Abstract
Mechanical stress caused by agitation is one of the factors that can affect hematopoietic stem cell expansion in suspension bioreactors. Therefore, we have investigated the effects of agitation on umbilical cord blood hematopoietic stem cell (UCB-HSC) growth and differentiation. A comparison was made between various agitation rates (20, 40 and 60 rpm) in spinner-flask and cells cultured in glass petri dish as a static culture. Moreover, the fluid dynamic at various agitation rates of spinner-flask was analyzed to determine shear stress. The spinner-flask contained a rotational moving mixer with glass ball and was kept in tissue culture incubator. To reduce consumption of cytokines, UCB-serum was used which widely decreased the costs. Our results determined that, agitation rate at 40 rpm promoted UCB-HSCs expansion and their colony forming potential. Myeloid progenitors were the main type of cells at 40 rpm agitation rate. The results of glucose consumption and lactic acid production were in complete agreement with colony assay and expansion data and indicated the superiority of culture in spinner-flask when agitated at 40 rpm over to other agitation speeds and also static culture. Cell viability and colony count was affected by changing the agitation speed. We assume that changes in cell growth resulted from the effect of shear stress directly on cell viability, and indirectly on signaling pathways that influence the cells to differentiate.
Similar content being viewed by others
References
Almici C, Carlo-Stella C, Wagner JE, Rizzoli V (1995) Umbilical cord blood as a source of hematopoietic stem cells: from research to clinical application. Haematologica 80:473–479
Andrade-Zaldívar H, Santos L, Rodríguez ADL (2008) Expansion of human hematopoietic stem cells for transplantation: trends and perspectives. Cytotechnology 56:151–160
Boehm D, Murphy WG, Al-Rubeai M (2010) The effect of mild agitation on in vitro erythroid development. J Immunol Methods 60:20–29
Brindley D, Moorthy K, Lee JH, Mason C, Kim HW, Wall I (2011) Bioprocess forces and their impact on cell behavior: implications for bone regeneration therapy. J Tissue Eng 2011:1–13
Broxmeyer HE, Srour E, Orschell C, Ingram DA, Cooper S, Plett PA, Mead LE, Yoder MC (2006) Cord blood stem and progenitor cells. Methods Enzymol 419:439–473
Cabral JMS (2001) Ex vivo expansion of hematopoietic stem cells in bioreactors. Biotechnol Lett 23:741–751
Cabrita GJM, Ferreira BS, da Silva CL, Gonçalves R, Almeida-Porada G, Cabral JMS (2003) Hematopoietic stem cells: from the bone to the bioreactor. Trends Biotechnol 21:233–240
Chisti Y (2000) Animal-cell damage in sparged bioreactors. Trends Biotechnol 18:420–432
Choi YS, Noh SE, Lim SM, Kim DI (2010) Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures. Biotechnol Lett 32:1969–1975
Collins PC, Miller WM, Papoutsakis ET (1998a) Stirred culture of peripheral and cord blood hematopoietic cells offers advantages over traditional static systems for clinically relevant applications. Biotechnol Bioeng 59:534–543
Collins PC, Nielsen LK, Patel SD, Papoutsakis ET, Miller WM (1998b) Characterization of hematopoietic cell expansion, oxygen uptake, and glycolysis in a controlled, stirred-tank bioreactor system. Biotechnol Prog 14:466–472
De Léon A, Mayani H, Ramírez OT (1998) Design, characterization and application of a minibioreactor for the culture of human hematopoietic cells under controlled conditions. Cytotechnology 28:127–138
Du Z, Jin H, Cai H, Yang S, Tan WS (2014) Hematopoietic repopulating ability of CD34+ progenitor cells ex vivo expanded with different cytokine combinations. Artif Cells Nanomed Biotechnol. [Epub ahead of print]
Gilbertson JA, Sen A, Behie LA, Kallos MS (2006) Scaled-up production of mammalian neural precursor cell aggregates in computer-controlled suspension bioreactors. Biotechnol Bioeng 94:783–792
Gluckman E, Broxmeyer HA, Auerbach AD, Friedman HS, Douglas GW, Devergie A, Esperou H, Thierry D, Socie G, Lehn P (1989) Hematopoietic Reconstitution in a patient with Fanconi’s anemia by means of umbilical-cord blood from an HLA-identical sibling. N Engl J Med 321:1174–1178
Gregoriades N, Clay J, Ma N, Koelling K, Chalmers JJ (2000) Cell damage of microcarrier cultures as a function of local energy dissipation created by a rapid extensional flow. Biotechnol Bioeng 69:171–182
