Vol.4, No.1, 8-12 (2014) Stem Cell Discovery
Copyright © 2014 SciRe s . OPEN ACCESS
Description of the technique of bone marrow
harvesting in the coxal tuberosity for isolation and
culture of mesenchymal stem cells of buffaloes
(Bubalus bubalis)
Carolina Nogueira de Moraes*, Amanda Jerôni m o Listoni, Leandro Maia,
Carla Martins de Queiroz, Flavia Caroline Destro, Eunice Oba,
Fernanda da Cruz Landim-Alvarenga
Department of Animal Reproduction and Veterinary Radiology, São Paulo State University, Botucatu, Brazil;
*Corresponding Author: carolnmoraes@ho tmail.co m
Received 14 November 2013; revised 5 December 2013; accepted 12 December 2013
Copyright © 2014 Carolina Nogueira de Moraes et a l. This is an open access art icle distribu ted under the Creative Co mmons Attri-
bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited. In accordance of the Creative Commons Attribution License all Copyrights © 2014 are reserved for SCIRP and the owner of
the intellectual property Caroli na Nogueira de Moraes et al. All Copyright © 2014 are guarded by law and by SCIRP as a guardian.
Several studies with mesenchymal stem cells
(MSCs) have been developed in many species
because of its ability to differentiate into other
mesoderm lineages, capacity of self-reg ener a-
tion, low immunogenicity, paracrine, anti -inflam-
matory, immunomodulatory and antiapoptotic
effects which make the m a promissory source to
be used in therapeutic strategies. The aim of this
study is to report the technique of harvest of
bone marr ow (BM) in the cox al tuber osity (CT) of
buffaloes and its processing and cu ltivation. For
this, after anesthetic block from the region cor-
responding to the CT, bone marrow harvesting
was performed with a myelograms needle. The
samples collected showed plastic adherence
with 96 hours a nd took approximately 32 days to
reach 80% confluence. And then differentiation
into adipogenic and osteogenic lineages was
performed. Samples showed morphological
changes during differentiation protocol, but not
all presented production of extracellular depo-
sits of calcium or intracellular fat droplets. The
anatomical site tested showed to be an alterna-
tive site of harvest of BM once provided with the
appropriate isolation and culture of the mono-
nuclear fraction. Moreover, the procedure was
performed without difficulty and with great se-
curity. Based on this, it can be concluded that CT
is an excellent anatomical site for isolation and
culture of MSCs and the proposed technique is
viable and feasible to be held in buffal o es.
Buffaloes; MSCs; Coxal Tuberosity; Culture
The ability of mesenchymal stem cells (MSCs) to dif-
ferentiate into other mesoderm lineages such as bone and
cartilage opened a variety of experimental strategies to
investigate the possibility of these cells to be used in
tissue engineering since MSCs derived from bone mar-
row (BM) have been applied in the treatment of muscu-
loskeletal disease in several species [1]. Besides the dif-
ferentiation capacity, those cells have the capacity of
self-regeneration, low immunogenicity, paracrine, an-
ti-inflammatory, immunomodulatory and antiapoptotic
effects which make these cells a great alternative to be
used in regenerative therapies.
Bone marrow-derived mesenchymal stem cells (BM-
MSCs) are the most studied source of MSCs and because
of this, they have received special attention and are best
characterized [2]. Bone marrow harvesting on coxal tu-
berosity (CT) has already been described in other species
such as horses [3,4], dog, cat [5], sheep [6], pig [7] and
humans [8 ].
In buffaloes, there are reports of studies with embryo-
nic stem cells [9], MSCs derived from adipose tissue [1]
and from amniotic fluid [10]. Based on this, this work
C. N. de Moraes et al. / Stem Cell Discov ery 4 (2014) 8-12
Copyright © 2014 SciRe s . OPEN ACCESS
aims to evaluate an alternative site of harvest of bone
marrow in the CT and the cultivation of mesenchymal
stem cells. The results o f thi s study ca n contrib ute to and
aid in the harvest of buffalo MSCs and maximize their
therapeutic use.
2.1. Animals
Six healthy adult female buffaloes aged 8 - 14 years
were selected and used. The experimental protocol (No.
