2.3. Cytotoxicity Assay According to ISO 10993-5

Cytotoxic effects of the transparent PLA material on keratinocytes and fibroblasts in their respective medium were investigated following the ISO standard 10993-5. This standard offers the choice of different assays to determine cytotoxicity. Here, the thiazolyl blue tetrazolium bromide (MTT) assay and visible validation were chosen.

Briefly, keratinocytes were seeded at a cell concentration of 10⁵ cells/mL, fibroblasts at 10⁵ cells/mL into a 96-well plate to reach a confluency of 50% the next day. The first and the last columns were spared to avoid edge effects. Two columns were only filled with media for non-cell control. At 24 h after seeding, 100 µL treatment media was applied to the wells. The treatment medium was created by placing sterilized inserts for 48 ± 2 h into a 6-well plate (Corning, Glendale, CA, USA) filled with 6 mL keratinocyte or fibroblast growth medium respectively. The treatment medium was applied in concentrations ranging from 0–100%. The 0% treatment media was equal to the fresh cell culture medium, 50% was a 1:1 mixture of fresh and 100% medium, that was incubated with the insert. Cells were incubated for 24 h with the various treatment media groups. Then, 25 mg of MTT powder (Thermo Fisher Scientific, Waltham, MA, USA) was dissolved in 5 mL sterile PBS to make a 5 mg/mL MTT stock solution. Next, the treatment medium was discarded. Then, 50 µL of MTT solution generated by diluting the stock solution in respective medium was added to each well. After an incubation period of 2 h at 37 °C, the reagent was removed from each well. Afterward, 100 µL isopropanol (Sigma-Aldrich, USA) was added, and plates were incubated for 30 min on an orbital shaker (VWR International, Radnor, PA, USA). Absorbance was then measured at 570 nm (reference wavelength 650 nm) on a plate reader (Polarstar, BMG Labtech, Ortenberg, Germany).