, Tehran, Iran. 3Department of Biomedical Engineering and Medical Physics, Faculty of Medicine,
Shahid Beheshti University (MC), 19395-4719, Tehran, Iran. 4Both the authors contributed equally to this work. Correspondence should be addressed to M.S.
(Soleim_m@modares.ac.ir) or S.N. (nadrisamad@sbmu.ac.ir or s.nadri@stemcellstech.com).

Published online 8 January 2009; doi:10.1038/nprot.2008.221

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

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 1C–5% CO2
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 1C). A purified population of MSCs can be obtained 3 weeks after the initiation of culture.

INTRODUCTION
The extensive definition of a stem cell is a population of primitive
cells with the ability to self-renew and differentiate into multiple
cell lineages1. Two stem cell populations with distinct progenies
are housed within adult BM, hematopoietic stem cells and MSCs2.
MSCs have been referred to in the literature by other names
such as colony-forming fibroblastic cells3, BM stromal stem cells4,
mesenchymal progenitor cells5 and BM stromal cells6. MSCs are
considered to be a potential source for cell and gene therapy
strategies7. Effectiveness of MSCs in hematopoietic recovery,
bone regeneration and in the treatment of patients with osteo-
genesis imperfecta, infracted myocardium or joint diseases has
been well documented7–13; for this reason, isolation of MSCs from
different species such as mouse and extensive preclinical studies
is required. A number of techniques have been developed to
fulfill this purpose14–20, such as exposure of cultures to cytotoxic
materials21,22, cell sorting23, low- and high-density culture14,18,24
and negative and positive selection14,16,17,19, which permits the
isolation of mouse mesenchymal stem cells (mMSCs) from mouse
BM. However, these methods have inconvenient effects on the
biological properties of stromal cells such as decline in proliferation
and differentiation potential of mMSCs.
For many years, our work focused on the isolation and charac-
terization of mMSCs, in particular from BM. mMSCs are usually
isolated and purified from BM by way of their physical propensity
to adhere to the plastic substrate of the cell culture plate. However,
the standard and unmodified method based on plastic adherence5
has been confirmed as unsuccessful for mMSCs because various
hematopoietic cell lineages proliferate on stromal layers and
therefore constitute a large percentage of the plastic adherent
population5,25. Although a range of methods have been illustrated
to eradicate contaminating hematopoietic populations from these
cultures such as exposure of cultures to cytotoxic materials21,22, cell
sorting23 and negative and positive selection14,16,17,19, they have not
gained widespread acceptance due to their effects on proliferation
and differentiation properties of isolated cells. Consequently, we
developed a method on the basis of frequent medium changes
during the initial phase of culturing and diminished duration
of trypsination to fractionate hematopoietic and endothelial
lineages from plastic adherent fibroblastoid (stromal) cells derived
from murine BM. To date, several approaches have been investigated
for the preparation of a more homogeneous cell population of BM-
derived mMSCs using other isolation protocols; results showed
that mMSCs purified by exposure of plastic adherent murine
BM cultures to cytotoxic materials do not have the potential to
differentiate into mesenchymal lineages21,22. Using a cell sorting
approach, the clonogenicity and osteogenic potential of mMSCs
were found to be reduced23, although negative selection methods
lead to downregulation of many of the genes involved in cell
proliferation, and granulocyte–monocyte lineage cells reappeared
following 1 week of culture17. Although cells isolated by positive
selection have shown higher proliferation ability than those
obtained by negative selection techniques, and could be maintained
for up to three passages without the need for growth and mitogenic
factors, this method to isolate mMSCs from all mouse strains failed
to prove effective due to the lack of CD34 antigens for direct
isolation of mMSCs from other mouse strains5,16,19; hence, it may
indicate that some characteristics of MSCs are strain specific.
We describe here a protocol for uncomplicated isolation of
mMSCs from BM. This protocol is derived from the original
description by Nadri et al.20. A purified population of MSCs
with spindle-shaped morphology is obtained in the first passages
(3 weeks after the initiation of culture). Moreover, no additional
growth factors are required for this protocol, which is advantageous
because many growth factors and related products can modify
protein synthesis and intracellular trafficking. Furthermore, this
method has proved successful in the isolatation of mMSCs from
BM of several mouse strains (Balb/c20, NMRI, C57Bl/6, FVB/N
(S.N., M.S. and Amir Atashi, data not published)). Previous
researchers have reported that mMSCs progressively lost their
multiple differentiation and colony-forming potential at increasing
passages26,27. Using this method, however, the purity, differentiation
and proliferation capacity of isolated cells are evaluated with