160/2012- CEUA) was approved by the ethics and wel-
fare committee of the Sao Paulo State University - Botu-
catu. All procedures were performed under the interna-
tional guidelines for the care and use of experimental
2.2. BM Harvesting
Buffalo BM harvesting was performed on selected
animals properly contained in a stock. The anatomical
site of right CT was properly identified by palpation and
it was performed trichotomy of the region. After this,
antisepsis was done with chlorhexidine and alcohol
(Riohex, Rioquímica—Brazil) followed by local anes-
thetic block with 10 ml of 2% lidocaine (Cristália, Bra-
zil). Past ten minutes, a myelograms needle (Lang®
Brasil) was introduced approximately 2 cm for bone
marrowharvesting at an angle of approximately 45 de-
grees relative to the animal in dorsoventral and crani-
ocaudal direction (Figure 1). Once the needle was firmly
fixed in the CT , the mand ril wa s re moved and proc eeded
BM aspiration (Figure 2) with a syringe of 20 ml prop-
erly identified containing 1 ml of heparin at 1000 IU/ml
(Hemofol®, Cristália, BRA) and 1 ml of PBS phos-
phate -saline, pH = 7.2 (PBS® 1x, LGC Biotechnology,
BRA). After BM harvesting, each sample collected was
manua ll y homo ge nized, identified, cooled and referred to
the laboratory for isolation, cultivation and characteriza-
tion from mononuclear fraction obtained from Histopa-
que® (1077Sigma ®, USA) gradient in a laminar flux
environ me nt.
2.3. Isolation, Culture and Differentiation of
After collection, the samples were filtered and centri-
fuged at 340 g for ten minutes. The supernatant was re-
moved and culture medium composed by DMEM low-
glucose (Invi trogen®, USA) was added .
Mononuclear fraction (MF) was isolated in a Histo-
paque ® (1077Sigma ®, USA) gradient (Figure 3),
trough 40 minutes centrifugation at 340 g. The MF was
washed twice and cultured in DMEM low glucose/F12
(1:1), supplemented with 10% fetal bovine serum,
Figure 1. Myelogram’s needle position for bone mar-
row harvesting on CT in buffaloes.
Figure 2. Bone marrow’s harvest on CT with the use
of a syringe of 20 ml.
Figure 3. Mononuclear fraction formed after centrifu-
gation on Histopaque® gradient.
C. N. de Moraes et al. / Stem Cell Discov ery 4 (2014) 8-12
Copyright © 2014 SciRe s . OPEN A CCESS
antibiotics (1%) and antimycotics (1.2%) (Invitrogen®,
USA) in humidified atmosphere containing 5% CO2.
Maintenance medium was changed at each two or three
days and when the cells reached 80% confluence, first
passage was performed by ressuspending the cells with
TrypLE Express® (Invitr o g e n®, USA, cat. number:
12604021), and transfer to 6-wells plates (Sarstedt®,
USA) for differentiation assay into osteogenic and adi-
pogenic lineage.
The differentiation protocol took about 10 days using
media and supplements prepared from the Adipogenesis
Differentiation Kit (Invitrogen®, USA, cat. number:
A10070-01) and the STEMPRO® Chondrogenesis media
and supplements prepared from the Adipogenesis.
Differentiation Kit (Invitrogen®, USA, cat. number:
A10070-01) and the STEMPRO® Chondrogenesis Dif-
ferentiation Kit (Invitrogen®, USA, cat. number:
A10071-01) according to manufacturers recommenda-
tion. The medium of differentiation was changed every
two days. At the end of differentiation period the mor-
phological changes of cells were analyzed and samples
were stained with 2% Alizarin Red (Sigma®, USA, pH =
4.2) and 0,5% Oil Red (Sigma®, US), for t he o bse rvati o n
of the deposit of calcium in the extracellular matrix and
the presence of intracytoplasmic lipids droplets, respec-
tivel y.
BM harvesting on CT was easily conduced and it was
not found any difficult or problem during or after the
collection. Furthermore, the needle, anesthetic protocol,
doses and MSCs processing was considerate appropriate
for the species under study.
From each animal it was collected on average 3 ml of
BM and this volume was sufficient for the processing of
the sample from these animals, promoting correct isola-
tion, culture and characterization of the cells. In one an-
imal, with advanced age, the volume collected was lower
(1 ml) and the culture failed. The process of cooling the
material during transport apparently did not affected the
quality of the samples once all samples were submitted
to culture.
The adhesion time for these cells was four days and
the period between the adhesion and 80% confluence
was on average 32 days (Figure 4). Morphological
changes in cells in culture were observed during the dif-
ferentiation s period (Figure 5). Of all samples collected,
the culture of only one animal (the older) failed. So of
the five sa mples, fo ur (80%) were able to d ifferentiate in
osteogenic lineage and two (40%) were able to differen-
tiate in the adipogenic lineage. All samples showed
morphological changes, but some presented no produc-
tion of extracellular deposits of calcium or intracellular
Figure 4. Buffalo MSCs during culture. A: beginning of cell
adhesion4 days; B: cell culture after 10 days; C: cell culture
after 25 days. D: cell culture after 50 days. 2 0× magnification.
