Sodium bicarbonate

Medical grade CS 95/1000 (with a molecular weight range between 200 and 500 kDa was) purchased from Heppe Medical (Berlin, Germany), TPP was obtained from Acros Organics (Geel, Belgium), 35% hydrochloric acid, sodium hydroxide palettes, and glacial acetic acid were all acquired from VWR (Prague, Czech Republic). The following solutions were freshly prepared by using deionized water from VWR (Prague, Czech Republic): 2 M HCl, 10 M NaOH, 1%, and 2% (v/v) acetic acid. GRS Taq DNA polymerase was purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA). TripleXtractor used in RNA extraction was obtained from GRISP (Porto, Portugal). DMEM-F12 and DMEM-HG were purchased from GIBCO (Waltham, MA, USA). Sodium bicarbonate was obtained from MP Biomedicals (Santa Ana, CA, USA). Agarose, MgCl₂ and GreenSafe were obtained from NZYtech (Lisbon, Portugal). All solutions were freshly prepared by using ultra-pure grade water, purified with a Milli-Q system from Millipore (Billerica, MA, USA).

Urothelial cells (UROtsa and T24) were diluted in their respective medium at a concentration of 50 million cells/ml. Consequently, the cells were mixed with a gel containing Type I collagen (84%; Advanced Bio Matrix), l-glutamine (7.8%; Sigma-Aldrich), sodium bicarbonate 7.5% (2.1%; MP Biomedicals LLC, Solon, Ohio, USA) and 10 × Minimum Essential Medium (10 × MEM; Invitrogen) 6.5%; to a final cell concentration in the gel of 500,000 cells/ml. Cell culture was performed in 12-filter culture dishes (Transwell Permeable Supports, Costar, USA; product # 3460). Before adding the cells to the culture dishes, the filters of the dishes were coated with cell-free Type I collagen gel suspension and were allowed to solidify at 37 °C; 250 μl of the cell-gel suspension was then added to the coated filters, and the culture dish was again placed in an incubator at 37 °C for the gel to solidify for 30 min. The cell-gel suspension was grown in DMEM (UROtsa) or McCoy’s 5a medium (T24) in an incubator at 37 °C (5% CO₂). Medium renewal occurred every 2–3 days.

Medical grade high molecular weight (HMW) chitosan, with a molecular weight range between 200 and 500 kDa, was purchased from Heppe Medical (Halle, Germany). Low molecular weight (LMW) chitosan, with a molecular weight range between 50 and 190 kDa, chitosan of 5 kDa molecular weight and GRS Taq DNA polymerase were purchased from Sigma Aldrich Chemicals (St. Louis, MO, USA). chitosan of 20 kDa molecular weight was purchased from Gletham Life Sciences (Corsham, UK). Sodium tripolyphosphate (TPP) was obtained from Across Organics (Geel, Belgium). TripleXtractor used in RNA extraction was obtained from GRISP (Porto, Portugal). DMEM-F12 was purchased from GIBCO (Waltham, MA, USA). MEM-α was purchased from HyClone classical media (Boston, MA, USA). Sodium bicarbonate was obtained from MP Biomedicals (Santa Ana, CA, USA). Agarose and GreenSafe was obtained from NZYtech (Lisbon, Portugal).