An improved protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow

Abstract

Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering and washing them. Our method is simply keeping the MSCs in their initial niche with minimal disturbance. (2) Our culture medium is not supplemented with any additional growth factor. (3) Our method does not need to separate cells using flow cytometry or immunomagnetic sorting techniques. (4) Our method has been carefully tested in several mouse strains and the results are reproducible. (5) We have optimised this protocol, and list detailed potential problems and trouble-shooting tricks. 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. Compared with the commonly used protocol, our protocol had higher success rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable, and alternative method for culturing MSCs from mouse bone marrow tissues.

Full text

ORIGINAL ARTICLE
An improved protocol for isolation and
culture of mesenchymal stem cells from
mouse bone marrow
Shuo Huang
a,b
, Liangliang Xu
a,b
, Yuxin Sun
a,b
, Tianyi Wu
a,b
,
Kuixing Wang
a,b,c
, Gang Li
a,b,c,d,e,
*
a
Department of Orthopaedics and Traumatology, Faculty of Medicine, The Chinese University of
Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China
b
Stem Cells and Regenerative Medicine Laboratory, Li Ka Shing Institute of Health Sciences,
The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong, China
c
Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences,
Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, China
d
The CUHK-ACC Space Medicine Centre on Health Maintenance of Musculoskeletal System,
The Chinese University of Hong Kong Shenzhen Research Institute, Shenzhen, Hong Kong, China
e
Lui Che Woo Institute of Innovative Medicine, Faculty of Medicine, The Chinese University of
Hong Kong, Hong Kong, China
Received 19 May 2014; received in revised form 17 July 2014; accepted 23 July 2014
KEYWORDS
bone marrow;
isolation;
mesenchymal stem
cells;
mouse;
protocol
Summary Mesenchymal stem cells (MSCs) from bone marrow are main cell source for tissue
repair and engineering, and vehicles of cell-based gene therapy. Unlike other species, mouse
bone marrow derived MSCs (BM-MSCs) are difficult to harvest and grow due to the low MSCs
yield. We report here a standardised, reliable, and easy-to-perform protocol for isolation
and culture of mouse BM-MSCs. There are five main features of this protocol. (1) After flushing
bone marrow out of the marrow cavity, we cultured the cells with fat mass without filtering
and washing them. Our method is simply keeping the MSCs in their initial niche with minimal
disturbance. (2) Our culture medium is not supplemented with any additional growth factor.
(3) Our method does not need to separate cells using flow cytometry or immunomagnetic sort-
ing techniques. (4) Our method has been carefully tested in several mouse strains and the re-
sults are reproducible. (5) We have optimised this protocol, and list detailed potential
problems and trouble-shooting tricks. Using our protocol, the isolated mouse BM-MSCs were
strongly positive for CD44 and CD90, negative CD45 and CD31, and exhibited tri-lineage differ-
entiation potentials. Compared with the commonly used protocol, our protocol had higher suc-
cess rate of establishing the mouse BM-MSCs in culture. Our protocol may be a simple, reliable,
and alternative method for culturing MSCs from mouse bone marrow tissues.
Copyright ª2014, Chinese Speaking Orthopaedic Society. Published by Elsevier (Singapore) Pte
Ltd. All rights reserved.
* Corresponding author. Room 904, 9/F, Li Ka Shing Institute of Health Sciences, Prince of Wales Hospital, The Chinese University of
Hong Kong, Shatin, Hong Kong, China.
E-mail address: gangli@cuhk.edu.hk (G. Li).
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
bone marrow, Journal of Orthopaedic Translation (2014), http://dx.doi.org/10.1016/j.jot.2014.07.005
http://dx.doi.org/10.1016/j.jot.2014.07.005
2214-031X/Copyright ª2014, Chinese Speaking Orthopaedic Society. Published by Elsevier (Singapore) Pte Ltd. All rights reserved.
Available online at www.sciencedirect.com
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Journal of Orthopaedic Translation (2014) xx,1e8

Introduction
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 [1,2]. MSCs are
easily accessible, expandable, immunosuppressive and they
do not elicit immediate immune responses [3,4]. Therefore,
MSCs are an attractive cell source for tissue engineering
and vehicles of cell therapy.
