To begin, maintain sterility by cleaning gloves, cartridges, and culture plates with 70%ethanol. Next, switch on the compressor for the bioprinter and select the initialized button to initialize the printer. Wipe down surfaces inside the printer with 70%ethanol.
Using the Print Run button, load the print file and open the Print Protocol PDF. Thaw bioink, activator fluids, and 50 milliliters of complete media at room temperature. Prepare the printing cartridge as instructed in the Print Protocol PDF, ensuring the correct volumes of reagents in specified compartments.
Next, add 1.2 milliliters of F3 to C1, 1.2 milliliters of F32 to C2, and 200 microliters of F261 to C4.Remove the polyethylenimine and laminin coated plate from the incubator. Set the plate holder compartment to a low profile plate. Insert the plate in the right hand plate holder compartment of the printer.
Remove the lid from the plate and place it in the lid holder. Place the print cartridge into the bioprinter and select the Print Inert Base button to start the print run. After thawing, centrifuge the glutamatergic neurons and astrocytes, aspirate the supernatant and resuspend both cell types separately in one milliliter of complete media.
Mix 20 microliters of the cell suspension with the same volume of trypan blue and count the cells to determine viable cell concentration for each cell type. Next, combine 3 million glutamatergic neurons with 1 million astrocytes in a 15 milliliter tube, and add complete media to make a final volume of eight milliliters. Centrifuge the cell suspension and aspirate the supernatant without disturbing the pellet.
Suspend the pellet in 200 microliters of activator fluid F299 by pipetting up and down. Place the lids on the cartridge and culture plate, and remove them from the bioprinter. In a biosafety cabinet, add 200 microliters of cell suspension to well C3 of the print cartridge.
Reinsert the cartridge into the bioprinter and remove the lid as demonstrated previously. Initiate the print models stage of the print run. Concurrently, replace the laminate media from the 2D control plate with complete media.
Insert the bioprinting targeting plate into the bioprinter and remove the lid. Begin the bioprint targeting process. When prompted, remove the targeting plate from the bioprinter.
Use the guide to identify locations where droplets can be observed on the plate. Reinsert the targeting plate into the bioprinter to repeat the droplet printing and selection process. When prompted, replace the targeting plate with a cell culture plate.
After printing, add complete media to all 3D co-culture wells, and incubate at 37 degrees Celsius and 5%carbon dioxide. Finally, dispose of cartridges and remaining liquids. according to lab protocols.
After seven days of printing, almost no single cells remained and interconnecting bundles of neurites and astrocytic projections appeared fortified. Neurite outgrowth increased linearly between 12 to 156 hours. During neurite outgrowth, cell body clusters also increased in size.
Cell viability analysis showed that approximately 72%of total cells were live on day four while 29%were dead. Immunostaining confirmed the healthy cell morphology of the bioprinted neurons and astrocytes with both cell types exhibiting outgrowth of cellular protrusions. The glutamatergic phenotype of the neurons was confirmed through immunostaining for glutamate receptor 2.
