Abstract
IntroductionRecent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Stimulatory signals can enhance proliferation and promote differentiation, whilst inhibitory signals can significantly limit culture output.MethodsNumerical models of various mitigation strategies are presented and applied to determine effectiveness of these strategies toward mitigation of paracrine inhibitory signalling inherent in these culture systems. The strategies assessed include mixing, media-exchange, fed-batch and perfusion.ResultsThe models predict that significant spatial concentration gradients exist in typical cell cultures, with important consequences for subsequent cell expansion. Media exchange is shown to be the most effective mitigation strategy, but remains labour intensive and difficult to scale-up for large culture systems. The fed-batch strategy is only effective at very small Peclet number, and its effect is diminished as the cell culture volume grows. Conversely, mixing is effective at high Peclet number, and ineffective at low Peclet number. The models predict that cell expansion in fed-batch cultures becomes independent of increasing dilution rate, consistent with experimental results previously reported in the literature. In contrast, the models predict that increasing the flow rate in perfused cultures will lead to increased cell expansion, indicating the suitability of perfusion for use as an automated, tunable strategy. The effect of initial cell seeding density is also investigated, with the model showing that perfusion outperforms dilution for all densities considered.ConclusionsThe models predict that the impact of inhibitory signalling in HSC cultures can be mitigated against using media manipulation strategies, with the optimal strategy dependent upon the protein diffusion time-scale relative to the media manipulation time-scale. The key messages from this study can be applied to any complex cell culture scenario where cell-cell interactions and paracrine signalling networks impact upon cell fate and cell expansion.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0048-7) contains supplementary material, which is available to authorized users.
Highlights
Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems
In this study, models of different strategies for mitigating paracrine inhibitory signalling on the expansion of HSC populations during culture are considered
The optimal inhibition strategy depends upon the diffusive time scale, measured by the Peclet number We demonstrate here that the relative importance of diffusion to the media manipulation of any particular mitigation strategy is defined by the Peclet number: Figure 6 Spatial variation of transforming growth factor beta (TGF-β) concentration within a perfused culture
Summary
Recent studies in the literature have highlighted the critical role played by cell signalling in determining haemopoietic stem cell (HSC) fate within ex vivo culture systems. Recent studies from Zandstra and colleagues have highlighted the influence of combinations of cell-synthesised inhibitory proteins present at subthreshold levels that significantly limit expansion of HSC and their immediate progeny [7,8,9,10]. These negative feedback regulatory loops are important in HSC cultures, especially those where cells are seeded at high density, resulting in minimal distance between precursor cells and/or their nascent progeny
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