Figure 5. Differentiation assay for osteogenic and adipogenic
lineages. A: beginning of osteogenic differentiation; B: red
drops represented the extracellular deposit of calcium stained
with Alizarin Red; C: beginning of adipogenic differentiation;
D: intracellular lipid droplets stained with Oil Red. 20× magni-
fat droplets, observed by staining with Alizarin Red and
Oil Red, respectively.
The collection of BM in the CT of buffalo is consi-
dered safe both for the veterinarian and for the animal.
Once held the correct conte nt i o n, fact was also reported
in hor s es [3].
Despite the possibility of obtaining MSCs from other
sources such as adipose tissue [1], umbilical cord blood
[8], peripheral blood [11], amniotic fluid [10], BM is
consi d ered the one that has the most abundant source of
MSCs [8].
C. N. de Moraes et al. / Stem Cell Discov ery 4 (2014) 8-12
Copyright © 2014 SciRe s . OPEN ACCESS
For BM harvesting in the CT of buffaloes, there is no
need for general anesthesia, once a simple anesthetic
block already allows the development of the technique.
Such a fact makes the technique advantageous since ad-
verse effects have been reported in human patients un-
dergoing general anesthesia such as pain at the site of
collection or even anemia [11]. Besides, this harve st can
be done with the live a ni mal, so there is no need to make
a surgical wo u n d . And the cells can be used in the same
In this experiment, the volume collected from all ani-
mals was considerate properly. However, in other species,
the volume collected was greater with data of 60 ml in
pigs [12], 10 ml in humans [8], 11 ml in horses [3] or 1
ml in do gs [13]. As already reported, the best sample for
isola tion and e xpansio n of MSCs i s obtai ned in the ea rly
stages of bone marrow aspiration. Furthermore, it is
known that the volume of material obtained decreases
along the samp les [8].
In one ani mal fro m t his s tud y, i t wa s ob serve d t hat the
volume material collected was lower (1 ml) and yello-
wish. This can be explained by the advanced age of this
animal in or der that the he matopoie tic tissue is graduall y
replaced by non-hematopoietic mesenchymal cells, also
called fatty bone marrow [14].
Compared with samples from other species processed
in the same laboratory, a longer primary culture time was
obs er ved d ur ing cultur e. Fo r exa mpl e, i n d o gs the ti me to
reach confluence is on average 10 days (unpublished
data) and in horses is on average 22 days [15].
With respect to differentiation, heterogeneity in re-
sponse to differentiation after induction was noted. Per-
haps this response is linked with an inadequacy of the
differentiation media used. However, it is important to
remember that intrinsic difference among the samples
canno t be ruled out. Since the population of mesenchym-
al stem cells contained in bone marrow environment is
heter o geneo us , it is committed to diverse differentiation
pro ce sses.
With this protocol proposed, it was evident that the
antisepsis, local block and site of harvest were efficient
on buffaloes and can be used in this species for bone
marrow harvesting from CT. The anatomical site tested
showed to be an alternative site of harvest of BM once
provided with appropriate isolation, culture and charac-
terization of mononuclear fraction. Besides, the proce-
dure was performed without difficulty and with great
security. Although the osteogenic potential was evident,
the adipogenic differentiation needs to be improved.
Based on the above, it can be concluded that CT is an
excellent anatomical site for isolation and culture of
MSCs and that the proposed technique is viable and
feasible to be held in buffaloe s and can b e used in futur e
The authors ensure that there are no conflicts of interest (including
specif i c fi nanc ial in terest and re l ation s hi ps an d aff il iatio n) .
[1] Hepsibha, P., Meenambigai , T.V., Mangalago wri, A., Pala-
nisamy, A., Stalin, A., Nithya, S. and Humanan, K. (2011)
Multipotent differentiation potential of buffalo adipose
tissue derived mesenchymal stem cells. Asian Journal of
Animal and Ve ter inary Adva nc es , 6, 772-788.
[2] Fortier, L.A. and Travis, A.J. (2011) St em cells in v eteri-
nary medicin e. Stem Cell Research, 2, 1-6.
[3] Delling, U., Lindner, K., Ribitsch, I., Jülke, H. and Brehm,
W. ( 2012) Comparison of the bone marrow aspiration at
the sternum and the tuber coxae in middle-aged horses.
Canadian Jour n al of Veterinary R esearch, 76, 52-56 .
