A Method for 3D Bioprinting Murine Cortical Astrocytes

1. Synthesis of gelatin methacryloyl (GelMA)

1. Weigh 10 g of gelatin obtained from porcine skin and dissolve in 100 mL of PBS by letting the solution stir on a heating plate at 240 rpm and 50 °C until complete dissolution.
2. Under a hood, add 2 mL of methacrylic anhydride (MA) for a low degree of functionalization, and let the gelatin emulsion stir at 240 rpm and 50 °C for 2 h.
   CAUTION: MA hazard statement: H302 + H332 (harmful if swallowed or inhaled), H311 (toxic in contact with skin), 314 (causes severe skin burns and eye damage), 315 (causes skin irritation), H317 (may cause an allergic skin reaction), H318 (causes serious eye damage), 331 (toxic if inhaled), H332 (harmful if inhaled), H335 (may cause respiratory irritation). Handling guidelines: P261 (avoid breathing dust/fume/gas/mist/vapours/spray), P305 + P351 + P338 + P310 (IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. Immediately call a POISON CENTER/doctor), P301 + P312 + P330 (IF SWALLOWED: Call a POISON CENTER/doctor if you feel unwell. Rinse mouth).
   NOTE: Add MA very slowly, drop by drop.
3. Dilute the gelatin-MA solution in 100 mL of preheated PBS (50 °C) to obtain 200 mL of final volume and let the solution stir at 240 rpm and 50 °C for 10 min.
4. Cut ~20 cm of dialysis membrane (molecular cutoff 12-14 kDa) and soak it in deionized water until it is soft.
   NOTE: Fill the membranes with deionized water to ensure no holes or defects.
5. Using a funnel, transfer the gelatin-MA solution to the membranes.
   NOTE: Close both sides leaving extra space inside to allow mixture.
6. Place the membranes containing the gelatin-MA solution in a container with 2 L of distilled water for dialysis, and let them stir at 40 °C for 5 days (500 rpm).
   NOTE: Cover the container to avoid water evaporation.
7. Change the distilled water two times a day. Each time, flip the membranes upside down for homogenization.
8. On the fifth day, mix 200 mL of preheated ultrapure water (40 °C) with the dialyzed gelatin-MA and stir for 15 min at 40 °C.
9. Transfer the gelatin-MA solution to 50 mL conical tubes up to 25 mL and let the tubes remain at -80 °C for 2 days.
   ​NOTE: Store the tubes horizontally to facilitate lyophilization.
10. Lyophilize the frozen solutions for 3-5 days and store the lyophilized GelMA protected from humidity.

2. Bioink preparation

NOTE: In order to obtain 1 mL of bioink, it is recommended to fabricate at least 3 mL of biomaterial solution, as there may be losses during filtration.

1. Preparation of fibrinogen solution​
   1. Prepare saline solution (NaCl 0.9%) in deionized water, and dissolve 10 mg of fibrinogen from bovine plasma in 1 mL of the saline solution to obtain a concentration of 10 mg/mL.
      NOTE: As fibrinogen adsorbs to glass, do not use glass flasks to prepare the fibrinogen solution.
   2. Leave the solution under agitation at 37 °C until complete dissolution of fibrinogen.
      ​NOTE: For fibrinogen dissolution, use a rotary system placed inside an oven at 37 °C. Magnetic agitation (180 rpm) of fibrinogen on a hot plate at 37 °C is also suitable. Under this condition, 10 mg/mL fibrinogen takes approximately 40 min to dissolve.
2. Preparation of gelatin/GelMA solution
   1. Weigh 0.12 g of gelatin and add it to 1.9 mL of preheated PBS (40 °C) to obtain a final concentration of 4% (w/v) gelatin. Vortex to facilitate dissolution.
   2. Keep the emulsion at 40 °C until complete dissolution.
   3. Weigh 0.06 g of lyophilized GelMA and transfer it to the gelatin solution to obtain a final concentration of 2% (w/v) GelMA. Vortex to facilitate dissolution.
   4. Keep the solution at 40 °C until complete dissolution.

