Inkredible

Collagen-Matrigel inks were incubated on ice for 1 h and then 3D printed at room temperature (~20°C) as described previously^{59 (link), 60} . 3D-printing experiments were completed using a microextrusion bioprinter (Inkredible+, CELLINK, Sweden) and conical polyethylene nozzles with a diameter of 254 μm (Nordson EFD, Robbinsville, NJ). The printing pressure and speed were ~30 kPa and 80 mm/s, respectively, and the distance of separation between the printing nozzle and the substratum was ~0.1 mm. Samples were 3D printed onto no. 1 glass coverslips and gelled in an incubator at 37°C and 5% CO₂. To prevent alignment of collagen fibers due to evaporation^{61 (link)}, ~300 μL of sterile phosphate-buffered saline (PBS) was added around the construct before incubation. Prior to printing, glass coverslips were treated in a UV/ozone (UVO) cleaner (Jelight Company, Irvine, CA) for 7 min and then silanized by exposure to 3,3,3-trifluoropropyl-trichlorosilane (Alfa Aesar, Haverhill, MA) under vacuum for 20 min.

Polydimethylsiloxane (PDMS) molds were sterilized in 70% ethanol and UV overnight, followed by a thorough washing step with sterile DPBS. The supporting astrocyte-laden bath was pipetted into the molds and kept at 4 °C for 5–8 min prior to printing. The bioink was loaded into 1 ml syringes (BD) capped with 30 G needles (blunt, BD) and mounted on the extruder of a Cellink Inkredible bioprinter. The tip of the needle was positioned at a designated point with respect to the position of the mold containing the supporting hydrogel, which represented the XYZ origin for all the printing configurations. A syringe pump (New Era Pump Systems) was used to extrude the bioink at 3 μl min⁻¹. After printing, the full structures were crosslinked under UV light (800 mW, 60 s) using an OmniCure S2000 machine. Next, the constructs were extracted from the molds, washed with DPBS and transferred to a well plate containing fresh MEM complete media (MEM + 10% FBS + 1% P/S). All printed constructs were incubated at 37 °C in 5% CO₂ in well plates immediately after crosslinking. Culture medium was changed the day after printing, and every 2 d after that until analysis was done at different time points.