Nu 5500e

In vitro safety and efficacy of AuNPs were assessed in murine healthy fibroblasts L-929 (CCL-1^TM, ATCC^®, Manassas, VA, USA), murine breast cancer 4T1 cells (CRL-2539™, ATCC^®, Manassas, VA, USA) and human breast cancer MCF-7 (HTB-22^TM, ATCC^®, Manassas, VA, USA) and MDA-MB-231 cells (HTB-26^TM, ATCC^®, Manassas, VA, USA). The MCF-7 cell line represents an estrogen receptor (ER) and progesterone receptor (PR) positive and HER2 negative cancer [61 (link)], whereas MDA-MB-231 and 4T1 cell lines represent triple-negative cancer [62 (link),63 (link)]. All cell lines were cultured in DMEM with high glucose (4500 mg/L) enriched with 10% of FBS (v/v), 100 IU/mL of penicillin and 100 µg/mL of streptomycin (henceforward, complete medium). Cells were kept in an incubator (NuAire NU-5500E, NuAire, Plymouth, MN, USA) at 37 °C and 5% CO₂ atmosphere, and every two days cell medium was changed when a confluence of 80% was reached.

The human bladder cancer cell line T24 was purchased from the Shanghai Institutes for Biological Sciences (Chinese Academy of Sciences, Shanghai, China). The human bladder cancer cell line 5637 was purchased from the Cell Bank of the Chinese Academy of Sciences. The normal uroepithelium cell line SV-HUC-1 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). The T24, 5637, and SV-HUC-1 cells were cultured in RPMI 1640 medium (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) containing 0.05 g/L streptomycin, 0.05 g/L penicillin, 0.8 g/L NaHCO₃, 3.6 g/L HEPES, and 10% fetal bovine serum (Hangzhou Sijiqing Biotechnology Materials Co., Ltd, Hangzhou, China). Cell culture was performed in a thermostatic incubator (NU-5500E; NuAire, Plymouth, MN, USA) at 37 °C with 5% CO₂ and saturated humidity. Cell suspensions were prepared by digesting the cells in the logarithmic growth phase with 0.25% trypsin (Shanghai Biotech Bioengineering Co., Ltd, Shanghai, China).

The DUC tissues were frozen at −80 °C overnight and were lyophilized in a freeze-drier (TrioScience, Istanbol, Turkey) for 24 h. The lyophilized DUC samples were then cryo milled to powder, and gel was prepared as previously described [34 (link)]. Briefly, the dried DUC powder from 4–5 donors was pooled and digested with 1 mg pepsin/mL (Sigma, USA; then, it was dissolved in 0.05 M HCl) per 10 mg DUC tissue under constant agitation for 48 h at room temperature. Ten and twenty milligram powdered tissue was exposed to UV light for 30 min inside the biosafety cabinet for disinfection, followed by the addition of 1 mL of pepsin/HCL (to make 10 mg/mL and 20 mg/mL hydrogel) in each of the vials for up to 48 h at room temperature with constant agitation to ensure complete digestion and formation of a pre-gel liquid which was then neutralized by the addition of ice-cold solution of 1 M NaOH (1/10th of original digest volume) and 10X PBS (1/10 of final neutralized volume) to attain a pH of 7.4. Neutralized pre-gel solution was allowed to gel by incubation at 37 °C (NU5500E, NuAire, USA) for 20–30 min to be completely turned into hydrogel. Gelation was confirmed either macroscopically or microscopically under phase contrast microscope (TE 2000S Eclipse, Nikon, Tokyo, Japan).