Artículos de investigación

 

Expansion of CD34⁺ human hematopoietic cells from umbilical cord blood using roller bottles

 

Expansión de células CD34⁺ de sangre de cordón umbilical en frascos giratorios

 

H. Andrade-Zaldívar^1,3, M. A. Kalixto-Sánchez², A. P. Barba de la Rosa¹ and A. De León-Rodríguez¹*

 

¹ Instituto Potosino de Investigación Científica y Tecnológica (IPICyT), División de Biología Molecular, Camino a la Presa San José, 2055 Col. Lomas 4a sección CP 78216. San Luis Potosí, S.L.P., México. *Corresponding author. E-mail: aleonr@ipicyt.edu.mx Tel. +52-444-8342044, Fax +52-444-8342010.

² Hospital General del ISSSTE, Carlos Diez Gutiérrez 915 Col. Julian Carrillo CP 78340. San Luis Potosí, S.L.P., México.

³ Centro de Biociencias de la Universidad Autónoma de San Luis Potosí, Km 14.5 Carretera S.L.P.-Matehuala, Ejido Palma de la Cruz, Soledad de Graciano Sánchez, S.L.P. México.

 

Received June 11, 2013.
Accepted February 24, 2014.

 

Abstract

Hematopoietic stem cell (HSC) transplantation is limited by the initial CD34⁺ cell content in cord blood units; the aim of this work was the in vitro expansion of HSC to overcome this issue. Supplemented IMDM roller bottle cultures of CD34⁺ cells from human umbilical cord blood were established for hematopoietic progenitor expansion. The maximum total colony forming cells (CFC) expansion was achieved after 5 days of culture, being 16.94±2.82 folds in roller bottles and 17.28±4.47 in static cultures. However, the maximum total cell fold expansion was attained after 10 days of culture. It was of 20.31±6.18 for the roller bottles and 26.45±9.89 for the static cultures. The efficacy of the roller bottles system for short-term cultures of CD34⁺ cells and expanding hematopoietic progenitors in IMDM was demonstrated; encouraging the use of this culture system for other experiments and may be used for future clinical applications.

Keywords: CD34⁺, expansion, hematopoietic stem cell, bone marrow transplantation, cell culture, roller bottles.

 

Resumen

El transplante de células madre hematopoyéticas está limitado por la cantidad de células CD34⁺ presentes en las unidades de sangre de cordón umbilical; el objetivo de este trabajo fue obtener la expansión in vitro de células madre hematopoyéticas. Se establecieron cultivos de células CD34⁺ de sangre de cordón umbilical en medio IMDM suplementado con citocinas para promover la expansión de progenitores hematopoyéticos. En 5 días de cultivo en frascos giratorios se obtuvo la máxima expansión de unidades formadoras de colonias (UFC) totales de 16.94±2.82 veces, mientras que en cultivo estático fue de 17.28±4.47. La máxima expansión de células totales fue de 20.31±6.18 veces en frasco giratorio y de 26.45±9.89 veces en cultivo estático a los 10 días de cultivo. Se demostró la eficacia del sistema de frascos giratorios con medio IMDM para el cultivo a corto plazo de células enriquecidas CD34⁺ provenientes de sangre de cordón umbilical y para la expansión de progenitores hematopoyéticos, potencializando el uso de este sistema para otros experimentos y aplicaciones clínicas a futuro.

Palabras clave: CD34⁺, expansión, células madre hematopoyéticas, trasplantes de médula ósea, cultivo celular, frascos giratorios.

 

DESCARGAR ARTÍCULO EN FORMATO PDF

 

Acknowledgments

This work was partially supported by CONACYT-Fondos Sectoriales Sector Salud Grant 13993. Dr. Antonio De León Rodríguez thanks the Marcos Moshinky Cathedra 2012 and Hera Andrade thanks the Consejo Nacional de Ciencia y Tecnologia (CONACyT) for the PhD scholarship 103403.

 

References

Adamo, L., Naveiras, O., Wenzel, P.L., McKinney-Freeman, S., Mack, P.J., Gracia-Sancho, J., Suchy-Dicey, A., Yoshimoto, M., Lensch, M.W., Yoder, M.C., García-Cardena, G., Daley, G.Q. (2009). Biomechanical forces promote embryonic haematopoiesis. Nature 459, 1131-1135.         [ Links ]

Andrade-Zaldívar, H., Santos, L. and De León Rodríguez, A. (2008). Expansion of human hematopoietic stem cells for transplantation: trends and perspectives. Cytotechnology 56, 151-60. doi: 10.1007/s10616-008-9144-1.         [ Links ]

Andrade-Zaldívar, H., Kalixto-Sánchez, M.A., Barba de la Rosa, A.P. and De León-Rodríguez, A. (2011). Expansion of Human Hematopoietic Cells from Umbilical Cord Blood Using Roller Bottles in CO(2) and CO(2)-Free Atmosphere. Stem Cells and Development 20, 593-598.         [ Links ]

Berson, R.E. and Friederichs, G. (2008). A self-feeding roller bottle for continuous cell culture. Biotechnology Progress 24, 154-157.         [ Links ]

Bortin M.M. (1970). A compendium of reported human bone marrow transplants. Transplantation 9, 571-87.         [ Links ]

