4.6.1. Cell Cultures

The L929 mouse fibroblast cell line and the National Collection of Type Cultures (NCTC) human keratinocyte cell line were obtained from the American Type Culture Collection (LGC Standards srl, Milan, Italy). Cells were cultured at 37 ◦C in a humidified, 5% CO2 atmosphere, in high glucose Dulbecco's modified Eagle's medium (D-MEM) with glutamax (Euroclone, Milan, Italy), which was supplemented with 10% FBS (Euroclone) with penicillin/streptomycin as antibiotics.

#### 4.6.2. Cell Growth and Cell Adhesion

To evaluate cell growth on SCM-coated plates, experiments were performed in quadruplicate on 96-well plates. Both L929 and NCTC cell lines were plated at a density of 5000 cells/well on 96-well plates pre-coated with F1, F2, F3, and F4. *C. reniformis* FSs extracts, prepared, as described in Section 4.4. Conversely, controls were grown onto rat tail standard collagen coated plates, prepared, as already described in the same paragraph. Cells were incubated for 3 days, 6 days, and 15 days at 37 ◦C in complete medium. At the end of the experiments cell viability was assayed by the MTT test (0.5 mg/mL final concentration), as already reported [81]. For the evaluation of cell adhesion on FS-coated wells, experiments were performed in duplicate on 24-well plates. Both L929 and NCTC cell lines were plated at a density of 50,000 cells/well on 24-well plates that were pre-coated with F1, F2, F3, F4 extracts, or rat tail standard collagen as control. Cells were allowed to adhere for 16 h at 37 ◦C in complete medium and then the MTT test was performed as well to estimate the attached cells when compared to control cells on uncoated wells. Data are means ± S.D. of four independent experiments.
