This work was supported by The S&#227;o Paulo Research Foundation (FAPESP), grant numbers 2018/23039-3 and 2018/12605-8; National Council for Scientific and Technological Development (CNPq), grant numbers 465656/2014-5 and 309679/2018-4; and Coordination for the Improvement of Higher Education Personnel (CAPES), financial code 001.

All the procedures involving animals followed international guidelines for animal use in research (http://www.iclas.org) and were approved by the Committee for Ethics in Research of Universidade Federal de S&#227;o Paulo (CEUA 2019/ 9292090519).
1. Mice brain dissection
<ol>
	<li>Transfer 10 mL of cold Hanks Buffered Salt Solution (HBSS) to a 100 mm culture dish and 1 mL to a 1.5 mL microtube. Prepare one microtube per animal. 
	NOTE: Both the culture dish and the microtube need to be kept on ice.</li>
	<li>Prepare astrocytes culture medium using DMEM F12 + 10% Fetal Bovine Serum (FBS), 2% glutamine, and 1% Penicillin-Streptomycin (P/S). Sterilize the medium by filtering using a 0.2 &#181;m filter.</li>
	<li>Euthanize C57Bl/6 mice pups (postnatal day 1) by decapitation using a sharp operating scissor. Using forceps, pull the skin and expose the skull. Ensure both scissors and forceps are sterilized with 70% ethanol.</li>
	<li>Cut the skull from the foramen magnum to the top of the head along the sagittal plane using a sharp curved tip scissor. 
	NOTE: Make sure the encephalic tissue is not damaged.</li>
	<li>Using a spatula previously sterilized with ethanol 70%, lift the brain from the cranial cavity and place it in the culture dish containing 10&#160;mL of cold HBSS.</li>
	<li>Place the culture dish containing the brain under the stereomicroscope, and using two blunt-tip forceps, remove the meninges from the brain (Figure 1).</li>
	<li>Separate the cortices from the rest of the brain by gently rolling them away from the median line of the brain using a spatula.</li>
	<li>Collect both cortices and immediately transfer them to the same microtube containing 1 mL of cold HBSS.</li>
</ol>
2. Astrocytes isolation and culture
<ol>
	<li>Under the laminar flow, cut the cortical tissue into small pieces using a curved micro scissor, and wash them with 1 mL of HBSS by pipetting up and down 3x. Wait for the tissue to settle down. Remove HBSS and add fresh HBSS, repeating the process two more times.</li>
	<li>Remove HBSS and incubate the tissue with 1 mL of 0.05% trypsin at 37 &#176;C for 5 min. 
	NOTE: Only trypsin digestion is sufficient at this point.</li>
	<li>Mechanically dissociate the tissue by gently pipetting up and down 15x. 
	NOTE: The complete dissociation of the tissue is observed by the increase in the suspension turbidity and by the absence of large fragments of tissue in the suspension.</li>
	<li>Transfer the solution to a 15 mL conical tube, neutralize trypsin activity by adding an equal volume of FBS, and filter the solution in a cell strainer filter of 0.4 &#181;m to remove non dissociated fragments.</li>
	<li>Wash the filter with 1 mL of astrocytes medium, collect the cell suspension that passed through the strainer, and centrifuge it for 5 min at 200 x g and 25 &#176;C. After centrifugation, discard the supernatant and suspend the pellet in 1 mL of astrocytes culture medium.</li>
	<li>Transfer the cell suspension to a T25 culture flask, make up the volume of the medium to 3.5 mL, and incubate the cells at 37 &#176;C and 5% CO2.</li>
	<li>Ensure that after 24 h, the cells are adherent. Then, replace the medium and change it every 3 days.</li>
	<li>After 7 days, remove microglia and oligodendrocytes from the culture by washing the cells with 2 mL of 1x PBS.</li>
	<li>Replace the PBS solution with the astrocytes culture medium and leave the culture flask in an orbital shaker at 180 rpm overnight. 
	&#8203;NOTE: Astrocytes form a confluent monolayer in approximately 10-12 days of culture.</li>
</ol>
3. Synthesis of gelatin methacryloyl (GelMA)
<ol>
	<li>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 &#176;C until complete dissolution.</li>
	<li>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 &#176;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.</li>
	<li>Dilute the gelatin-MA solution in 100 mL of preheated PBS (50 &#176;C) to obtain 200 mL of final volume and let the solution stir at 240 rpm and 50 &#176;C for 10 min.</li>
	<li>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 make sure there are no holes or defects.</li>
	<li>Using a funnel, transfer the gelatin-MA solution to the membranes. 
	NOTE: Close both sides leaving extra space inside to allow mixture.</li>
	<li>Place the membranes containing the gelatin-MA solution into a container with 2 L of distilled water for dialysis, letting them stir at 40 &#176;C for 5 days (500 rpm). 
