Hanks balanced salt

Cell culture and seeding. SH-SY5Y cells were cultured in complete media at 37 °C and 5%
CO2 in tissue culture-treated 75 cm 2 -flasks (Nunc). For cell passage, cells were detached from the flasks by incubation at 37 °C with trypsin-EDTA solution 1X (2.5 g porcine trypsin and 0.2 g EDTA•4Na per liter of Hanks′ Balanced Salt, Sigma) and spun at 1000 RCF for 5 min; the obtained pellet was resuspended in 1 ml of complete media and disaggregated by driving the suspension several times through a 25 gauge syringe needle. For cell counting, the cell suspension was serially diluted 1:10 in PBS and 1:2 in the exclusion dye Trypan Blue solution (0.4% in 0.81% sodium chloride and 0.06% potassium phosphate, dibasic, Sigma). 10 μL of the diluted cell suspension was counted in a haemocytometer chamber under transmitted light in an inverted microscope (DMIL, Leica). Inmediately, before cell seeding all the 3D scaffolds were sterilized under UV light and transferred to 8 well glass slides (Millicell EZ Slide, Millipore). Then, 350 µl complete media containing 50,000 SH-SY5Y cells was added to the wells and left still to deposit for 20 min.
The slides were incubated for 3, 7 or 14 days at 37 °C and 5% CO2.

All the cell lines used in this study were cultured in complete Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI) at 37 °C in humidified atmosphere of 5 % CO2 in tissue culture-treated 75 cm 2 -flasks (from Nunc). For cell passage, adherent cells were lifted from the flasks by incubation at 37 °C with trypsin-EDTA solution 1x (2.5 g porcine trypsin and 0.2 g EDTA • 4 Na per liter of Hanks' Balanced Salt from Sigma), span at 1000 g for 5 min and the pellet re-suspended in 1 ml media. For cell counting, the cell suspension was serially diluted 1:10 in PBS and 1:2 in the exclusion dye Trypan Blue solution (0.4 % in 0.8 % sodium chloride and 0.1 % potassium phosphate, dibasic from Sigma Aldrich), 10.0 μL of the diluted cell suspension was counted in a haemocytometer chamber under transmitted light in an inverted microscope (DMIL, Leica).

Type I atelocollagen was extracted from the swim bladder of Bester sturgeon as described previously [28] .
Porcine pepsin (EC 3.4.23.1, 1:10,000, Wako Pure Chemical Industries Ltd., Osaka, Japan) at a concentration of 0.1% was used for removing telopeptides of collagen molecules. For comparison, porcine skin type I collagen (Cellmatrix type I-C, Nitta Gelatin Inc., Osaka, Japan) was used. Cell culture media and penicillin/streptomycin (P/S) solution were obtained from Thermo Fisher Scientific (Waltham, MA).
Hank's balanced salt was obtained from Sigma-Aldrich (St. Louis, MO). Other chemicals were obtained from Wako Pure Chemical Industries Ltd unless otherwise stated. Cell culture wares were from Corning, NY. Cells were obtained from RIKEN Cell Bank (Tsukuba, Japan) and Japanese Collection of Research Bioresources (Tokyo, Japan).