MSCs can be isolated from various sources such as adi-
pose tissue, tendon, peripheral blood, and cord blood
[5e7]. Bone marrow (BM) is the most common source of
MSCs. MSCs have been successfully isolated and charac-
terised from many species including mouse, rat, rabbit,
dog, sheep, pig, and human [8e12]. 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 [13]. How-
ever, 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
[1,14,15].
Two main stem cell populations and their progenies,
haematopoietic stem cells and BM-MSCs, are the main
residents of bone marrow [1,15]. BM-MSCs are usually iso-
lated and purified through their physical adherence to the
plastic cell culture plate [16]. Several techniques have
been used to purify or enrich MSCs including antibody-
based cell sorting [17], low and high-density culture tech-
niques [18,19], positive and negative selection method
[20], frequent medium changes [21], and enzymatic
digestion approach [22]. However, they all had some short
falls: the standard MSCs culture method based on plastic
adherence has been confirmed to have lower successful
rate [23]; whereas the cell sorting approach reduced the
osteogenic potentials of MSCs [17]. Negative selection
method leads to granulocyteemonocyte lineage cells
reappearing after 1 week of culture [24]. 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 [25]. Frequent
medium change method is inconvenient because it is
required to change the culture medium every 8 hours dur-
ing the first 72 hours of the initial culture [21]. Therefore,
an easy and effective protocol for isolation of mouse BM-
MSCs is needed.
Materials and methods
Reagents
Reagents used included: 0.25% trypsineEDTA (1) with
phenol red; penicillinestreptomycineneomycin (PSN; Life
Technologies, Carlsbad, CA, USA) antibiotic mixture; foetal
bovine serum, qualified, heat-inactivated (Life Technolo-
gies); minimal essential medium (MEM) a, nucleosides,
powder (Life Technologies); and NaHCO
3
(SigmaeAldrich, St
Louis, MO, USA).
Reagent setup
Stock of a-MEM was made up with 1 bag of a-MEM powder
(1 L) and 2.2 g NaHCO
3
in 1000 mL of Milli-Q water, adjusted
to pH 7.2, filtered to sterilise, and stored for 1e2 weeks at
4C. Complete a-MEM medium was a-MEM medium stock
supplemented with 15% foetal bovine serum and 1% PSN,
stored at 4C. Phosphate-buffered saline (PBS) included:
NaCl 8.0 g, KCl 0.2 g, KH
2
PO
4
0.24 g, and Na
2
HPO
4
1.44 g in
1 L Milli-Q water (pH 7.4, sterilised and stored at 4C).
Animals
In this study, two mouse strains (ICR and C57) with different
ages (4 weeks and 8 weeks, males and females) were tested
using our protocol. All mice were purchased from and
housed in a designated and government approved animal
facility at The Chinese University Hong Kong, Hong Kong
SAR, China, in according to The Chinese University Hong
Kong’s animal experimental regulations. All efforts were
made to minimise animal suffering.
BM-MSCS culture protocol
Isolation and culture of mouse BM-MSCs
Mice aged 4 weeks or 8 weeks are terminated by cervical
dislocation and placed in a 100-mm cell culture dish (Bec-
ton 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). Four claws
are dissected at the ankle and carpal joints, and incisions
made around the connection between hindlimbs and trunk,
forelimbs, and trunk. The whole skin is then removed from
the hind limbs and forelimbs by pulling toward the cutting
site of the claw. Muscles, ligaments, and tendons are
carefully disassociated from tibias, femurs, and humeri
using microdissecting scissors and surgical scalpel. Tibias,
femurs, and humeri are dissected by cutting at the joints,
and the bones are transferred onto sterile gauze. Bones are
carefully scrubbed to remove the residual soft tissues
(Fig. 1B), and transferred to a 100-mm sterile culture dish
with 10 mL complete a-MEM medium on ice (Fig. 1C). All
samples are processed within 30 minutes following animal
death to ensure high cell viability. The soft tissues are
completely dissociated from the bones to avoid
contamination.
In a biosafety cabinet, the bones are washed twice with
PBS containing 1% PSN to flush away the blood cells and the
residual soft tissues, then bones are transferred into a new
100-mm sterile culture dish with 10 mL complete a-MEM
medium. The bone is held with forceps and the two ends
excised just below the end of the marrow cavity using
microdissecting scissors (Fig. 1D). A 23-gauge needle
attached to a 5 mL syringe is used to draw 5 mL complete a-
MEM medium from the dish; then the needle is inserted into
the bone cavity. The marrow out is slowly flushed and the
bone cavities washed twice again until the bones become
pale (Fig. 1E). All the bone pieces are removed from the
dish using forceps, leaving the solid mass in the medium,
and the dish is incubated at 37C in a 5% CO
2
incubator for 5
days (Fig. 1F). In order to obtain enough marrow cells, the
2 S. Huang et al.
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
bone marrow, Journal of Orthopaedic Translation (2014), http://dx.doi.org/10.1016/j.jot.2014.07.005

bone cavities are flushed repeatedly until the bones appear
to be pale.