Broxmeyer, H.E., Cord blood hematopoietic stem cell transplantation (2010). StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.52.1, http://www.stembook.org.         [ Links ]

Capmany, G., Querol, S., Cancelas, J.A. and Garcia, J. (1999). Short-term, serum-free, static culture of cord blood-derived CD34+ cells: effects of FLT3-L and MIP-1alpha on in vitro expansion of hematopoietic progenitor cells. Haematologica 84, 675-682.         [ Links ]

Case, J., Rice, A. and Vowels, M. (1997). Cytokine mediated expansion of human umbilical cord blood CD34⁺ cells: comparison of the use of partially purified and pure CD34⁺ target cells. Hematology and Cell Therapy 39, 193-197.         [ Links ]

De León, A., Mayani, H. and Ramirez, O. (1998) Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions. Cytotechnology 1-3,127-138        [ Links ]

Espinosa-Ayala, G.E., Rivas-Morales, C., Arévalo-Niño, K., Oranday-Cardenas, A., Cruz-Vega, D.E., Castro-Garza, J. and Carranza-Rosales, P. (2007). Diseño de un medio de cultivo para células de mamífero utilizando fuentes alternativas de nitrógeno y vitaminas. Revista Mexicana de Ingeniería Química 6, 243-249.         [ Links ]

Fietz, T., Berdel, W.E., Rieder, H., Reufi, B., Hopp, H., Thiel, E. and Knauf, W.U. (1999). Culturing human umbilical cord blood: a comparison of mononuclear vs CD34⁺ selected cells. Bone Marrow Transplant 23, 1109-1115.         [ Links ]

Flores-Guzman. P., Gutierrez-Rodriguez, M. and Mayani, H. (2002). in vitro proliferation, expansion, and differentiation of a CD34⁺ cell-enriched hematopoietic cell population from human umbilical cord blood in response to recombinant cytokines. Archives of Medical Research 33, 107-114.         [ Links ]

Gluckman, E., Broxmeyer, H.A., Auerbach, A.D. Friedman, H.S., Douglas, G.W., Devergie, A., Esperou, H., Thierry, D., Socie, G., Lehn, P., Cooper, S., English, D., Kutzberg, J., Bard, J. and Boyse, E.A. (1989). Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling. The New England Journal of Medicine 321, 1174-1178.         [ Links ]

Gordon, M.Y., Lewis, J.L. and Marley, S.B. (2002). Of mice and men...and elephants. Blood 100, 4679-80.         [ Links ]

Koller, M.R. and Palsson, B.O. (1993). Review: tissue engineering: reconstitution of human hematopoiesis ex vivo. Biotechnology Bioengineering 42, 909-930.         [ Links ]

Liu, Y., Liu, T., Fan, X., Ma, X. and Cui, Z. (2006). Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel. Journal of Biotechnology 124, 592-601.         [ Links ]

McAdams, T.A., Winter, J.N., Miller, W.M. and Papoutsakis, E.T. (1996). Hematopoietic cell culture therapies (Part II): Clinical aspects and applications. Trends in Biotechnology 14, 388-396.         [ Links ]

McKenna, D. and Sheth, J. (2011). Umbilical cord blood: current status & promise for the future. Indian Journal of Medical Research 134, 261-9.         [ Links ]

Nimgaonkar, M.T., Roscoe, R.A., Persichetti, J., Rybka, W.B., Winkelstein, A. and Ball, E.D. (1995). A unique population of CD34⁺ cells in cord blood. Stem Cells 13, 158-166.         [ Links ]

Piacibello, W., Sanavio, F., Garetto, L., Severino, A., Dane, A., Gammaitoni and L., Aglietta, M. (1998). Differential growth factor requirement of primitive cord blood hematopoietic stem cell for self-renewal and amplification vs proliferation and differentiation. Leukemia 12, 718-727.         [ Links ]

Querol, S. (2003). Ex vivo production of cord blood CD34⁺ derived myeloid precursors after serum-free static culture. Methods in Molecular Biology 215, 351-362.         [ Links ]

Rubenstein, P., Carrier, C., Scaradavou, A., Kurtzberg, J., Adamson, J., Migliaccio, A.R., Berkowitz, R.L., Cabbad, M., Dobrila, N.L., Taylor, P.E., Rosenfield, R.E. and Stevens, C.E. (1998) Outcomes among 562 recipients of placental-blood transplants from unrelated donors. The New England Journal of Medicine 339, 1565-77.         [ Links ]

Takizawa, H., Schanz, U., Manz, M.G. (2011). Ex vivo expansion of hematopoietic stem cells: mission accomplished? Swiss Medical Weekly 2011 141, w13316. doi: 10.4414/smw.2011.13316.         [ Links ]

Unsworth, B.R., Lelkes, P.I. (1998). Growing tissues in microgravity. Nature Medicine 4, 901-907.         [ Links ]

Van Haute, I., Lootens, N., De Smet, S., De Buck, C., Verdegem, L., Vanheusden, K., Pinxteren, J. and Vandekerckhove, B. (2004). Viable CD34⁺ stem cell content of a cord blood graft: which measurement performed before transplantation is most representative? Transfusion 44, 547-554.         [ Links ]