2.4.1. Cell Culture Studies

Before using the samples for in vitro cell culture, it is essential to remove the DMF/MEK mixed solvents because of the possible cytotoxicity. All of the samples were washed with distilled water for 48 h and dried in oven at 80 ◦C overnight. Then, the nanofibers and films were cut into round shapes with a diameter of 10 mm, with three replicates per sample prepared. Sterilization was performed by deeply soaking the samples in 70% ethanol aqueous solution in a multi-well tissue culture polystyrene (TCPS) dish for 1 h, followed by rinsing three times in phosphate buffer saline (PBS) to remove all traces of ethanol.

NIH3T3 mouse embryonic fibroblasts were used to measure the cell adhesion and proliferation. For the cell adhesion test, 50,000 cells (in 1 mL of medium) were mixed well into the sample. After 3 h, the cells were harvested in 1 mL of 0.5% Triton X-100/PBS solution, and evaluated by LDH assay for adhesion evaluation of the cells.

The cell proliferation test was a quantitative investigation of the capacity for the cells to grow on the electrospun nanofibers and films. At the seeding step of the proliferation culture, 5000 cells were added into the sample. The experiment lasted for a total of seven days, and the results of the first, third, fifth, and seventh days were compared.

The LDH activity was immediately measured by ultraviolet absorption at a wavelength of 340 nm, using the Thermo Scientific Multiskan FC microplate photometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) with a recorder. The enzyme activity of LDH can be measure from chemical reaction of LDH, when it is released into the cells' medium from the damaged or dead cells, due to cell membrane damage. LDH converts lactate using NAD as a coenzyme, and produces pyruvic acid and NADH. The number of cells was calculated from the calibration curve obtained by the relation between the known number of cells, and the absorbance value at 340 nm of NADH in the assay supernatant.

The shape of the cells was observed by SEM to qualitatively investigate the cell reactions when in contact with the electrospun nanofibers. After each incubation period, the sample was fixed with paraformaldehyde (PFA) as a cross-linking fixation agent, to stop the proliferation of cells, and to preserve their shapes. The sample was further dehydrated by serially using ethanol gradient solutions of 50, 70, 95, and 99.5% for 30 min each, over a continuous process. Then, the sample was coated with platinum for SEM analysis.