The initial spindle-shaped cells appear on Day 3 in
phase-contrast microscopy, and then culture becomes more
confluent and reaches 70e90% confluence within only 2
days. Cells are washed with PBS twice, and digested with
2.5 mL of 0.25% trypsin for 2 minutes at 37C, then the
trypsin neutralised with 7.5 mL complete a-MEM medium.
The bottom of the plate is flushed using pipet-aid and the
cells transferred to a 15 mL Falcon tube (Becton Dickinson),
which is centrifuged at 800gfor 5 minutes, and the cells
resuspended in a 75 cm
2
cell culture flask (Corning Inc,
Corning, NY, USA) at a split ratio of 1:3. Note: Washing the
cells with PBS prior to digestion is important, as it removes
the residual medium and cell secretion and loosens the
adhesive force of MSCs to the dish. The digestion should be
limited to 2 minutes, as longer digestion is harmful for
MSCs, and could lift non-MSCs from the dish.
Passaging should be performed every 4e6 days at a split
ratio of 1:3. Normally, the cells at Passage 3 contain fewer
macrophages and blood cells, and less fat than those at
Passages 1 and 2, and can be readily used for experiments.
Challenges and possible solutions in mouse BM-MSCs culture
are summarised in Table 1.
The commonly used protocol for establishing (mouse)
BM-MSC culture
The commonly accepted isolation strategy for BM-MSCs was
initially reported by Nadri et al. [16]. Briefly, a mouse is
terminated; tibias, femurs, and humeri are dissected. Both
ends of the bones are removed with sharp scissors. Bone
marrow is flushed out from the bone cavity using plain
culture medium, then filtered through a 70-mm filter mesh,
washed, and resuspended. The dish is then incubated at
37C in a 5% CO
2
incubator. Nonadherent cells are removed
24e72 hours later by changing the medium. When culture
reaches 70e90% confluence, cells are subcultured at a split
ratio of 1:3.
Phenotypic characterisation and cell growth rate of the
mouse BM-MSCs
BM-MSCs at Passage 3 were used for characterisation.
Mesenchymal stem cell markers CD44, CD90, endothelial
cell marker CD31 and haematopoietic marker CD45 were
examined by flow cytometry according to a previously
published paper [26]. The trilineage differentiation abilities
were tested according to previous published protocols
[26,27]. We have used Alizarin red, Oil Red O, and toluidine
blue staining as indicators for osteogenic, adipogenic, and
chondrogenic differentiation according to our previously
published methods [27].
In order to compare the differences between our pro-
tocol and the commonly used protocol for mouse BM-MSCs
culture, we used 16 mice of two different strains (ICR and
C57) with two different ages (4 weeks and 8 weeks, males
and females) and assigned them into four groups as shown
in Table 2. Left/right side of the femurs were chosen for
isolation of BM-MSCs using our protocol and the contralat-
eral femur was then used for isolation of BM-MSCs using the
commonly used method. To calculate the cell growth rate,
when the cultured cells reached about 90% confluence,
they were trypsinised, cell numbers were counted using a
handheld automatic cell counter (Scepter 2.0 Cell Counter,
Merck Millipore, Darmstadt, Germany) and recorded. The
passaged cells were then subcultured into new flasks at a
split ratio of 1:3.
Figure 1 Illustrations of mouse bone marrow cell collection procedures. (A) The mouse was terminated by cervical dislocation,
placed in a 100-mm culture dish, and washed with 70% (vol/vol) ethanol for 2 minutes. (B) Tibias, femurs, and humeri were
dissected; muscles, ligaments, and tendons were removed and the bones transferred onto sterile gauzes. (C) Bones were trans-
ferred to a 100-mm sterile culture dish with 10 mL complete a-minimal essential medium on ice. (D) The dish was transferred into
the biosafety cabinet and washed twice to flush away impurities; the two ends just below the end of the marrow cavity were
excised with microdissecting scissors. (E) A 23-gauge needle was inserted into the bone cavity and used to slowly flush the marrow
out. The bone cavities were washed twice again until the bones became pale. (F) All the bone pieces were removed from the dish
and the fat mass was left in the medium. Then the dish was incubated at 37C in a 5% CO
2
incubator.