	NOTE: Cover the container to avoid water evaporation.</li>
	<li>Change the distilled water two times in a day. Each time, flip the membranes upside down for homogenization.</li>
	<li>On the fifth day, mix 200 mL of preheated ultrapure water (40 &#176;C) to the dialyzed gelatin-MA, and let it stir for 15 min at 40 &#176;C.</li>
	<li>Transfer the gelatin-MA solution to 50 mL conical tubes up to 25 mL and let the tubes remain at -80 &#176;C for 2 days. 
	&#8203;NOTE: Store the tubes horizontally to facilitate lyophilization.</li>
	<li>Lyophilize the frozen solutions for 3-5 days and store the lyophilized GelMA protected from humidity.</li>
</ol>
4. 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.
<ol>
	<li>Preparation of fibrinogen solution
	<ol>
		<li>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.</li>
		<li>Leave the solution under agitation at 37 &#176;C until complete dissolution of fibrinogen. 
		NOTE: For fibrinogen dissolution, use a rotary system placed inside an oven at 37 &#176;C. Magnetic agitation (180 rpm) of fibrinogen on a hot plate at 37 &#176;C is also suitable. Under this condition, 10 mg/mL fibrinogen takes approximately 40 min to dissolve.</li>
	</ol>
	</li>
	<li>Preparation of gelatin/GelMA solution
	<ol>
		<li>Weigh 0.12 g of gelatin and add it to 1.9 mL of preheated PBS (40 &#176;C) to obtain a final concentration of 4% (w/v) gelatin. Vortex to facilitate dissolution.</li>
		<li>Keep the emulsion at 40 &#176;C until complete dissolution.</li>
		<li>Weigh 0.06 g of lyophilized GelMA and transfer to the gelatin solution to obtain a final concentration of 2% (w/v) GelMA. Vortex to facilitate dissolution.</li>
		<li>Keep the solution at 40 &#176;C until complete dissolution.</li>
	</ol>
	</li>
	<li>Preparation of astrocytes-laden gelatin/GelMA/fibrinogen bioink
	<ol>
		<li>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.</li>
		<li>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&#160;at 40 &#176;C protected from light to avoid PI degradation. 
		NOTE: Stock the bioink at 4 &#176;C for a maximum 24 h.</li>
		<li>Under the laminar flow, filter the solution using a 0.2 &#181;m filter into a sterile 15 mL conical tube. 
		NOTE: The biomaterial solution should be at 37-40 &#176;C to allow filtration.</li>
		<li>Transfer 980 &#181;L of the biomaterial solution to a 15 mL conical tube.</li>
		<li>Dilute laminin in saline solution to obtain a stock solution of 100 &#181;g/mL.</li>
		<li>Pipette 20 &#181;L of laminin and transfer to the tube containing the bioink to obtain a final concentration of 2 &#181;g/mL laminin.</li>
		<li>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 &#176;C until it is mixed with the cells.</li>
		<li>Trypsinize primary astrocytes with 0.05% trypsin for 5 min. 
		NOTE: Use astrocytes from passages 1 to 3.</li>
		<li>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.</li>
		<li>Count the cells and transfer 1 x 106 cells to a different conical tube. Centrifuge it at 200 x g for 5 min.</li>
		<li>Remove the supernatant leaving a small volume (~200 &#181;L) to suspend the cell pellet, by gently tapping the bottom of the conical tube.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
5. Preparation of the crosslinker solution
<ol>
	<li>Thrombin reconstitution
	<ol>
		<li>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 &#176;C. 
		NOTE: As thrombin adsorbs to glass, do not use glass flasks to prepare the stock solution or store the aliquots.</li>
	</ol>
	</li>
	<li>Preparation of thrombin-CaCl2 solution
	<ol>
		<li>Pipette 100 &#181;L of thrombin stock solution and transfer to a 50 mL conical tube containing 8.9&#160;mL of sterile deionized water to obtain a final concentration of 1 U/mL thrombin.</li>
		<li>Prepare a 10% (w/v) CaCl2 solution in deionized water and sterilize using a 0.2 &#181;m filter.</li>
		<li>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). 
		&#8203;NOTE: Prepare the crosslinker solution at the volume to be used in the experiment, avoiding storage.</li>
	</ol>
	</li>
</ol>
6. Bioprinting astrocytes-laden bioink using an extrusion-based bioprinter
<ol>
	<li>Design of the neural tissue
	<ol>
		<li>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).</li>
	</ol>
	</li>
	<li>Bioprinter set up
	<ol>
		<li>Expose the machine to UV light for 15 min, and then wipe it down with ethanol 70%.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Preparation of the bioprinting syringe
	<ol>
		<li>Transfer the astrocytes-laden gelatin/GelMA/fibrinogen bioink to a 5 mL plastic syringe using a 1,000 &#181;L pipette. 
		NOTE: Transfer slowly to avoid bubble formation.</li>
		<li>Connect a sterile 22 G blunt needle to the syringe. 
		NOTE: Leave the syringe at 4 &#176;C for 2 min.</li>
		<li>Connect the syringe to the bioprinter printhead and manually flush the bioink to remove the remaining bubbles.</li>
	</ol>
	</li>
	<li>Bioprinting 
	NOTE: The bioprinting was performed outside the laminar hood.
