Research paper The effect of mild agitation on in vitro erythroid development Daniela Boehm a , William G. Murphy b , Mohamed Al-Rubeai a, ⁎ a School of Chemical and Bioprocess Engineering, and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland b Irish Blood Transfusion Service, Dublin, Ireland and School of Medicine and Medical Science, University College Dublin, Ireland article info abstract Article history: Received 26 February 2010 Received in revised form 21 May 2010 Accepted 25 May 2010 Available online 10 June 2010 The cultivation of erythroid cells at large scale would have to be performed in suitable bioreactors which would most likely employ some mode of agitation to ensure optimal mass and gas transfer and prevent culture inhomogeneity. The effect of low agitation at 15–20 rpm on ex vivo erythropoiesis of PB CD34+ derived cultures was investigated and found to have significant impact on erythroid development. Agitated cultures showed a reduced lag phase and increased cell expansion during the early stages of culture. Additionally, agitation accelerated erythroid differentiation as seen by the loss of early development markers, acquisition of late erythroid markers and premature cell cycle arrest, although not yielding higher fractions of terminally differentiated cells in comparison to stationary culture. However, agitation at 20 rpm led to significantly increased loss of cell viability after day 15 in culture, an effect that could be reduced by decreasing the agitation rate to 15 rpm. On the one hand these results imply that agitation may improve cell yields and reduce expensive cytokine-dependent early culture stages but on the other hand it might introduce the risk of increased cell death in large scale culture. © 2010 Published by Elsevier B.V. Keywords: Ex vivo erythropoiesis Agitation Erythroid differentiation Apoptosis 1. Introduction While haematopoietic cells are still most commonly expanded in static culture systems such as well-plates, T-flasks or gas-permeable culture bags, particularly when co-cultured with stromal cells (Cabrita et al., 2003; Collins et al., 1998a; Sardonini and Wu, 1993; Yang et al., 2008), these systems have serious limitations due to their non-homogenous nature in terms of concentration gradients of pH, dissolved gas, nutrients, cytokines and metabolites (Collins et al., 1996, 1998a,b). Further draw-backs of static systems for large-scale expansion applications lie in lower process reproducibility, reduced possibilities of on-line monitoring and control, and the limitations of available surface area which can restrict produc- tivity, and eventually limit the possibility of developing a suitable process that can be approved by FDA (Cabrita et al., 2003; Collins et al., 1998a). Studies have been performed on the use of different types of bioreactors for the expansion of haematopoietic stem cells (HSCs), the main types being stirred tank, hollow fibre perfusion, rotating and packed bed reactors (Cabrita et al., 2003; Nielsen, 1999). Hydrodynamic forces present in agitated bioreactors are known to affect growth, viability, metabolism, cell cycle, cell size and surface marker expression (Al-Rubeai et al., 1993, 1995a,b; Lakhotia et al., 1992, 1993; McDowell and Papoutsakis, 1998). While hydrodynamic stress has been found to reduce the concentrations of cellular surface receptors (Al-Rubeai et al., 1993; Lakhotia et al., 1993), certain receptors have been reported to be up-regulated with increasing shear forces (McDowell and Papoutsakis, 1998). Cis-acting shear stress responsive elements have been identified in the pro- moter region of several endothelial genes, which are known to be shear-inducible (Resnick et al., 1993; Resnick and Gimbrone, 1995), and could be involved in the regulation of other genes. Overall, agitation can act on cell surface receptors in several ways: by preventing the binding of receptors to their respective ligands, thus preventing cell-adhesion mediated signal transduction (Prosper and Verfaillie, 2001), or by affecting expression of cell surface receptors causing either up- or down-regulation, which can be mediated at the level of gene expression (Carswell and Papoutsakis, 2000). Journal of Immunological Methods 360 (2010) 20–29 ⁎ Corresponding author. Tel.: + 353 17161862; fax: + 353 17161177. E-mail address: m.al-rubeai@ucd.ie (M. Al-Rubeai). 0022-1759/$ – see front matter © 2010 Published by Elsevier B.V. doi:10.1016/j.jim.2010.05.007 Contents lists available at ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim