It was determined whether BCM, created using the toxic ATCC® 29213™
S.aureus strain, affected the viability of 3T3 fibroblasts. Fibroblasts were
incubated with increasing concentrations of BCM for 24 or 48 hours, and then their viability, compared to controls, was assessed using MTT assays (figure 4.1. A and B). The MTT assay is a colorimetric technique often used to assess viability of cells, as only live cells with active NAD(P)H-dependent oxidoreductase enzymes are capable of reducing yellow MTT to insoluble blue formazan crystals; the extent of blue coloration is measured by absorbance at 570 nm and reflects the number of viable cells (Meerloo et al. 2011). A trend was apparent, particularly at the 48 hour time point, whereby increasing concentrations of BCM were associated with decreasing absorbance. At both the 24 and 48 hours incubation with 75% and 100% BCM caused a significant reduction in the absorbance reading, with the effect greatest after 48 hours.
Although MTT assays are normally used as an indicator of the viability of a population of cells, changes in metabolic activity can also influence the results; a reduction in both metabolic activity and the number of viable cells will reduce the production of formazan from MTT (Sumantran 2011). Therefore to support the results of the MTT assays, cell counts were also taken of controls and of fibroblasts incubated with 100% BCM, at 0, 24 and 48 hours (figure 4.1.C). Incubation with 100% BCM resulted in significantly fewer cells than in controls at both 24 and 48 hours. Moreover, the population of control fibroblasts more than doubled between 0 and 24 hours (2.7±0.4 fold), and doubled again between 24 and 48 hours (1.9±0.1 fold), whereas fibroblasts incubated with 100% BCM proliferated slower, growing 1.7±0.3 fold between 0 and 24 hours, but barely proliferating between 24 and 48 hours (1.08±0.004 fold increase in cell count).
Figure 4.1. Incubation with biofilm conditioned medium (BCM) reduces cell number. (A, B) Graphs illustrating MTT absorbance at 24 (A) and 48 (B) hours when 3T3 fibroblasts were
incubated with increasing concentrations of BCM. A one-way ANOVA showed significant differences between the groups at 24 hours (p<0.05) and 48 hours (p<0.001). A Tukey’s post hoc revealed that incubation with 75% and 100% BCM caused significant differences from controls at both time points. N=4. (C) Graph showing the number of cells counted in controls, or after incubation with 100% BCM, over 48 hours. Independent Student’s t-tests showed that there were significantly less cells in the 100% BCM group compared to controls at both 24 and 48 hours. N=3. All error bars are SEM. *p<0.05, **p<0.01.
Cell death is usually accompanied by a change in cell volume: apoptosis causes cell shrinking, while necrosis results in swelling (Orrenius & Sten 2001). The cell counts used to assess cell viability when incubated with 100% BCM were made using a Scepter™ handheld automated cell counter, which creates histograms of the cell’s volumes and diameters. However, no statistically significant differences were observed in the volume or diameter of fibroblasts incubated with 100% BCM (figure 4.2.A-B), suggesting that the cells exposed to BCM were not dying. As neither of the techniques previously used to access viability (MTT assays or cell counts) allow a distinction to be made between cytostatic and cytotoxic effects it was possible that the fibroblasts were not dying, but instead failing to proliferate. To investigate this, the nuclei of cells incubated with 100% BCM for 24 hours were visualised by staining with Hoechst, and then observed for signs of apoptosis or necrosis. Apoptosis normally causes nuclear fragmentation and chromatin condensation, and necrotic cells exhibit nuclear swelling (Orrenius & Sten 2001). However, the nuclei of cells incubated with 100% BCM for 24 hours appeared healthy, exhibiting no morphological signs of either necrosis or apoptosis (figure 4.2.C). This agrees with the data showing cell volume and diameter are not altered by incubation with 100% BCM, and indicates that the reductions seen in cell counts and in absorbance in the MTT assays were not the result of reduced viability, but of reduced overall cell number. To determine if this was the case fibroblasts were incubated with 100% BCM or control media and imaged hourly over 24 hours (figure 4.2.D-E). The cell’s nuclei were labelled with Hoechst, which enabled the rate of cell proliferation to be assessed by comparing the total number of cells at each time point. Throughout the experiment the medium also contained propidium iodide (PI). This dye is excluded by viable cells but readily enters cells that have lost membrane integrity, and so it is often used to assess cell viability (Johnson et al. 2013). By imaging both Hoechst (total number of cells per image) and PI labelled cells (non-viable cells), the changing percentage of non-viable cells in the population could be determined. By observing the fold change in cell number (figure 4.2.D), it was evident that incubation with 100% BCM significantly reduced the proliferation of 3T3 fibroblasts. The growth rate in the BCM group began diverging from controls at around 8 hours; the fold change of the controls remained exponential, whereas the fold change of BCM incubated cells began to plateau. At 11 hours
Figure 4.2. Incubation with 100% biofilm conditioned medium (BCM) prevents cell proliferation but doesn’t reduce cell viability. (A-B) Graphs demonstrating the (A) diameter
and (B) volume control and 100% BCM incubate cells at 0, 24 and 48 hours. (C) Confocal images of nuclei from 3T3 fibroblasts controls (left) and fibroblasts incubated with 100% BCM for 24 hours (right). The images are representative of 3 experiments. Scale bars indicate 50 µm.
(D) Graph presenting the fold change in cell number over 24 hours as determined by live
imaging of Hoechst labelled cells. There were significant differences in proliferation between control and 100% BCM at every time point after 11 hours (p<0.05 or <0.01). (E) The number of propidium iodide positive cells, as a percentage of the whole population over 24 hours is shown for control 3T3 fibroblasts, and fibroblasts incubated with 100% BCM. Mixed design ANOVAs or independent Student’s t tests were used to test statistical differences as appropriate. A-B, N=3; D-E, N=4. All error bars are SEM. *p<0.05, **p<0.01.
significant differences between control and BCM incubated groups emerged, and the groups differed significantly at every time point thereafter. By 12 hours the fold change in cell number no longer increased in the BCM incubated group, suggesting that the cells had stopped proliferating, and had entered cell cycle arrest. The viability of the population was also assessed, by determining the percentage of the total population that were positive for PI (figure 4.2.E). This was low in both controls and cells incubated with 100% BCM. Less than 1% of cells were PI positive, and there were no differences between the groups