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 Jerônimo Listoni, Leandro Maia, Carla Martins de Queiroz, Flavia Caroline Destro, Eunice Oba, Fernanda da Cruz Landim-Alvarenga

Department of Animal Reproduction and Veterinary Radiology, Sao Paulo State University, Botucatu, Brazil.

DOI: 10.4236/scd.2014.41002                

Abstract

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-regeneration, low immunogenicity, paracrine, anti-inflammatory, immunomodulatory and antiapoptotic effects which make them a promissory source to be used in therapeutic strategies. The aim of this study is to report the technique of harvest of bone marrow (BM) in the coxal tuberosity (CT) of buffaloes and its processing and cultivation. For this, after anesthetic block from the region corresponding to the CT, bone marrow harvesting was performed with a myelogram’s needle. The samples collected showed plastic adherence with 96 hours and 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 deposits of calcium or intracellular fat droplets. The anatomical site tested showed to be an alternative site of harvest of BM once provided with the appropriate isolation and culture of the mononuclear fraction. Moreover, the procedure was performed without difficulty and with great security. 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 buffaloes.

Keywords

Buffaloes; MSCs; Coxal Tuberosity; Culture

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Hepsibha, P., Meenambigai, T.V., Mangalagowri, A., Palanisamy, 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 Veterinary Advances, 6, 772-788. http://dx.doi.org/10.3923/ajava.2011.772.788
[2]	Fortier, L.A. and Travis, A.J. (2011) Stem cells in veterinary medicine. 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 Journal of Veterinary Research, 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 eqüinos para isolamento e cultivo de células tronco mesenquimais. Procedings of XII Conferência Anual da ABRAVEQ, Campinas, 117.
[5]	Townsend, F.I. (2008) Bone marrow aspiration in dogs and cats. Laboratory Animals, 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 Buiatrics Congress, Lisbon, 3 June 2012, 323.
[7]	Abukawa, H., Phelps, M., Jackson, P., Smith, R.M., Vacanti, 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 Maxillofacial Surgery, 67, 2412-2417. 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 affecting mesenchymal stromal cells yield from bone marrow 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) Isolation and characterization 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, culturing and characterization of feeder-independent amniotic fluid stem cells in buffalo (Bubalus bubalis). Research of Veterinary Science, 93, 743-748. http://dx.doi.org/10.1016/j.rvsc. 2011.09.007
[11]	Styczynski, J., Balduzzi, A., Gil, L., Labopin, M., Hamladji, R.M., Marktel, S., Yesilipek, M.A., Fagioli, F., Ehlert, 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 during hematopoietic stem cell collection in pediatric sibling donors: A prospective European group for blood and marrow transplantation pediatric diseases working party study. Blood, 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: Collection and separation. Microscopy Research and Technique, 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) Conversion of red bone marrow into yellow—Cause and mechanisms. Medical Hypotheses, 69, 531-536. http://dx.doi.org/10.1016/j.mehy.2007.01.052
[15]	Maia, 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 mesenchymal stem cells from equine bone marrow. Microscopy Research and Technique, 76, 618-624. http://dx.doi.org/ 10.1002/jemt.22208