Description of the technique of bone marrow harvesting in the coxal tuberosity for isolation and culture of mesenchymal stem cells of buffaloes (<i>Bubalus bubalis</i>)

doi:10.4236/scd.2014.41002

Paper Menu >>

Journal Menu >>

Vol.4, No.1, 8-12 (2014) Stem Cell Discovery

http://dx.doi.org/10.4236/scd.2014.41002

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

bution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

AB STRAC T

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 myelogram’s 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.

KEYWORDS

Buffaloes; MSCs; Coxal Tuberosity; Culture

1. INTRODUCTION

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

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. MAT ERIALS AND METHODS

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

animals.

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 myelogram’s 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® (1077—Sigma ®, USA) gradient in a laminar flux

environ me nt.

2.3. Isolation, Culture and Differentiation of

MSC

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 ® (1077—Sigma ®, 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

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 manufacturer’s 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.

3. RESULTS

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

adhesion—4 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-

fication.

fat droplets, observed by staining with Alizarin Red and

Oil Red, respectively.

4. DISCUSSION

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

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

animal.

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

researches.

ACKNO WLE DGE MENTS

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) .

REFERENCE S

[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.

http://dx.doi.org/10.3923/ajava.2011.772.788

[2] Fortier, L.A. and Travis, A.J. (2011) St em cells in v eteri-

nary medicin e. Stem Cell Research, 2, 1-6.

http://dx.doi.org/10.1186/scrt50

[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.

http://dx.doi.org/10.1038/laban1108-497.

[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.

http://dx.doi.org/10.1016/j.joms.2009.05.434

[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.

http://dx.doi.org/10.1016/j.rvsc.2011.09.007

C. N. de Moraes et al. / Stem Cell Discov ery 4 (2014) 8-12

[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.

http://dx.doi.org/10.1182/blood-2011-04-349688

[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.

http://dx.doi.org/10.1111/vcp.12036

[14] Gurevitch, O., Slavin, S. and Feldman, A.G. (2007) C on -

version of red bone marrow into yellow—Cause and me-

chani sms. Medical Hypotheses, 69, 531-536.

http://dx.doi.org/10.1016/j.mehy.2007.01.052

[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.

http://dx.doi.org/10.1002/jemt.22208