A Method for 3D Bioprinting Murine Cortical Astrocytes

Take an astrocyte suspension.

Add a bioink solution containing gelatin, photocrosslinkable gelatin derivatives, photoinitiators, fibrinogen, and laminin.

Transfer the suspension to a syringe with a blunt needle.

Chill the syringe for bioink gelling. Connect the syringe to the bioprinter's printhead.

Position the needle appropriately to the culture plate. Initiate bioprinting with set parameters and the design.

Astrocyte-containing bioink is pushed out of the needle, and is deposited in layers onto the plate, forming a three-dimensional structure with trapped astrocytes.

Expose the bioprinted construct to ultraviolet light. The photoinitiator aids in gelatin derivative crosslinking, enhancing bioconstruct stability.

Treat with thrombin and calcium ions.

Thrombin, with calcium ions, converts fibrinogen into fibrin, assembling into a stable 3D biopolymer network.

Discard the solution and wash with buffer to remove the crosslinking agents.

Add astrocyte media and incubate. Laminin aids astrocyte adhesion and spreading, forming a 3D neural-like tissue rich in astrocytes.

To obtain a final concentration of 1 x 10⁶ cells per milliliter, transfer 1 milliliter of gelatin-gelatin-methacryloyl-fibrinogen solution to the tube containing the cells, and homogenize by gently pipetting up and down. Use a 1000-microliter pipette to slowly transfer the astrocytes laid in gelatin-gelatin-methacryloyl-fibrinogen bioink solution to a 5-milliliter plastic syringe, avoiding bubble formation. Connect a sterile 22-gauge blunt needle to the syringe.

Expose the bioprinter to UV light for 15 minutes, and then wipe the bioprinter with 70% ethanol. Then connect the syringe to the bioprinter print head, and manually flush the bioink to remove the remaining bubbles.

To conduct bioprinting, place a 35-millimeter culture dish on the bioprinter table. Position the needle 0.1 millimeters away from the culture dish surface to allow movement of the needle, and press the Print button. Once the bioprinting is over, ensure that the syringe moves away from the dish, and close the culture dish. Place the culture dish under UV light for gelatin-methacryloyl cross-linking.

Use a sterile spatula to transfer the bioprinted construct to a 24-well plate, add 500 microliters of thrombin calcium chloride solution, and leave for 30 minutes to allow fibrin cross-linking. After removing the cross-linking solution, wash the construct with 2 milliliters of PBS. Then, replace the PBS with 1 milliliter of astrocyte culture medium. Incubate at 37 degrees Celsius and 5% carbon dioxide, and change the medium every three days.