PRESENTACIÓN DE LA UNIDAD 6 Título

Following F-actin staining of RCE cells cultured on different PDMS blends, the number of RCE cells attaching to each PDMS surface and the area coverage of cells across the sample surface were measured using the Nikon BioStation CT, an automated imaging system in which experiments can be performed without having to remove samples from the incubator environment. The methods used are described in Chapter 2, Section 2.6.3.

The F-actin stained RCE cell – PDMS sample images were then analysed using the Nikon CL-Quant automated image analysis software which uses preconfigured standard assays and also allows the user to create analysis assays specific to their data, allowing batch processing of large image data sets. The method of analysis used was a set of procedures customised to the RCE cell – PDMS sample images to ensure the correct measurement of RCE cells on the surface of the PDMS blends. The methods used are described in more detail in Chapter 2, Section 2.6.3.

Figure 40 shows the percentage coverage of RCE cells on the surface of the different PDMS blends following 24 hours in culture, fixation and staining of PDMS – RCE cell samples. Samples were imaged in the BioStation CT and tiling images of the whole well contain a sample were taken at 10X magnification in phase contrast and fluorescence in order to image the whole PDMS surface to further investigate the effectiveness of the PDMS surface properties in promoting cell adhesion and to compare this across the PDMS blends.

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Figure 37 –Percentage RCE cell coverage of RCE cells cultured on PDMS blends after 24 hours of culture and fixation. RCE cells were stained with phalloidin (F-actin) and DAPi (nuclei). Images were quantified using CL Quant (Nikon) and the area measured for each well was 9.6 cm2 _{or the whole well in a 6-well plate, covering}

the whole PDMS sample size of 2 cm2 _{(mean ± SD, n = 3). * shows a statistically significant increase in % cell}

coverage in plasma treated PDMS 184 compared to untreated PDMS 184 (p ≤ 0.02). ***shows significant increase in % cell coverage after plasma treatment for PDMS 1:1 p ≤ 0.001 (one-way ANOVA using Tukey analysis, Minitab).

The images taken were used to quantify the attachment profiles of the RCE cells on each PDMS blend by using a modified analysis method (, Chapter 2, Section 2.6.3) to mask the cell areas using the fluorescence to differentiate the RCE cells from the PDMS in the background. This gave the percentage coverage of RCE cells when subtracted from the total area.

Untreated and plasma treated PDMS – RCE cell samples were compared and it was found that there were significant differences between PDMS 184 when comparing untreated to plasma treated samples and also between 1:1 PDMS samples in terms of percentage cell coverage observed. PDMS 184 showed a significant increase in the percentage RCE cell coverage after plasma treatment from 67.1 ± 17.6 % on untreated PDMS 184 to 98.6 ± 0.3 % on plasma treated PDMS 184, which followed the trend observed in the fluorescent staining images (Figures 35-39). This data further supported the data shown in Figure 21 (Chapter 3) that PDMS 184 was the more successful PDMS blend used in

0 10 20 30 40 50 60 70 80 90 100

Untreated PDMS Plasma treated PDMS

Per ce n tage R CE c e ll co ve rag e ( % ) PDMS 184 PDMS 10:1 PDMS 5:1 PDMS 1:1 PDMS 1:5 *

*

***

130 terms of the uptake of surface modification (Chapter 3, Section 3.2.1.2) and also had increased attachment of RCE cells to the PDMS surface when compared to other PDMS blends. This may have been linked to the stiffness data obtained in Chapter 3 (Section 3.2.2), which showed that in the AFM data, PDMS 184 had the highest Young’s modulus compared to PDMS 10:1, PDMS 5:1 and PDMS 1:1. However, this was not the case for the tensile testing results and ESPI results obtained and PDMS 184 Young’s modulus from these mechanical tests was not significantly different to the other PDMS samples tested (Chapter 3, Figures 31-33).

It was also observed that for PDMS 1:1, there was a significant increase in the percentage RCE cell coverage after plasma treatment from 44.3 ± 17.8 % on untreated PDMS 1:1 to 98.3 ± 0.6 % on plasma treated PDMS 1:1. However, the percentage RCE cell coverage observed for PDMS 1:1 on the untreated PDMS sample was significantly lower than the other PDMS blends measured. This result did not correlate with the trends observed in the surface and bulk mechanical properties (Chapter 3) as PDMS 1:1 had a lower Young’s modulus when compared to PDMS 184, PDMS 10:1 and PDMS 5:1 in two of the three mechanical testing methods used, tensile testing and AFM. However, PDMS 1:1 had the highest Young’s Modulus when compared to PDMS 184, PDMS 10:1 and PDMS 5:1 using the ESPI measurement technique. The stiffness of PDMS 1:1 was also significantly less when compared to PDMS 184 in the AFM results described previously (Chapter 3, Figure 33).

PDMS 1:5 showed lower percentage cell coverage after plasma treatment when compared to the other plasma treated PDMS samples (Figure 40). This was thought to be due to the material properties of PDMS 1:5 as discussed in Chapter 2, Section 2.1.2, supported by the RCE cell – PDMS sample staining images shown for PDMS 1:5 (Figure 39). The morphology of the RCE cells cultured on PDMS 1:5 appeared to be very different from the rest of the PDMS blends, with a more rounded shape and markedly less RCE cell spreading and F-actin visualisation (Figure 39). The results obtained in the work so far are closely linked when PDMS 1:5 was investigated as it did not provide a preferential surface for RCE cell attachment and spreading and this was thought to be strongly related to the bulk mechanical properties and polymer chemistry of PDMS 1:5 as described previously.

Overall, the data obtained in Figure 40 did support key observations in particular regarding the stiffest and softest PDMS blends, PDMS 184 and PDMS 1:5 respectively. It showed that there was an increase in the percentage coverage of RCE cells on all PDMS blends after plasma treatment, which further supported the success of the plasma treatment used. It also highlighted the lower percentage RCE cell coverage for PDMS 1:5 after plasma treatment, which was expected. The methods used required further testing and calibration using several more images in order to

131 accurately determine the number of RCE cells attached to the whole PDMS surface rather than percentage coverage and to reduce the error in the measurements. Percentage coverage was a good indication and estimation of RCE cell attachment over the surface of the PDMS samples but did not provide an exact number of cells attached to each PDMS sample.

Experiments carried out from this point in the work did not use PDMS 1:5 as a substrate blend. This was decided based on the results obtained for bulk mechanical properties, surface characterisation, actin staining and the percentage RCE cell coverage experiments. PDMS 184, PDMS 10:1, PDMS 5:1 and PDMS 1:1 were still used and this was thought to provide a good range of Young’s moduli for testing with RCE cells.