Cytotoxicity (MTT assay)

The culture of adherent mouse L929 fibroblasts (CCL-1, ATCC) was established and carried out in RPMI 1640 medium (Biological Industries, Israel), with the addition of 10% FCS (Biological Industries, Israel) and supplemented with: penicillin–streptomycin-L-glutamine solution (100 U/mL, 100 µg/mL, 0.292 mg/mL, respectively; Gibco, United States), in culture flasks (growth area of 25 cm²; TPP, Switzerland). The cells, after their detachment from the growth surface (trypsinization with the 0.25% trypsin-1 mM EDTA solution; Gibco, United Kingdom), were used for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, United States) assay. Briefly, after preparing a cell suspension with the density of 1 × 10⁵/mL, it was portioned by 100 µL into the wells of a 96-well flat bottom tissue culture plate (TPP, Switzerland) and incubated for 24 h (37° C, 5% CO₂). The culture medium was removed and replaced with a medium containing the specified concentrations of test extracts (100 µL/well). After 24, 48 and 72 h of incubation (under the conditions as above), the medium with the added extracts was removed and replaced for 2 h with the solution of MTT (1 mg/mL; 50 µL/well) in Opti-MEM medium with no phenol red (Gibco, United Kingdom), during which the live cells reduced the MTT salt to formazan crystals. After discarding the media from the cells, the crystals were dissolved in 100 µL of isopropanol. Finally, the absorbance was measured at 570 nm using a spectrophotometer Multiskan EX (LabSystems, Finland).

The viability of the cells was evaluated colorimetrically based on the amount of reduced MTT. Various concentrations of the tested moss extracts, ranging from 1.9 to 1000 µg/mL were added to the L929 fibroblasts. Incubation continued for 24, 48, and 72 h. The concentration of the extracts’ solvent (Et-OH) did not exceed 2,5% and did not disturb fibroblast growth. The cell survival was calculated as follows:

As—absorbance of test sample (the cells exposed to the extract), Ac—absorbance of negative control (the cells in medium alone), Ab—absorbance of blank control (cell-free samples – background absorption).

The multiplied results (n = 9) were statistically analyzed using the ANOVA statistical method.