[4] Maia, L., Venturini, R.M., Taffarel, M.O., Freitas, N.P.P.,
Monteiro, G.A., de Vita, B., Monteiro, B.A., Landim-
Alvarenga, F.C. and Amorim, R.M. (2011) Técnica de
colheita da medula óssea na tuberosidade coxal de eqüi-
nos para isolamento e cultivo de células tronco mesen-
quimais. Procedings of XII Conferência Anual da
ABRAVEQ, Campinas , 117.
[5] Townsend, F.I. (2008) Bone marrow aspiration in dogs
and cat s. Labor atory A n i m als , 37, 497-498.
[6] Amorim, R.M., Nascimento, G.D., Maia, L., Listoni, A.J.,
Paola, B.S., Ferreira, D.L.O., Cavalcanti, R.M. and Landim-
Alvarenga, F.C. (2012) Bone marrow mesenchymal stem
cells from sheep. Proceedings of the XXVII World Buia-
trics Congress, Lisbon, 3 J une 2012, 323.
[7] Abukawa, H., Phelps, M., Jackson, P., Smith, R.M., Va-
canti, J.P., Kaban, L.B. and Troulis, M.J. (2009) Effect of
ibuprofen on osteoblast differentiation of porcine bone
marrow-derived progenitor cells. Journal of Oral and
Maxi ll ofac ial Surgery, 67, 2 412-2417.
[8] Li, J., Wong, W.H.S., Chan, S., Chim, J.C.S., Cheung,
K.M.C., Lee, T.L., Au, W.Y., Ha, S.Y., Lie, A.K.W., Lau,
Y.L., Liang, R.H.S. and Chan G.C.F. (2011) Factors af-
fecting mesenchymal stromal cells yield from bone mar-
row aspiration. Chinese Journal of Cancer Research, 23,
43-48. http://dx.doi.org/10.1007/s11670-011-0043-1
[9] Verma, V., Gautam, S.K., Singh, B., Manik, R.S., Palta, P.,
Single, S.K., Goswami, S.L. and Chauhan, M.S. (2007)
Isolat ion and characterizat ion of embryonic stem cell-like
cells from in vitro-produced buffalo (Bubalus bubalis)
embryos. Molecular Reproduction and Development, 74 ,
520-529. http://dx.doi.org/10.1002/mrd.20645
[10] Dev, K., Gautam, S.K., Giri, S.K., Kumar, A., Yadav, A.,
Verma, V., Kumar, P. and Singh, B. (2012) Isolation, cul-
turing and characterization of feeder-independent amni-
otic fluid stem cells in buffalo (Bubalus bubalis). Re-
search of Veterinary Science, 93, 743-748.
C. N. de Moraes et al. / Stem Cell Discov ery 4 (2014) 8-12
Copyright © 2014 SciRe s . OPEN A CCESS
[11] Styczynski, J., Balduzzi, A., Gil, L., Labopin, M., Ham-
ladji, R.M., Marktel, S., Yesilipek, M.A., Fagioli, F., Eh-
lert, K., Matulova, M., Dalle, J.H., Wachowiak, J., Miano,
M., Messina, C., Diaz, M.A., Vermylen, C., Eyrich, M.,
Badell, I., Dreger, P., Gozdzik, J., Hutt, D., Rascon, J.,
Dini, G. and Peters, C. (2012) Risk of complications dur-
ing hematopoietic stem cell collection in pediatric sibling
donors: A prospective European group for blood and
marrow transplantation pediatric diseases working party
study. Blo od , 19, 2935-2941.
[12] Branco, E., Cabral, R., Gomes, B.D., Kfoury Jr., J.R. and
Miglino, M.A. (2012) Bone marrow cells of swine: Col-
lection and separation. Microscopy Research and Tech-
nique, 7, 5917-920. http://dx.doi.org/10.1002/jemt.22013
[13] Defarges, A., Abrams-Ogg, A., Foster, R.A. and Bienzle,
D. (2013) Comparision of sterna, iliac, and humeral bone
marrow aspiration in Beagle dogs. Veterinary Clinical
Pathology, 42, 170 -176.
[14] Gurevitch, O., Slavin, S. and Feldman, A.G. (2007) C on -
version of red bone marrow into yellowCause and me-
chani sms. Medical Hypotheses, 69, 531-536.
[15] Mai a, L., Landim-Alvarenga, F.C., Mota, L.S.L.S., Golim,
M.A., Laufer-Amorim. R., de Vita, B., Barberini, D.J.,
Listoni, A.J., Moraes, C.N., Heckler, M.C.T. and Amorim,
R.M. (2013) Immunophenotypic, immunocytochemistry,
ultrastructural, and cytogenetic characterization of me-
senchymal stem cells from equine bone marrow. M icro-
scopy Research and Technique, 76, 618-624.