3D bioprinting of astrocytes represents an advance in neuro tissue engineering, allowing the biofabrication of in vitro models that are useful to understand the mechanisms involved in neurological disease. The main advantage of 3D bioprinting is to biofabricate cell adjunct structures that mimic the tissues'biochemical and mechanical features, providing a better understanding of cell dynamics in health and disease. This protocol can be applied to the biofabrication of other soft tissues, as it is based on the 3D bioprinting of a bio-ink composed of natural polymers and extracellular matrix components.
To begin with, cut the cortical tissue isolated from the euthanized mouse into small pieces with a curved micro scissor. Wash the tissue pieces three times with one milliliter of HBSS by pipetting up and down. After removing HBSS added for the third time, add one milliliter of 0.05%trypsin and incubate the tissue for five minutes at 37 degrees Celsius.
For mechanical tissue dissociation, gently pipette the tissue trypsin mixture up and down 15 times. Next, transfer the dissociated mixture to a 15 milliliter conical tube and add an equal volume of FBS to neutralize trypsin activity. Filter the suspension through a 0.4 micrometer cell strainer filter to remove non-dissociated fragments.
Wash the filter with one milliliter of astrocytes medium. Pellet down the filtered cell suspension by centrifugation. After discarding the supernatant, suspend the cell pellet in one milliliter of astrocytes culture medium.
Transfer the cell suspension to a T25 culture flask. Make up total medium volume of 3.5 milliliters and incubate the cells at 37 degrees Celsius and 5%carbon dioxide. For obtaining the fibrinogen at the final concentration of three milligrams per milliliter, transfer 0.9 milliliters of 10 milligrams per milliliter fibrinogen solution to the gelatin-gelatin methacryloyl solution.
For obtaining the photo initiator at the final concentration of 0.5%weight by volume, add 0.015 grams of photo initiator to the prepared gelatin-gelatin methacryloyl fibrinogen solution. After vortexing, keep the solution at 40 degrees Celsius, protected from light to avoid PI degradation. Next, filter the solution through a 0.2 micrometer filter into a sterile 15 milliliter conical tube.
Transfer 980 microliters of the biomaterial solution to a 15 milliliter conical tube. For obtaining the laminin at the final concentration of two micrograms per milliliter, add 20 microliters of diluted laminin to the tube containing bio-ink. Mix gently by pipetting up and down, avoiding bubbles and keep the bio-ink solution at 37 degrees Celsius until ready to get mixed with the cells.
Trypsinize the primary astrocytes with 0.05%trypsin for 5 minutes and neutralize the trypsin activity with FPS at a ratio of one to one. Then, transfer the cells to a 15 milliliter conical tube and centrifuge at 200 times G for five minutes. After counting the cells, transfer 1 times 10 to the 6 cells to a different conical tube and centrifuge as demonstrated.
Leave only 200 microliters of the supernatant in the tube and suspend the cell pellet by gently tapping the bottom of the conical tube. To obtain a final concentration of 1 times 10 to the 6 cells per milliliter, transfer one 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 bio-ink solution to a five 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 bio-ink to remove the remaining bubbles. To conduct bioprinting, place at 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 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 two milliliters of PBS, then replace the PBS with one milliliter of astrocytes culture medium. Incubate at 37 degrees Celsius and 5%carbon dioxide and change the medium every three days.
Use a spatula to transfer the bioprinted construct to a 35 millimeter culture dish. Wash the construct with one milliliter of PBS. Deposit 100 microliters of the live dead reagent over the construct and keep it at 37 degrees Celsius for 30 minutes, keeping it protected from light.
After removing the live dead reagent, wash the construct with PBS as demonstrated. Transfer the sample to a confocal dish and observe the cells within the construct under a confocal microscope using 488 and 570 nanometers excitation for image acquirement. After 3D bioprinting, the integrity of the bioprinted scaffold was estimated and the formation of a well-defined structure was observed with cells entrapped within the biomaterial.
After bioprinting and cross-linking processes, most cells presented round morphology when the construct was incubated with an astrocyte medium. The bioprinted scaffolds maintained integrity after seven days of incubation, and although some round cells were observed, many astrocytes spread throughout the construct, presenting astrocytic morphology and interconnection. The cell viability was evaluated right after bioprinting and results showed that at the lower speed, viable cells represented up to 74%of total cells, being significantly higher than cells bioprinted at higher speed.
The viability of bioprinted astrocytes was normalized to 2D cultured astrocytes and the results indicated that on the seventh day, the bioprinted astrocytes had significantly increased viability. Immediately after the bioprinting, the astrocytes showed round morphology and live dead assay. After one week, the astrocytes spread throughout the construct and presented a distinct morphology of cells, identical to 2D culture.
The 3D bioprinted astrocytes were characterized by immunofluorescence. After seven days of incubation in the bioprinted construct, highly dense astrocytes with a star-like morphology were observed. The bioprinted astrocyte cells expressed the specific astrocytes marker glial fibrillary acidic protein indicates retainment of astrocytic phenotype after seven days of bioprinting, and these results indicate that the bio-ink composition provided a biocompatible microenvironment to promote astrocytes'adhesion, spread and growth.
The evaluation of specific genes and cell markers expression by the bioprinted astrocytes would provide evidence of the neuro tissue-like function, such the astrocytary reactivity following specific stimuli. This protocol paves the way for the biofabrication of more complex neuro-like structures composed of different types of cells in biomolecules, allowing the mimicking of specific neurogenic niches.
