## 3 Results and Discussion
### Method Validation
#### 3.2.8 Cell Viability Results  
Initial investigation of exposing HEI-OC1 cells for 24 h to diabetic conditions (25 mM glucose, HG; 2.5 mM metforminin, M2.5; 5 mM metforminin, M5; and 10 mM metforminin, M10) as well as the combined treatments (HG + M2.5, HG + M5 and HG + M10) did not reveal any changes to the cell viability detected by CellTitre-Blue assay. The decision was made to expose these auditory cell lines to all treatment conditions over a few days (longer duration) to allow the cells to metabolise the high glucose added and to investigate whether metforminin could mitigate the stressing conditions created by the simulated diabetic state. This constitutes a more clinically relevant setting of hyperglycaemia. In general, treatment type (drug dose) results were considered highly significant (\(p<0.0001\)). Metformin was not cytotoxic up to the concentration 10 mM to HEI-OC1 after 4 days of exposure (Figure 6). After 2 days of HEI-OC1 exposure to all treatments, all cells showed more than 96.5% (\(\pm\)2.52) viability. Cells exposed to 25 mM glucose (HG treated cells) showed slightly higher viability 99.75% (\(\pm\)1.71). All other treated cells showed cell viability ranging between 96.5% (\(\pm\)2.52) and 100.5% (\(\pm\)3). Neither the experimental day results nor the treatment type after 2 days of treatment were considered significant. The Bonferroni post hoc test revealed that only (HG versus HG + M10) and (M10 versus HG + M10) results were statistically significant, where the \(p\) value was \(<\)0.05 (Figure 6A). Day 3 results post treatment revealed that cells exposed to 25 mM glucose (HG treated cells) had the lowest viability of all treatments: 68% (\(\pm\)7.26). Cells exposed to metforminin 5 mM (M5) showed the highest cell viability of all treatments: 103% (\(\pm\)3.50). The cell viability of M2.5 and M10 were 99% (\(\pm\)2.58) and 102% (\(\pm\)2.83), respectively. Metformin seemed to mitigate the stressing effect of HG added. We noticed that, when metformin was co-added into HG treated cells, viability was improved to 95.5% (\(\pm\)1.92) in HG + M2.5, to 98% (\(\pm\)1.63) in HG + M5 and to 90.5% (\(\pm\)1.0) in HG + M10 (Figure 6B). Figure 6: Cont. Figure 6: **(A,B). ** HE-OCI cell viability results. (A) After 2 days and (B) after 3 days of treatment with: no treatment (CTRL), high glucose, 25 mM (RG), metformin 2.5 mM (M2.5), metformin 5 mM (M5), metformin 10 mM (M10) and combination treatments (HC + M2.5), (HG + MS) and (HC + M10). (**C,D**). HE-OCI cell viability results. (C) After 4 days and (D) after 5 days of treatment with: no treatment (CTRL), high glucose, 25 mM (HG), metformin 2.5 mM (M2.5), metformin 5 mM (M5), metformin 10 mM (M10) and combination treatments (HC + M2.5), (HC + MS) and (HC + M10). (**C,D**). HE-OCI cell viability results. (C) After 4 days and (D) after 5 days of treatment with: no treatment (CTRL), high glucose, 25 mM (HG), metformin 2.5 mM (M2.5), metformin 5 mM (M5), metformin 10 mM (M10) and combination treatments (HC + M2.5), (HC + MS) and (HC + M10). (**C,D**) and (**C,D**) represent level of statistical significance of results. (\(\uparrow\)) when \(p\)-value \(<\) 0.05, statistical results are significant. (\(\uparrow\)) when \(p\)-value \(<\) 0.01, statistical results are highly significant and more significant than (\(\uparrow\)) when \(p\)-value \(<\) 0.001, statistical results are highly significant and more significant than (\(\uparrow\)) and (\(\uparrow\)). Likewise, day 4 results post treatment revealed that cells exposed to 25 mM glucose (HG-treated cells) appeared more stressed and had a viability of 48.75% (\(\pm\)4.79). However, all metformin-treated cells looked healthy and attached to each other. Cells exposed to metformin 5 mM (M5) showed the highest cell viability of all treatments: 101.75% (\(\pm\)1.26). The cell viability of M2.5 and M10 were 97.5% (\(\pm\)1.91) and 99% (\(\pm\)2.0), respectively. Metformin at the concentrations 2.5 mM and 5 mM seemed to mitigate the deteriorating effect of HG in HEI-OC1 cells.