Improved isolation and culture of mouse BM-MSCs 3
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
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Statistical analysis
All quantitative data were transferred to statistical
spreadsheets and analysed by a commercially available
statistical program SPSS version 16.0 (IBM SPSS Inc., Chi-
cago, IL, USA), one-way analysis of variance were used for
comparison of mean values with p<0.05 considered sta-
tistically significant.
Results
Morphological features of cultured mouse BM-MSCs
Morphological features of the cultured mouse BM-MSCs
using our protocol were similar to those of the BM-MSCs
cultures using the commonly used method. On Day 1, most
of the cells were still mononuclear cells and fat droplets
were frequently seen (Fig. 2A). On Day 2, some spindle-
shaped cells (arrows) appeared among the mononuclear
cells and fat droplets (Fig. 2B). On Day 3, the number of
spindle-shaped cells continued increasing (Fig. 2C). On Day
4, the spindle-shaped cells reached about 60e80% conflu-
ence (Fig. 2D), the cell growth was slower in the flasks using
the commonly used method compared to the new method.
On Day 5, the spindle-shaped cells already formed cell
layers (Fig. 2E) using the new protocol, but this is not seen
in the flasks under the commonly used protocol. Using our
protocol, on Day 5, fibroblast-like cell grew out from a
dense cell nodule (Fig. 2F), and the cells were passaged on
this day; on Day 7, cells reached 100% confluence when left
without passaging, multiple cell layers and dense cell
nodules were formed in some areas of the culture dish
(Fig. 2G and H). By contrast, the cells cultured using the
commonly used method only reach 60e70% confluence at
Day 7. After the cells reached 90% confluence, they were
passed and split; after Passage 3, the cells had uniform
fibroblast-like morphology (Fig. 2I) regardless of the initial
cell culture protocol. Using our protocol, we could obtain
approximately 5 10
7
e1.5 10
8
BM-MSCs from one mouse
bone marrow preparation in about 2 weeks, whereas it
would take at least 3 weeks for the BM-MSCs to reach a
similar cell number using the commonly used protocol.
Confirmation of phenotype and differentiation
capacities of mouse BM-MSCs
Once established in the culture dishes and passaged three
times, the MSCs retain similar phenotypes or differentiation
Table 1 Challenges and possible solutions in mouse BM-MSCs culture.
Problem Possible cause Solution
Few harvested cells
from bone marrow
Incomplete bone marrow
cavity flushing
Repeatedly flush bone cavities until the bones appear to be pale
The bone was broken and
cells leaked out
Carefully dissect bones and dissociate soft tissue from bones
Cells were dead during
harvesting
Prepare the bone marrow within 30 min following animal death,
and keep bones in complete a-MEM medium on ice
Microbial contamination Contaminated during bone
sample harvesting
Wash the mouse body with 70% ethanol for at least 2 min
Avoid bones touching the mouse skin during dissection
Keep bones in complete a-MEM medium with 1% PSN
Contaminated during cell
culture period
Wipe the dish with 70% ethanol prior to transferring it into the
cabinet
Wash bones twice using pipet-aid to flush away impurities,
blood cells and residual soft tissue that slightly connect to
the bone with complete a-MEM medium containing 1% PSN
Cells are not digested
off by trypsin
Cells not washed with PBS
prior to digestion
Wash the cells twice with PBS prior to digestion to remove
any residual serum
Cells grow slowly after
passaging
Trypsin cells for >2 min Digest cells with trypsin for <2 min
The initial total MSC
numbers are low
Do not disturb the cells for the first 3 days and do not passage
the cells until they reach at least 70% confluence
Table 2 Animal group details.
Groups
(Our protocol)
Strain Numbers Age (wk) Femur used
a
Control groups
(Standard protocol)
Femur used
a
I4 ICR 4 4 Random I4-C Contralateral
I8 ICR 4 8 Random I8-C Contralateral
C4 C57 4 4 Random C4-C Contralateral
C8 C57 4 8 Random C8-C Contralateral
a
Left or right side of the femur was randomly used for cell culture using our protocol, and the contralateral femur was then used for
cell culture using the standard protocol.
