PRESENTACIÓN DE LA UNIDAD 8 Título

Cell migration assays were performed on RCE cell – PDMS samples and on RCE cells cultured on tissue culture plastic as described in Chapter 2, Section 2.7 in more detail. This was done in the form of scratch wound assays on a confluent layer of RCE cells cultured on a PDMS substrate. The confluent RCE cell layer was physically scratched or wounded using a pipette tip and the samples were then imaged using the BioStation CT, an automated imaging system and incubator. Following this the rate of closure of the scratch wound was measured using a customised wound healing recipe available on the Nikon CL-Quant analysis software. For each condition, measurements over 12 hours were used and the change in the wound to image size area ratio were compared to show the change in the percentage wound closure over time.

Figure 40 – The percentage wound closure between 0 to 12 hours in culture following the scratch wound assays on RCE cells cultured on TCP and plasma treated PDMS blends. ** showed a significant decrease in % wound closure of RCE cells on PDMS 1:1 when compared to TCP (p ≤ 0.004). * showed a significant decrease in % wound closure of RCE cells on PDMS 1:1 when compared to PDMS 10:1 (p ≤ 0.05) (mean ± SD, n=3) (One-way ANOVA using Tukey analysis, Minitab).

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 RCE cells on TCP PDMS 184 PDMS 10:1 PDMS 5:1 PDMS 1:1 Per ce n tage wo u n d c lo su re o ve r 0 - 12 h o u rs (% )

**

_*

137 The percentage scratch wound closure was compared between TCP and PDMS 184, PDMS 10:1, PDMS 5:1 and PDMS 1:1 (Figure 43). In order to standardise the images collected of the scratch wound closure over time, they were limited from 0 to 12 hours for the image analysis, with 0 being the start of the assay following the scratch wound procedure. The percentage wound closure values

obtained for the scratch assays were; TCP = 36.4 ± 1.3 %, PDMS 184 = 17.8 ± 11.2 %, PDMS 10:1 = 27.3 ± 11.7 %, PDMS 5:1 = 20.4 ± 3.6 % and PDMS 1:1 = 8 ± 5.8 %.

The overall trend observed in the RCE scratch wound assays was that in the control condition on TCP, the RCE cells had the highest percentage wound closure compared to the PDMS blends. This was expected as the differing Young’s moduli of the PDMS blends have already been shown to impact on the RCE cell structure and adhesion behaviours. PDMS 184 had a lower percentage wound closure rate when compared to the other PDMS blends. This was not expected as PDMS 184 was found to have the highest Young’s modulus compared the rest of the PDMS blends in Chapter 3 (Figure 33) where the bulk mechanical properties were tested. However this was not the case when other techniques were used such as tensile testing and ESPI (Figures 31 and 32). The lower percentage wound closure rate for PDMS 184 could have been due to other factors such as variation generated by using the scratch wound assay method as a manual scratch was created in the RCE cell layer (Kramer et al., 2013) or the variability observed in the material mechanical properties.

However, the trend observed in Figure 43 for PDMS 10:1 to PDMS 1:1 showed that with decreasing Young’s modulus, the percentage RCE cell wound closure over 12 hours also decreased. A significant decrease was observed for PDMS 1:1 when compared to TCP and PDMS 10:1, and PDMS 1:1 was the softest PDMS blend and had the lowest Young’s modulus. This trend correlated with the change in stiffness of the PDMS substrates and it was apparent that the substrate mechanical properties had an effect on the motility of RCE cells. The data in Figure 43 showed that as the PDMS blend became softer, the motility of the RCE cells decreased and began to inhibit the physical behaviour of the RCE cells. The significant decrease in percentage wound closure of the softest PDMS blend PDMS 1:1 when compared to the control on TCP showed the impact of the lower Young’s modulus on RCE cell migratory behaviour and their response to mechanical changes in their surrounding environment. Although the result for PDMS 10:1 was higher than expected (Figure 43) it was still lower than the control condition of TCP. The results determined that the lower the Young’s modulus of the PDMS substrate, the slower the rate of percentage wound closure when data was compared over a 12 hour period.

