*2.4. Cell Survival Analysis*

The goblet cells were trypsinized after 14 days of cultivation, counted, and replated in a 96-well plate with a cell density of 25,000 cells/cm<sup>2</sup> for LDH and 50,000 cells/cm2 for the MTT assay. Cells were incubated for an additional four to five days at 37 ◦C and 5% CO2, to ensure adhesion before they were treated for 30 min with eye drops that were diluted at 1:7 (*v*/*v*) in media. After 30 min of treatment, the eye drops were removed, fresh media was added, and the cells were incubated for various time periods, depending on the assay performed thereafter. The LDH assay was performed 20 h after treatment with eye drops. LDH solution was prepared immediately before the experiment, added to the cells, and incubated at room temperature in the dark for 15–20 min before the stop solution (10% HCL, *v*/*v*) was added. Concerning the MTT assay, 12 mM MTT (*w*/*v*) in PBS was added to the cells, immediately after treatment with the diluted eye drops. The cells were incubated at 37 ◦C and 5% CO2 for one hour, followed by adding 0.01% (*v*/*v*) HCL in 10% (*w*/*v*) SDS, in PBS solution. The cells were then incubated in the dark for 18 h at

room temperature. A SpectraMax i3X multi-mode microplate reader (Molecular Devices, San Jose, CA, USA) with an absorbance of 490 nm for LDH and 560 nm for MTT was applied. To secure the reproducibility of the results, a minimum of three batches from a minimum of four different donors was required for analysis. For every experiment, a control treated with only RPMI media was included. The cell survival data was calculated as the mean percentage change in absorbance, compared to the control ± standard deviation (SD).

### *2.5. Immunohistochemical Staining*

The goblets cells were cultivated on slides and treated with RPMI media or diluted eye drops 1:7 (*v*/*v*) for 30 min at 37 ◦C 5% CO. With the use of paraformaldehyde 4% (*v*/*v*), the slides were fixated and stored at 4 ◦C. The cell membranes of the goblet cells were permeated using 0.1% *v*/*v* Triton X-100 in PBS, and by using 3% (*w*/*v*) bovine serum albumin in PBS, unspecific binding was blocked. The cells were treated with the primary antibodies Cytokeratin-7 (anti-cytokeratin7, 1:500 *v*/*v*) and monoclonal anti-human gastric mucin (anti-mucin, 1:200 *v*/*v*), diluted in 0.25% bovine serum albumin/0.1% saponin in PBS and washed with PBS thereafter. The fluorescent secondary antibodies, Alexa488 (anti-rabbit, 1:500 *v*/*v*) and Texas red (anti-mouse, 1:200 *v*/*v*), both diluted in 0.25% bovine serum albumin/0.1% saponin in PBS, were added. Then, 0.3 μM of DAPI in PBS stained the nuclei of the goblet cells. Imaging was performed using an Axioskop 2 (Zeiss; Göttingen, Germany) with an Axio Cam MRm camera (Zeiss; Göttingen, Germany) and an HXP 120 lighting unit (Zeiss; Göttingen, Germany). Image scaling and the merging of pictures were conducted using ImageJ 1.52q (Wayne Rasband, National Institute of Health, Bethesda, MD, USA).

#### *2.6. Statistics*

The software program, GraphPad Prism 9 (GraphPad Software, San Diego, California USA), was used for the statistical analyses and graphs. Descriptive statistics and a comparative one-way analysis of variance (ANOVA) were chosen as the statistical analyses for all data sets. To estimate the differences in cell survival, osmolality, pH value, and surface tension among the treatments, Tukey's multiple comparison test was applied. All results were expressed as mean ± SD, and a *p*-value of ≤ 0.05 was considered statistically significant.