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 PROTOCOL

fluorescence-activated cell sorting, specific staining and CFU-F
assay20, and a purified population of mMSCs with high proliferation
and differentiation potential can be achieved in the first passage.
In summary, the isolation of MSCs from whole BM through
their preferential attachment to tissue culture plastic is very efficient
using this method. Compared with other mMSCs isolation meth-
ods, our method for the isolation of mMSCs through frequent
medium changing and diminished trypsinization time is simple
and straightforward. It can be easily followed by a researcher
without previous experience.

MATERIALS
REAGENTS
. DMEM with 2 mM L-glutamine and without ribonucleosides and
ribonucleotides (GIBCO, cat. no. 12800-116)
. FBS (GIBCO, cat. no. 10270-106)
. 0.25% trypsin/1 mM ethylenediaminetetraacetic acid (GIBCO, cat. no.
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols
. Incubator with both temperature and gas composition controls
. Optical microscope with phase-contrast equipment
. A procedure for material sterilization (e.g., Poupinel oven)
. 10-ml syringes with 27-gauge needles
. Surgical forceps (straight and curved)

27250-018) . Surgical scissors

. Hank’s balanced salt solution (Sigma, cat. no. H 4034) . Pipette (ARS, cat. no. 537-503/200)
. Streptomycin (Sigma, cat. no. 1277) . Filter mesh (70 mm; BD, Falcon, cat. no. 1520012)
. L-glutamine (GIBCO, cat. no. 35050-038) . 6-well culture dishes (100 mm; Orange Scientific, cat. no. 5530500)
. Penicillin (Sigma, cat. no. 4687) . 25-cm2 flask (Orange Scientific, cat. no. 70025)
. NaHCO3 (Sigma, cat. no. S 5761) REAGENT SETUP
. FBS (GIBCO, cat. no. 10270-106) Harvest buffer Mix Hank’s balanced salt solution and 100 mg ml 1
EQUIPMENT streptomycin. Harvest buffer can be stored for 2 weeks at 4 1C.
. Hood for cell culture with vertical laminar flow and equipped with UV light Complete DMEM medium (with 15% FBS) Adjust the volume to 100 ml
for decontamination by adding DMEM ‘stock’ to a solution containing 2 mm of L-glutamine,
. Water bath with temperature control 100 mg ml 1 penicillin and 100 mg ml 1 streptomycin, 3.7 g liter 1 NaHCO3 and
. Centrifuge (no temperature control is needed) 15% of FBS. ‘Complete DMEM medium’ can be stored for 1 week at 4 1C.

PROCEDURE
Isolation and culture of mesenchymal stem cells from Murine BM
1| To isolate marrow, kill mice (Balb/c, 6–8 weeks old) by cervical dislocation. Then, rinse the animal skeleton freely in 70%
ethanol, make an incision around the perimeter of the hind limbs where they attach to the trunk and remove the skin by pulling
toward the foot, which is cut at the anklebone. This eliminates further contact of the hind limb with the animal’s fur, which is a
source of contaminating bacteria. Then, dissect the hind limbs from the trunk of the body by cutting along the spinal cord with
care not to damage the femur. Store limbs on ice in DMEM supplemented with 1 penicillin/streptomycin while awaiting further
dissection.
? TROUBLESHOOTING