3. Preparation of astrocytes-laden gelatin/GelMA/fibrinogen bioink

1. Pipette 0.9 mL of the 10 mg/mL fibrinogen solution and transfer to the gelatin/GelMA solution to obtain a final concentration of 3 mg/mL of fibrinogen.
2. Weigh 0.015 g of photoinitiator (PI) and transfer to the gelatin/GelMA/fibrinogen solution to obtain a final concentration of 0.5% (w/v) PI. Mix the solution by flipping the tube up and down and keep it at 40 °C protected from light to avoid PI degradation.
   NOTE: Stock the bioink at 4 °C for a maximum 24 h.
3. Under the laminar flow, filter the solution using a 0.2 µm filter into a sterile 15 mL conical tube.
   NOTE: The biomaterial solution should be at 37-40 °C to allow filtration.
4. Transfer 980 µL of the biomaterial solution to a 15 mL conical tube.
5. Dilute laminin in saline solution to obtain a stock solution of 100 µg/mL.
6. Pipette 20 µL of laminin and transfer to the tube containing the bioink to obtain a final concentration of 2 µg/mL laminin.
7. Mix gently by pipetting up and down, avoiding bubbles. If any bubbles persist, centrifuge the conical tube at 200 x g for 2 min. Keep the bioink at 37 °C until it is mixed with the cells.
8. Trypsinize primary astrocytes with 0.05% trypsin for 5 min.
   NOTE: Use astrocytes from passages 1 to 3.
9. Neutralize the trypsin activity with FBS at a ratio of 1:1 and transfer the cells to a 15 mL conical tube. Centrifuge it at 200 x g for 5 min.
10. Count the cells and transfer 1 x 106 cells to a different conical tube. Centrifuge it at 200 x g for 5 min.
11. Remove the supernatant leaving a small volume (~200 µL) to suspend the cell pellet, by gently tapping the bottom of the conical tube.
12. Transfer 1 mL of gelatin/GelMA/fibrinogen solution to the tube containing the cells and gently pipette up and down to homogenize, obtaining a final concentration of 1 x 10⁶ cells/mL.

3. Preparation of the crosslinker solution

1. Thrombin reconstitution​
   1. Prepare a stock solution of thrombin 100 U/mL in sterile deionized water with 0.1% (w/v) bovine serum albumin (BSA) in a 15 mL conical tube. Stock in microtubes at -20 °C.
      ​NOTE: As thrombin adsorbs to glass, do not use glass flasks to prepare the stock solution or store the aliquots.
2. Preparation of thrombin-CaCl₂ solution
   1. Pipette 100 µL of thrombin stock solution and transfer to a 50 mL conical tube containing 8.9 mL of sterile deionized water to obtain a final concentration of 1 U/mL thrombin.
   2. Prepare a 10% (w/v) CaCl₂ solution in deionized water and sterilize using a 0.2 µm filter.
   3. Transfer 1.1 mL of the 10% CaCl₂ solution to the conical tube containing thrombin in order to obtain a final ratio of 1:9 (CaCl₂ to thrombin).
      ​NOTE: Prepare the crosslinker solution at the volume for the experiment, avoiding storage.

4. Bioprinting astrocytes-laden bioink using an extrusion-based bioprinter

1. Design of the neural tissue​
   1. Using the G-code: construct a grid of 6 x 6 mm (squared shape) with 1 mm of distance between each bioprinted line on the X and Y-axis, and 6 layers on the Z-axis (0.2 mm between each line); set the extrusion (E) to 0.01 mm, increasing 0.001 mm at each new layer of the Z-axis; and set the printing speed (F) to 400 mm/min (Supplemental Information).
2. Bioprinter set up
   1. Expose the machine to UV light for 15 min, and then wipe it down with ethanol 70%.
   2. Turn on the bioprinter using the power switch. Connect the machine to the computer through a USB cable. Open the controlling software to connect it to the bioprinter and load the file design.
3. Preparation of the bioprinting syringe
   1. Transfer the astrocytes-laden gelatin/GelMA/fibrinogen bioink to a 5 mL plastic syringe using a 1,000 µL pipette.
      NOTE: Transfer slowly to avoid bubble formation.
   2. Connect a sterile 22 G blunt needle to the syringe.
      NOTE: Leave the syringe at 4 °C for 2 min.
   3. Connect the syringe to the bioprinter printhead and manually flush the bioink to remove the remaining bubbles.
4. Bioprinting
   NOTE: The bioprinting was performed outside the laminar hood.
   1. Place a 35 mm culture dish on the bioprinter table and position the needle 0.1 mm away from the culture dish surface to allow needle movement.
      ​NOTE: Use one 35 mm culture dish for each bioprinting.
   2. Press the Yazdır button.
   3. Once the bioprinting is over, ensure that the syringe moves away from the dish. Then, close the culture dish and prepare for the cross-linking process.
      ​NOTE: The bioprinting of one construct takes approximately 1 min and 10 s.
5. Crosslinking the bioprinted construct and culture
   1. Place the culture dish under UV light at 2 mW/cm2 for 2 x 60 s (up and down) for GelMA crosslinking.
   2. Under the laminar flow, transfer the bioprinted construct to a 24-well plate using a sterile spatula.
   3. Add 500 µL of thrombin/CaCl₂ solution and leave for 30 min to allow fibrin crosslinking.
   4. Remove the crosslinking solution and wash the construct with 2 mL of PBS 1x. Then, replace the PBS with 1 mL of astrocyte culture medium and incubate at 37 °C and 5% CO₂. Change the medium every 3 days.