*3.2. The Sea-Buckthorn Seed Oil Showed Pro-Proliferative Effects at Low Concentrations*

We tested the effect of sea-buckthorn seed oil on viability of normal and dysplastic skin cells using serial dilutions starting at 1/2000 (*v*/*v* in complete cell medium). The starting concentration was chosen following previous experiments, in which higher concentrations were cytotoxic for both cell types (data not shown). Further dilutions showed no cytotoxicity when reported on non-treated controls. Cell viability increased with subsequent dilution up to 1/8000, and then it started to decrease for both cell types (Figure 2). As a result, this concentration was selected for further tests.

**Figure 2.** Assessment of toxicity of sea-buckthorn seed oil. Normal human keratinocytes (HEK) and dysplastic keratinocytes were seeded at a density of 10,000 cells/well in 96 well plates and treated for three days with the indicated dilutions of sea-buckthorn seed oil. Cytotoxicity was assessed by lactate dehydrogenate (LDH) release in the cell medium and viability by MTS assay. LDH control is represented by the lysis control (100% mortality). The MTS control was represented by cells maintained in standard cell culture conditions. The highest non-toxic and pro-proliferative concentrations (highlighted in red) were chosen for further tests. Bars represent average of triplicates, calculated as percentage to control.

#### *3.3. Sea-Buckthorn Seed Oil Inhibits Cell Migration of Dysplastic Keratinocytes*

Next, in real-time microscopy we assessed the ability of cells to proliferate and migrate in a scratch-wound assay, following 24 h treatment with sea-buckthorn oil at 1/8000 dilution. After 24 h, both treated and untreated dysplastic cells failed to migrate and close the denuded area, and the oil treatment impaired migration even further (Figure 3). Normal keratinocytes were also less effective at closing the gap in the presence of diluted sea-buckthorn oil, but the effect was less impaired than that of dysplastic cells.

**Figure 3.** Real-time assessment of cell migration and proliferation of normal and dysplastic keratinocytes, treated (+) with sea-buckthorn seed oil. Treated cells (both normal and dysplastic) were less efficient in covering the denudated area. The effect was more prominent for dysplastic cells.

To this point, we noticed that there is a contrast between the effects of sea-buckthorn seed oil on cell proliferation versus cell migration, which might indicate a possible deleterious effect of such treatment on the migration of epidermis cell populations. We wanted to further confirm these findings and address other literature-reported effects of seabuckthorn oil, such as protection against UVA irradiation and use of a real-time assessment of cell proliferation.