Hunt MM, Meng G, Rancourt DE, Gates ID, Kallos MS (2014) Factorial experimental design for the culture of human embryonic stem cells as aggregates in stirred suspension bioreactors reveals the potential for interaction effects between bioprocess parameters. Tissue Eng Part C Methods 20:76–89
Jing Q, Cai H, Du Z, Ye Z, Tan WS (2013) Effects of agitation speed on the ex vivo expansion of cord blood hematopoietic stem/progenitor cells in stirred suspension culture. Artif Cells Nanomed Biotechnol 41:98–102
Kowalczyk M, Waldron K, Kresnowati P, Danquah M (2011) Process challenges relating to hematopoietic stem cell cultivation in bioreactors. J Ind Microbiol Biotechnol 38:761–767
Kwon J, Kim B-S, Kim M-J, Park H-W (2003) Suspension culture of hematopoietic stem cells in stirred bioreactors. Biotechnol Lett 25:179–182
Liu Y, Liu T, Fan X, Ma X, Cui Z (2006) Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel. J Biotechnol 124:592–601
Luni C, Zagallo M, Albania L, Piccoli M, Pozzobon M, De Coppi P, Elvassore N (2011) Design of a stirred multiwell bioreactor for expansion of CD34+ umbilical cord blood cells in hypoxic conditions. Biotechnol Prog 27:1154–1162
Ma CYJ, Kumar R, Xu XY, Mantalaris A (2007) A combined fluid dynamics, mass transport and cell growth model for a three-dimensional perfused bioreactor for tissue engineering of haematopoietic cells. Biochem Eng J 35:1–11
Mardikar SH, Niranjan K (2000) Observations on the shear damage to different animal cells in a concentric cylinder viscometer. Biotechnol Bioeng 68:697–704
Meissner P, Schröder B, Herfurth C, Biselli M (1999) Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells. Cytotechnology 30:227–234
Nielson LK (1999) Bioreactors for hematopoietic cell culture. Annu Rev Biomed Eng 1:129–152
O’Connor KC, Papoutsakis ET (1992) Agitation effects on microcarrier and suspension CHO cells. Biotechnol Tech 6:323–328
Palsson BO, Paek S-H, Schwartz RM, Palsson M, Lee G-M, Silver S, Emerson SG (1993) Expansion of human bone marrow progenitor cells in a high cell density continuous perfusion system. Nat Biotech 11:368–372
Papoutsakis ET (1991) Fluid-mechanical damage of animal cells in bioreactors. Trends Biotechnol 9:427–437
Ratcliffe E, Glen KE, Workman VL, Stacey AJ, Thomas RJ (2012) A novel automated bioreactor for scalable process optimisation of haematopoietic stem cell culture. J Biotechnol 161:387–390
Rodrigues CAV, Fernandes TG, Diogo MM, Silva CLD, Cabral JMS (2011) Stem cell cultivation in bioreactors. Biotechnol Adv 29:815–829
Scadden DT (2006) The stem-cell niche as an entity of action. Nature 441:1075–1079
Shayan N, Ebrahimi M, Beiki B, Janzamin E (2012) A non-rotational, computer-controlled suspension bioreactor for expansion of umbilical cord blood mononuclear cells. Biotechnol Lett, 1–7
Sucosky P, Osorio DF, Brown JB, Neitzel GP (2004) Fluid mechanics of a spinner-flask bioreactor. Biotechnol Bioeng 85:34–46
Yang S, Cai H, Jin H, Tan W-S (2008) Hematopoietic reconstitution of CD34<sup>+</sup> cells grown in static and stirred culture systems in NOD/SCID mice. Biotechnol Lett 30:61–65
Yao C-L, Liu C-H, Chu IM, Hsieh T-B, Hwang S-M (2003) Factorial designs combined with the steepest ascent method to optimize serum-free media for ex vivo expansion of human hematopoietic progenitor cells. Enzyme Microbial Technol 33:343–352
Zandstra PW, Eaves CJ, Piret JM (1994) Expansion of hematopoietic progenitor cell populations in stirred suspension bioreactors of normal human bone marrow cells. Nat Biotechnol 12:909–914
Acknowledgments
The authors thank Mr. Fazel S. Sahraneshin for his excellent technical assistance and helpful advices at flow cytometry data. This study was funded by a grant provided from Royan Institute.
Conflict of interest
The authors declare that they have no competing interests.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Hosseinizand, H., Ebrahimi, M. & Abdekhodaie, M.J. Agitation increases expansion of cord blood hematopoietic cells and promotes their differentiation into myeloid lineage. Cytotechnology 68, 969–978 (2016). https://doi.org/10.1007/s10616-015-9851-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10616-015-9851-3