2| Perform further dissection of the hind limbs under the hood. Bisect each hind limb by cutting through the knee joint.
Remove the muscle and connective tissue from both the tibia and the femur by scraping the diaphysis of the bone clean then
pulling the tissue toward the ends of the bone. After cleaning, store the bones in DMEM supplemented with 1 penicillin/
streptomycin on ice.
? TROUBLESHOOTING

3| Harvest the BM in a hood using proper sterile technique. Cut the ends of the tibia and femur just below the end of the
marrow cavity using a pair of sharp rongeur. Insert a 27-gauge needle attached to a 10-ml syringe containing complete media
into the spongy bone exposed by removal of the growth plate. Flush the marrow plug out of the cut end of the bone with
1 ml of complete media and collect in a 10-ml tube on ice.
m CRITICAL STEP Strong flushing is necessary during marrow cell preparation.
? TROUBLESHOOTING
4| Filter the cell suspension through a 70-mm filter mesh to remove any bone spicules or muscle and cell clumps. Determine
the yield and viability of cells by Trypan blue exclusion and counting on a hemocytometer as described in ref. 27.
m CRITICAL STEP Typically, we obtain B70106 BM cells from one donor.

5| Culture BM cells in 95-mm culture dishes in 1 ml of complete medium at a density of 25106 cells ml 1 (on the basis of
the cell count obtained in Step 4). Incubate the plates at 37 1C with 5% CO2 in a humidified chamber without disturbing them.
After 3 h, remove the nonadherent cells that accumulate on the surface of the dish by changing the medium and replacing with
fresh complete medium.
? TROUBLESHOOTING

6| After an additional 8 h of culture, replace the medium with 1.5 ml of fresh complete medium. Thereafter, repeat this step
every 8 h for up to 72 h of initial culture.

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m CRITICAL STEP The medium must be changed slowly (rapidly a b

changing medium may cause MSCs to be lifted together with
non-mMSCs (hematopoietic cell lineages)).
7| Wash the adherent cells (passage 0) with phosphate-
buffered saline, and add fresh medium every 3–4 d. The initial
adherent spindle-shaped cells appear as individual cells on 500 um 500 um
the third day in phase-contrast microscopy (Fig. 1a). Within
4–8 d, the culture becomes more confluent and reaches
c d
65–70% confluence within 2 weeks. At this stage, the
cultures typically exhibit two characteristics: first, plates
may contain distinct colonies of fibroblastic cells that vary
© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

in size; and second may contain very small numbers of

hematopoietic cells interspersed between or on the colonies
500 um 500 um
(Fig. 1b and c).
Figure 1 | Morphological features of mMSCs. (a) Spindle-shaped morphology
8| After 2 weeks of initiating culture, wash the cells with of marrow cells that appear at day 1. (b,c) The number and size of colony
phosphate-buffered saline and lift cells by incubation in appears to gradually increase on days 4–10. (d) More confluent mMSCs at
0.5 ml of 0.25% trypsin/1 mM ethylenediaminetetraacetic 21 days of culturing marrow cells.
acid for 2 min at room temperature. Neutralize the trypsin
by adding 1.5 ml of complete medium, and culture all lifted cells in a 25-cm2 flask. Discard the nonlifted cells.
m CRITICAL STEP The time and temperature of passaging is very important (if the time and temperature were higher than 2 min
and 25 1C, respectively, non-MSCs together with mMSCs would be lifted from plastic culture dishes).
? TROUBLESHOOTING

9| Change the culture medium every 3 d (replacing with 6 ml of medium each time). Typically, cell confluence is achieved
in 7 d (Fig. 1d).

