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PRESENTACIÓN DE LA UNIDAD 7 Título
Following investigations into RCE cell attachment and cytoskeletal changes after 24 hours in culture and quantifying the percentage RCE cell coverage after this period, the viability and the proliferation of the RCE cells cultured on the different PDMS blends was measured. This was done using PrestoBlue® cell viability reagent, which could be used as a live cell assay. The viability and proliferative capacity of the RCE cells was tested using a continuous live culture over 8 days to see the effects of PDMS blends on RCE cells over a longer period of time. The methods used for this assay are described in detail in Chapter 2, Section 2.6.4.
Figure 41 shows the different PDMS blends used and the control culture on tissue culture plastic and the general trend in RCE cell viability over 8 days in culture can be observed.
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Figure 38 - A comparison of the average percentage reduction of PrestoBlue® cell viability reagent by RCE cells cultured over 8 days on different plasma treated PDMS blends and on tissue culture plastic (mean ± SD, n=3).
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 1 2 3 4 5 6 7 8 Per ce n tage R e d u ction o f Pr e sto B lu e ® b y R CE c e lls (% )
Time Points (Days)
PDMS 184 PDMS 10:1 (184:527) PDMS 5:1 (184:527) PDMS 1:1 (184:527) Tissue Culture Plastic
133 In Figure 41 the overall trend observed in the cell viability of RCE cells over 8 days in culture was that there was an increase in RCE cell viability and culture on the PDMS blends did not have a cytotoxic or detrimental effect on the RCE cell viability. It has been shown in the literature that Sylgard 184 and Sylgard 527 do not have cytotoxic effects on cells and Sylgard 184 in particular has been extensively used in research to produce substrates for cell culture (Palchesko et al., 2012; Dow Corning, 2014a, 2014b; Lycans et al., 2014). However, to test the PDMS samples used in this work with the RCE cell line, viability and proliferation studies were carried out.
Cell viability and proliferation were measured in the assay by the percentage reduction of the PrestoBlue reagent as the metabolising and therefore living RCE cells would reduce the component in PrestoBlue called Resazurin (blue, non-fluorescent), which changed to Resorufin (red, fluorescent). The colour change in the RCE cell culture medium was then detected by fluorescence optical intensity. The tissue culture plastic control showed that up to day 4 in culture, the RCE cells showed a greater increase in viability and proliferation, which indicated that they were in the growth phase, with metabolically active cells. By day 5, cell proliferation rate had slowed and then from days 6 – 8, plateaued by the end of the culture period. This trend was not observed in the PDMS blends shown in Figure 41. Overall, the viability and proliferation of the RCE cells increase over the 8 day culture period but at a much slower rate compared to tissue culture plastic. This was expected due to the change in the culture substrate and also the differences in mechanical and surface properties of the PDMS blends, as discussed previously.
By day 8, the percentage reduction of PrestoBlue by RCE cells was found to be very similar for PDMS 10:1, PDMS 5:1 and PDMS 1:1, at 22.4 %, 24.9 % and 23.6 %. For PDMS 184, the percentage reduction of RCE cells cultured on this substrate and therefore the RCE cell viability was highest by day 8 in culture when compared to the other PDMS blends measured at this time point at 28.9 %. This showed that PDMS 184 was the most appropriate cell culture substrate in terms of RCE cell viability and promoted cell adhesion, proliferation and spreading of the RCE cells on its surface. This was also supported by the data obtained in Chapter 3 for the bulk mechanical properties of the different PDMS blends and is related to the preference of cells to attach to stiffer substrates, which has been reported in the literature (Lo et al., 2000; Discher, Janmey and Wang, 2005; Rehfeldt et al., 2007; Schwarz and Gardel, 2012).
