Recombinant Spider Silk Enhances Neural Stem Cell Proliferation

Mouse neural stem cells (NSCs) were used for the cell culture test. NSCs derived from the brains of adult mice were cultured in Ham’s F-12 and HG-DMEM (1:1), with 10% fetal bovine serum (FBS; Caisson), 1% penicillin-streptomycin-amphotericin (PSA; Caisson), and 400 mg/mL G418 (Invitrogen), in a humid incubator containing 5% CO₂ at 37 °C. The culture medium was refreshed every day. The recombinant spider silk-coated surface (area of 1.9 cm²), in a 24-well plate, was immersed in 75% ethanol for 1 h and irradiated with UV light for 1 h before NSC inoculation (cell density 4 × 10⁴ cells/cm²). The proliferation of cells on the surface was evaluated by the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich) assay [27 (link)]. The reacted CCK-8 solution was mixed gently to ensure uniformity and then collected into a 96-well plate. The absorbance, at a wavelength of 450 nm, was detected by a plate reader (SpectraMax M5, Molecular Devices, San Jose, CA, USA). Statistical differences between the experimental groups were performed with the student’s t-test. The result was considered statistically significant when the p-value was smaller than 0.05.