 TIMING
Day 1, Steps 1–5: harvesting, seeding and change of medium of marrow cells
Day 2–3, Step 5: slowly change medium of culture
Day 4–14, Steps 6 and 7: change medium every 3–4 d
Day 14, Step 8: subculturing adherent marrow cells
Day 15–21, Step 9: obtain purify population of mesenchymal stem cells with spindle-shaped morphology

? TROUBLESHOOTING
Contamination
Hematopoietic lineage cells can contaminate mMSC culture. mMSCs isolated by plastic adherence are heterogeneous and
contaminated by hematopoietic cells5 due to the fact that the fraction of hematopoietic cells is higher in both initial and
passage cultures of murine marrow cells6,28. mMSCs support the growth and proliferation of hematopoietic cells by secreting
growth factors, such as interlukin (IL)-6, IL-7, IL-8, IL-11, IL-12, IL-14 and IL-15, macrophage-CSF, LIF, FLt-3 ligand and stem
cell factor29,30. This problem was solved using frequent medium change that may prevent adherence of many of the non-MSCs
and hematopoietic cells to the culture dishes. See also Table 1 for further troubleshooting advice.

TABLE 1 | Troubleshooting table.

Steps Problem Possible cause and repair
1–3 Low yield of harvested marrow cells Increased age of mice and bad harvesting of marrow cells from end of bones; select mice at
6–8 weeks of age and insert needle into epiphysis of bones and use strong flushing during
marrow cell preparation

5 No adherent cells 24 h after seeding Bad conditions in incubator (hypoxia, high CO2 level, no water); verify incubator conditions

No adherent cells 72 h after seeding Bacterial contamination or too rapid changing of medium; check for contamination and slowly
change medium

8 Contamination with non-MSCs Bad subculturing (passaging); trypsinize at room temperature for up to 2 min

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a CD44 Sca-1 Thy1.2 CD135 CD31

Events
Events
Events

Events
Events

FL2 FL1
FL2 FL1 FL1

CD34 CD45 CD11b C-Kit Vcam-1

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Events
Events

Events
Events
Events

FL1 FL1 FL1

FL1 FL1

Figure 2 | Cell surface markers and differentiation capacity of mouse mesenchymal stem cells (mMSCs). (a) Histogram analysis of cell surface markers
showed that mMSCs were not contaminated by hematopoietic cell lineages. (b) The cells differentiated into (left) mineralizing cells stained with alizarin red,
(middle) adipocytes stained with oil red or (right) chondrocytic lineage cells stained with Toluidine blue.

ANTICIPATED RESULTS
During the isolation of mesenchymal stem cells from mouse BM and after flushing, we are usually able to observe many
hematopoietic cells in the harvested marrow cells. After 72 h of initiation culture, we can observe a dramatic decrease in
hematopoietic stem cell lineages that adhere to plastic culture dishes (Step 8). In the third week of culture, essentially all cells
have spindle-shape morphology (Figs. 2a and 1d). We achieve about 2105 mMSCs from one donor (S.N., M.S. and Amir Atashi,
data not published). These cells can be readily differentiated into osteocyte, adipocyte and chondrocyte cells by culturing in
appropriate induction media20. Endothelial, myeloid and hematopoietic cell lineage-specific antigens, such as CD31, Vcam-1,
CD34, C-Kit, CD135, CD11b and CD45, were not expressed in these cells20 (Fig. 2a). mMSCs isolated using this protocol are not
contaminated with hematopoietic cell lineages, readily differentiated into mesenchymal lineages (more than 95%) (Fig. 2b) and
more than 70% of cells have colony-formation capacity20. Furthermore, the cells maintained these capacities up to high passages20
(passage 10). As isolated cells maintain proliferation and differentiation potential up to high passage, large-scale and cost-effective
mMSC isolation can be achieved using this protocol, without a requirement for specific cell surface antibodies and equipment.

ACKNOWLEDGMENTS This study was supported by Iran Stem Cell Technology
Institute.
Published online at http://www.natureprotocols.com/
Reprints and permissions information is available online at http://npg.nature.com/
reprintsandpermissions/
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