4 S. Huang et al.
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
bone marrow, Journal of Orthopaedic Translation (2014), http://dx.doi.org/10.1016/j.jot.2014.07.005

potentials regardless of the protocol initially used. Results
showed that the established cells were strongly positive for
MSC markers CD44, CD90 (Fig. 3A and B), and negative for
endothelial cell marker CD31 (Fig. 3C), and the haemato-
poietic cell marker CD45 (Fig. 3D). The isotype control was
negative.
The differentiation capacities of BM-MSCs obtained using
either protocol remain similar. Alizarin red staining
demonstrated that mineralised nodules formed in the BM-
MSCs after 4 weeks under the osteogenic induction
(Fig. 3E). Intracellular Oil-red-O staining showed lipid-rich
vacuoles formation of the mouse BM-MSCs after 2 weeks
adipogenic induction (Fig. 3F). After 3 weeks chondrogenic
induction, the cell pellet was sectioned and stained with
toluidine blue; the positive acidic proteoglycan indicated
the chondrocyte-like cells formation (Fig. 3G).
Comparison of the cell growth rate using the new
protocol versus the commonly used protocol
As shown in Table 3, the successful isolation rate of MSCs
was not strain- or sex- dependent. Cells are able to reach
70e90% confluence from Passage 0e1 using both protocols;
however, cells in Groups I4, I8, C4, and C8 grew significantly
faster than that in control groups, it only takes 5 days when
cells reached 70e90% confluence from P0 to P1 and from P1
to P2 compared with 9 days, and the cell numbers are also
significantly higher than those using the standard culture
protocol (pZ0.037). After the first passage, cells in about
one in four of the cell culture dishes in the control groups
(using the commonly used cell culture method) grew very
slowly or stopped growing, and they were not able to reach
70e90% confluence even after culture for 1 month. These
cells accumulated internal fatty vacuoles, and displayed
the typical large and flat senescent morphology.
Discussion
Isolation of MSCs from bone marrow is far more challenging in
mouse than other species. We compared several reported
isolation strategies and developed a new protocol for stand-
ardised, reliable and easy-to-perform isolation of mouse
MSCs from bone marrow. There are five main features of our
method: First, after flushing bone marrow out of the marrow
cavity, we cultured the cells with fat mass without filtering
them. Our experience showed that the initial phase of culture
is crucial for later yieldof cells. The number of MSCs in mouse
Figure 2 Morphological features of the cultured mouse bone marrow mesenchymal stem cells using our protocol. (A) On Day 1,
most of the cells were still mononuclear and fat droplets (arrows) were frequently seen. (B) On Day 2, some spindle-shaped cells
(arrows) appeared among the mononuclear cells and fat droplets. (C) On Day 3, the numbers of spindle-shaped cells (arrows)
continued increasing. (D) On Day 4, the spindle-shaped cells reached about 60e80% confluence. (E) On Day 5, the spindle-shaped
cells formed cell layers (the circle). (F) On Day 5, fibroblast-like cells (arrows) grew out from a dense cell nodule, and the cells were
passaged on this day. (G, H) On Day 7, cells reached 100% confluence when left without passaging; multiple cell layers and dense
cell nodules were formed in some areas of the culture dish (the circle). (I) At Passage 3, the cells have uniform fibroblast-like
morphology. Scale bar Z200 mm.