It was observed that PDMS 10:1 had a lower percentage reduction of PrestoBlue overall across the time points after day 5 in culture when compared to the other PDMS blends. PDMS 184 had lower PrestoBlue reduction than all other PDMS blends but by day 6 in culture, had increased percentage reduction by RCE cells over all other PDMS blends for the remaining time points. PDMS 5:1 and 1:1
134 also had higher PrestoBlue reduction between days 1-5 in culture and then started to decrease between day 5 and 6. Due to the nature of the PrestoBlue data, the exact cell number is not calculated and PrestoBlue reduction by RCE cells is measured by fluorescence using the FLUOstar Omega plate reader. The percentage PrestoBlue reduction is worked out by dividing the fluorescence reading from a sample of a particular PDMS blend seeded with RCE cells by the fluorescence reading for the positive control (100% reduced PrestoBlue reagent in RCE cell culture medium) and multiplying by 100. This was done for all RCE cell – PDMS samples used in the experiment and is described in more detail in Chapter 2, Section 2.6.4.
A PrestoBlue calibration curve for RCE cells was carried out before conducting the experiments and this was then used to predict the number of RCE cells present at each time point. A regression analysis was performed using Minitab and the methods used to derive the predicted cell number have been described in Chapter 2, Section 2.8. The data in Figure 41 was then plotted using the predicted cell numbers to further investigate how the PrestoBlue percentage reduction translated to cell number, as shown in Figure 42.
Figure 39 – Predicted cell number from the PrestoBlue cell viability assay data showing the estimated cell number calculated using regression analysis. Predicted cell numbers are shown across the PDMS blends and tissue culture plastic over 8 days in culture. Regression analysis performed using Minitab, p value for quadratic model ≤ 0.005. 0 100000 200000 300000 400000 500000 600000 700000 800000 900000 1 2 3 4 5 6 7 8 Pr e d ic te d c e ll n u m b e r
Time points (days)
PDMS 184 PDMS 10:1 PDMS 5:1 PDMS 1:1 Tissue culture plastic
135 Figure 42 showed that the estimated RCE cell numbers cultured on PDMS blends were similar on day 1 and were around 40,000 – 50,000 RCE cells, except for PDMS 184 and PDMS 10:1, which were lower and had predicted cell numbers of approximately 27,000 and 35,000 respectively on day 1. The seeding density used for the these RCE cell - PDMS samples was 8.3 x 103 RCE cells/ cm2, which
was approximately 80,000 RCE cells per PDMS sample. Therefore the predicted cell numbers were closer to the actual seeding densities used on the RCE cell – PDMS sample for the PrestoBlue viability assay. This was due to the methodology used to prepare the RCE cell – PDMS samples for the assay after seeding. To ensure that only RCE cells cultured on the surfaces of the PDMS samples were measured for the 8 day viability and proliferation assay, after RCE cell seeding and 24 hours incubation, the RCE cell – PDMS samples were then removed from the well plate using sterile tweezers and were transferred to a new sterile 6 well plate (see Chapter 2, Section 2.6.4). In doing so, some of the RCE cells would have remained in the original plate that the PDMS sample was seeded in as low attachment plates were not used. This is why there was a reduction in the number of predicted RCE cells at the start of the assay. However, a similar trend in Figure 42 was observed in the RCE cells on TCP and PDMS blends when compared to the trends in Figure 41, as the relative fluorescence (RFU) was used from the PrestoBlue calibration curve to determine estimated cell numbers. When looking at the predicted cell number (Figure 42), all of the PDMS blends showed very similar predicted RCE cell numbers until day 6, where differences between the PDMS blends were more apparent when compared to Figure 41. The predicted RCE cell numbers for PDMS 184 were highest out of all the PDMS blends from day 6 (Figure 42), which was also found when percentage reduction of PrestoBlue was compared (Figure 41). PDMS 5:1 and 1:1 followed the expected trend of decreasing predicted cell numbers but PDMS 10:1 had the lowest predicted RCE cell numbers after day 6 when compared to the other PDMS blends (Figure 42).
Following the PrestoBlue viability assay, it was decided that based on this data the culture period used would be less than 72 hours of RCE cell culture on PDMS samples. This was implemented in further experiments because from the viability experiments, this time period was identified as having less variation in the viability and proliferation of RCE cells cultured on PDMS and in the predicted RCE cell number obtained. Also in the control condition, RCE cells cultured on TCP, the RCE cells were observed to be in the growth phase of the culture period and therefore between day 1 and day 3 (24 and 72 hours) of RCE cell culture was the optimum time to test the RCE cell – PDMS samples.
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