3.7. Cytotoxicity Assays

Cytotoxicity of PUF and PUF@Cu-BTC was assessed as described in prior papers [58]. MEFs were maintained at 37 °C in a humidified incubator with 5% CO₂ in T75 flasks containing DMEM (supplemented with 10% (v/v) FBS, 200 IU·mL⁻¹ penicillin, and 200 μg·mL⁻¹ streptomycin). On the collagen type I-coated glass slide (1.25 cm × 1.25 cm), MEFs were seeded at a density of 5 × 10⁴ cells·cm⁻¹ and cultured for 3 h. Non-adherent cells were rinsed in PBS, transferred to a well plate, and cultured. Each PUF and PUF@Cu-BTC (1.5 cm diameter) was carefully placed on the cell monolayer at the next day. The incubation period was 24 h. The live/dead assay (ThermoFisher Scientific, Waltham, MA, USA) was used to examine the cells, with the live cells fluorescing green and the dead cells fluorescing red. Inverted fluorescence microscopy (IX83, Olympus, Center Valley, PA, USA) was used to examine the labeled cells after they had been washed in PBS. The ratio of living cells to the total number of cells was used to determine cell viability. In addition, the viability of cells was quantified using an MTS test, which assesses the metabolic rates. PUF and PUF@Cu-BTC were incubated in a cell culture medium for 24 h to obtain the extract solutions, and the cells were seeded on a 24-well plate at a density of 5 × 10⁴ cells per well, as stated in earlier reports. The culture media in each well was changed with DMEM containing the extract solution (200 μL) after 24 h of incubation at 37 °C [59]. The DMEM-containing extract solution was carefully removed after 24 h of incubation, and each well was subsequently filled with an MTS cell proliferation assay kit solution (20 μL) and a fresh media (200 µL). The absorbance at a wavelength of 490 nm was measured using a microplate reader after an additional 4 h of incubation (Synergy H1, BioTek, Winooski, VT, USA). The number of proliferating cells was determined using the following equation and a standard curve of cells (Equation (2)):

where the absorbance of the wells containing extract solution was OD sample, the absorbance of the wells containing only culture media was OD control, and the absorbance of the wells without cell was OD blank.