Improved isolation and culture of mouse BM-MSCs 5
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
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bone marrow is much lower than that of rat or human MSCs
[1], if we filtered the bone marrow, some MSCs attached to
the fat masses will be entrapped onto the filter, thus further
reducing the MSCs yields. Furthermore, BM-MSCs are main-
tained in their niche composing of stromal cells, extracellular
matrix elements, and secreting factors to nurture and regu-
late MSCs self-renewal and differentiation [28]. Our method
is simple, but keeping the MSCs in their initial niche with
minimal disturbanceto allow the initial adjusting time for the
MSCs in culture. Second, the culture medium is not supple-
mented with any additional growth factor. It is reported that
supplement of growth factors in culture may modify MSC
protein synthesis and intracellular trafficking, and affect MSC
proliferation and differentiation potentials [21]. Third, our
method does not need to separate the haematopoietic stem
cells, which are preferentially localised in the endosteal re-
gions of the bone [29] using flow cytometry or immuno-
magnetic sorting techniques. Our method simply use the
culture medium to select the plastic adherent MSCs. Fourth,
this method has been tested in several mouse strains (ICR,
FVB/N, CMV-Luc, C57) as well as in mice with different ages
(2e8 weeks, males and females) and the results are repro-
ducible in all tested mice. The successful isolation rate of
MSCs was not strain- or sex-dependent. Fifth, we have
Figure 3 Confirmation of mesenchymal stem cell (MSC) surface markers and differentiation capacities of mouse bone marrow
(BM)-MSCs. (A, B) Flow cytometry analysis results showed that these cells were positive for MSC markers CD44 (A) and CD90 (B). (C)
Cells were negative for endothelial cell marker CD31. (D) Cells were negative for haematopoietic cell marker CD45. (E) Alizarin red
staining demonstrated that mineralised nodules formed in the BM-MSCs after 4 weeks under the osteogenic induction. (F) Intra-
cellular Oil-red-O staining showed lipid-rich vacuole formation of the mouse BM-MSCs after 2 weeks, adipogenic induction. (G) After
3 weeks’ chondrogenic induction, the cell pellet was sectioned and stained with toluidine blue; the positive acidic proteoglycan
indicated the chondrocyte-like cell formation. Scale bar Z1 mm (F and H) and 100 mm (G).
Table 3 Comparison of cell growth rate using the two protocols among different mice.
Groups Passage 0e1 Passage 1e2
Cells (10
6
) Duration
a
Success rate (%)
b
Cells (10
6
) Duration (d)
a
Success (%)
b
I4-C 2.27 0.06 9 100 3.43 0.08 9 75
I4 2.82 0.11 5 100 4.20 0.15 5 100
I8-C 2.26 0.10 9 100 3.31 0.10 9 75
I8 2.77 0.09 5 100 4.27 0.09 5 100
C4-C 2.27 0.03 9 100 3.32 0.09 9 50
C4 2.85 0.09 5 100 4.21 0.09 5 100
C8-C 2.23 0.10 9 100 3.38 0.06 9 75
C8 2.83 0.16 5 100 4.32 0.09 5 100
a
Duration: defined as the time needed for cells to reach 70e90% confluence after passage.
b
Success rate of cell culture: defined as cells reaching 70e90% confluence after passage.
6 S. Huang et al.
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
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optimised our culture protocol, and listed detailed problems
that may arise in cell culture and trouble-shooting tricks
(Table 1). The cells established using our culture method are
mesenchymal stem cells, which are not contaminated with
haematopoietic cell lineages and endothelial cells, and are
able to differentiate into osteoblasts, adipocytes and chon-
drocytes when respectively cultured with osteogenic, adi-
pogenic, and chondrogenic medium.
The current study focuses on the development of a simple
cell culture protocol for mouse BM-MSCs. We used minimal
manipulations during the initial cell harvest (no filtering, no
enzyme digestion). Once the MSCs established in either using
our protocol or the commonly used protocol, the MSCs
remain similar phenotypes or differentiation potentials.
In conclusion, compared with the commonly used mouse
BM-MSCs culture protocol, our protocol results in a higher
success rate of MSCs isolation and establishment in culture;
the cells displayed higher growth rate and maintained the
multipotent differentiation potentials. Our protocol may be
a simple, reliable, and alternative method for culturing
MSCs from mouse bone marrow tissues.
Conflicts of interest
All contributing authors declare no conflicts of interest.
Acknowledgements
This work was supported by a grant from the Hong Kong
Government Research Grant Council, General Research Fund
(CUHK470813), and a grant from the China Shenzhen City
Science and Technology Bureau under the Shenzhen City
Knowledge Innovation Plan, Basic Research Project
(JCYJ20130401171935811) to G.L. This study was also sup-
ported in part by the SMART program, Lui Che Woo Institute of
Innovative Medicine, Faculty of Medicine, The Chinese Uni-
versity of Hong Kong. This research project was made possible
by resources donated by Lui Che Woo Foundation Limited.
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Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
bone marrow, Journal of Orthopaedic Translation (2014), http://dx.doi.org/10.1016/j.jot.2014.07.005

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+MODEL
Please cite this article in press as: Huang S, et al., An improved protocol for isolation and culture of mesenchymal stem cells from mouse
bone marrow, Journal of Orthopaedic Translation (2014), http://dx.doi.org/10.1016/j.jot.2014.07.005