Confluency at 24h decreased with Young’s modulus (p<0.005). (B) Analysis of cell confluency from phase contrast images of four-day partially-differentiated cells attached for 24h onto 3 different GXG materials and TCP. Gelatin-coated TCP is shown as a control, representing current best practice in the laboratory. Numbers of attached cells observed decreases as GXG Young’s modulus increases. (A) Phase contrast images of four-day partially-differentiated cells (mESC’s differentiated for four days in N2B27 on TCP) seeded for 24h upon varying elasticities of GXG. N=3.Īttachment of neural precursors is maximised on low modulus substrates. No significant difference was found between any of the substrates or across the sample population as a whole, when one-way ANOVA was carried out, followed by Tukey post hoc correction. (D) The ratio between confluency at 72h and 24h was calculated in order to assess “fold expansion” of colonies post-attachment. (C) Analysis of cell confluency from phase contrast images of six-day partially-differentiated neurons attached for 72h onto 3 different GXG materials and tissue culture polystyrene. (B) Analysis of cell confluency from phase contrast images of six-day partially-differentiated neurons attached for 24h onto 3 different GXG materials and TCP. (A) Phase contrast images of six-day partially-differentiated neurons (mESC’s differentiated for six days in N2B27 on TCP) seeded for 24h upon varying elasticities of GXG. Published by Elsevier Ltd.Īttachment of immature neurons is unaffected by Young’s modulus. Conversely, the impact of Young's modulus on changes in phenotype increased as cells became more mature.Ĭopyright © 2015. Taken together our findings imply that the impact of Young's modulus on attachment diminishes as neuronal cells become more mature. 2 kPa increased the proportion of cells that differentiated from immature into mature neurons. Stiffness had no effect upon phenotypic changes during differentiation for mESC's and neural precursors. Expansion was independent of stiffness for all cell types, implying that the proliferation of cells during this differentiation process was independent of Young's modulus. The attachment of immature neurons was unaffected by stiffness. Attachment was found to decrease with increasing stiffness for both ESC's (with a 95% decrease on 35 kPa compared to 2 kPa) and neural precursors (with a 83% decrease on 35 kPa). Stiffness was varied from 2 kPa to 18 kPa to finally 35 kPa. In this investigation, three effects of stiffness on cells were considered attachment, expansion and phenotypic changes during differentiation. Our aim was to understand how stiffness impacts neuronal differentiation of embryonic stem cells (ESC's), and how this varies at three specific stages of the differentiation process. There is substantial evidence that cells produce a diverse response to changes in ECM stiffness depending on their identity.
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