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4.3. General discussion and conclusions

Overall the results obtained in this Chapter showed that as the stiffness or Young’s modulus of the PDMS blends used decreased, changes in RCE cell attachment and morphology were observed in terms of the F-actin and stress fibre formation and RCE cells cultured on the softest PDMS blends appeared more rounded and less spread onto the PDMS surface. This was thought to be due to the cell-matrix interactions on softer substrates as this contact is critical for anchorage – dependence and other cell functions (Rehfeldt et al., 2007). The adhesion of cells to substrates allows the cell to probe the surrounding ECM and sense the mechanical changes in the surrounding environment. The conversion of the mechanical force sensed by the cell into biochemical signals within the cell is important in several cellular processes and it is the integrin receptors that play a major role in cell adhesion and material interactions with substrates (Fusco et al., 2015). Integrins can either act as mechanotransducers directly transmitting forces from the ECM to the cell or transmitting force to other components. They can also be intermediate receptors in other pathways that stimulate the integrins (Ross et al., 2013). The cell attachment through contractile stress fibres is key for adhesion and maintaining cell attachment. The F-actin staining images showed that as the Young’s modulus of the PDMS substrate decreased, less F-actin and stress fibres were visible, suggesting that the RCE cells were unable to maintain attachments to the softer PDMS substrates. RCE cell attachment was also quantified and showed a decrease in the number of RCE cells attached to the softest PDMS blend PDMS 1:1, supporting the RCE cell F-actin staining images. This method required further optimisation to establish accurate counts of RCE cells attached to the PDMS substrates and more images were required for the analysis to reduce the variability in the data.

The viability and proliferation of the RCE cells cultured on the PDMS blends increased over an 8 day culture period and although the viability was lower than RCE cells cultured on TCP, it showed that overall the PDMS blends did not cause any cytotoxic effects to the RCE cells during the culture period. The predicted cell numbers from the PrestoBlue viability assays were calculated using a regression analysis from the PrestoBlue calibration curve. The viability assays and predicted RCE cell numbers showed that up to 72 hours in culture, RCE cell – PDMS samples were at the optimum growth phase as observed by the viability assay on RCE cells cultured on TCP. The cell numbers were not as variable between the different PDMS blends in the first 3 days of culture whereas after longer culture periods, differences in RCE cell viability were more apparent. Some process modification would be required going forward as there was a reduction in the predicted RCE cell number at the start of the PrestoBlue assay from the seeding density used.

139 RCE cell response to changes in the substrate stiffness was further investigated by using scratch wound assays and the results showed that as the Young’s modulus decreased from PDMS 10:1 to PDMS 1:1, the percentage wound closure also decreased over a 12 hour period. Although PDMS 184 had a lower percentage wound closure compared to PDMS 10:1, it was also found to have a lower percentage wound closure compared to TCP. This was observed across all PDMS blends when they were compared to TCP. Factors affecting cell migration include changes in substrate stiffness and generally it has been observed that some cell types will migrate towards a stiffer surface (A. Kim et

al., 2012). Here the RCE cells did not have a stiffness gradient and so the effects of the different

PDMS blends with a range of Young’s modulus could be tested on certain cell behaviours that are known to be affected by substrate stiffness.

The results observed in this Chapter combined with the results in Chapter 3 showed that the PDMS blends produced formed substrates with a range of different Young’s modulus. By culturing these substrates with RCE cells, the physical responses of the RCE cells could be observed in terms of how the macroscale or bulk mechanical properties affected RCE cell behaviour. The key observations were that significant differences could be seen in RCE cells cultured on the softest PDMS blend, PDMS 1:1, which seemed to be distinctly different in most conditions investigated except for the PrestoBlue viability assays. This showed that the lower Young’s modulus had more of an impact on RCE cell adhesion, proliferation and migration.

Following the macroscale studies and the response of RCE cells to the bulk mechanical properties of the PDMS blends, micro- and nano-scale investigations were carried out on RCE cells cultured on the different PDMS blends to investigate the forces in play at the cell sensing level.

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