	<ol>
		<li>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 movement of the needle. 
		NOTE: Use one 35 mm culture dish for each bioprinting.</li>
		<li>Press the Print button.</li>
		<li>Once the bioprinting over, ensure that the syringe moves away from the dish. Then, close the culture dish and prepare for the crosslinking process. 
		&#8203;NOTE: The bioprinting of one construct takes approximately 1 min and 10 s.</li>
	</ol>
	</li>
	<li>Crosslinking the bioprinted construct and culture
	<ol>
		<li>Place the culture dish under UV light at 2 mW/cm2 for 2 x 60 s (up and down) for GelMA crosslinking.</li>
		<li>Under the laminar flow, transfer the bioprinted construct to a 24-well plate using a sterile spatula.</li>
		<li>Add 500 &#181;L of thrombin/CaCl2 solution and leave for 30 min to allow fibrin crosslinking.</li>
		<li>Remove the crosslinking solution and wash the construct with 2 mL of PBS 1x. Then, replace the PBS with 1 mL of astrocytes culture medium and incubate at 37 &#176;C and 5% CO2. Change the medium every 3 days.</li>
	</ol>
	</li>
</ol>
7. Assessment of astrocytes viability
<ol>
	<li>Viability of bioprinted astrocytes
	<ol>
		<li>Transfer the bioprinted construct to a 35 mm culture dish using a spatula.</li>
		<li>Wash the construct with 1 mL of 1x PBS.</li>
		<li>Deposit 100 &#181;L of the Live/Dead reagent over the construct and keep it at 37 &#176;C for 30 min, keeping it protected from light.</li>
		<li>Remove the Live/Dead reagent and wash the construct with 1 mL of 1x PBS.</li>
		<li>Transfer the sample to a confocal dish using a spatula, and observe the cells within the construct under a confocal microscope using 488 and 570 nm excitation for images acquirement. 
		NOTE: Use a magnification of 10x for an overall visualization of the cells within the construct.</li>
		<li>Make sure the sample is sitting flat. If necessary, place a coverslip over the sample to increase the flatness. 
		NOTE: During imaging, ensure the confocal dish is well sealed to prevent the sample from drying out.</li>
		<li>Calculate the number of viable (green) and dead (red) using a computational software.</li>
	</ol>
	</li>
	<li>Viability of 2D astrocytes culture
	<ol>
		<li>Seed 0.5 x 106 astrocytes (passage 1-3) in a 35 mm confocal dish, add astrocytes medium, and incubate them at 37 &#176;C and 5% CO2.</li>
		<li>When cells are confluent, remove the culture medium and wash with 1 mL of 1x PBS.</li>
		<li>Deposit 200 &#181;L of the Live/Dead reagent, and keep the dish at 37 &#176;C for 30 min, protected from light.</li>
		<li>Remove the Live/Dead reagent and wash the cells with 1 mL of 1x PBS.</li>
		<li>Take the dish to a confocal microscope coupled with a digital camera, and use 488 and 570 nm excitation for image acquirement.</li>
		<li>Calculate the number of viable (green) and dead (red) using a computational software.</li>
	</ol>
	</li>
</ol>
8. Immunostaining of astrocytes
<ol>
	<li>Glial fibrillary acidic protein (GFAP) staining of 3D bioprinted astrocytes 
	NOTE: To investigate the presence of other cell markers, change the primary antibody accordingly.
	<ol>
		<li>Remove the medium from the well and wash the construct with 1 mL of 3x PBS.</li>
		<li>Add 4% paraformaldehyde (PFA) in PBS to the well until the construct is completely covered and leave it for 2 h at 4 &#176;C.</li>
		<li>Remove the PFA and wash the construct with 1 mL of 3x PBS. 
		NOTE: This procedure can be paused for several months if stored at 4 &#176;C in PBS. Make sure the well plate is sealed to avoid PBS evaporation.</li>
		<li>Treat the sample with glycine 0.1 mol/L for 5 min.</li>
		<li>Wash with 1 mL of 1x PBS for 5 min.</li>
		<li>Permeabilize the sample with PBS containing 0.1% Triton X-100 and 10% FBS for 1 h at 25 &#176;C under orbital agitation.</li>
		<li>Incubate the construct with chicken anti-GFAP (primary antibody dilution 1:500) at 4 &#176;C overnight.</li>
		<li>Aspirate the primary antibody and wash the sample with 1 mL of PBS for 5 min, 3 times. 
		NOTE: Primary antibody can be reused for several times when stored at 4 &#176;C.</li>
		<li>Incubate the sample with Alexa fluor 488-conjugated anti-chicken (secondary antibody dilution 1:500) and 1 &#181;g/mL DAPI for 1 h at 25 &#176;C under orbital agitation. 
		NOTE: Keep the sample protected from light.</li>
		<li>Wash the sample with 1 mL of PBS for 5 min, 3 times.</li>
		<li>Transfer the construct to a 35 mm confocal dish. 
		NOTE: Make sure the dish is well sealed to prevent the sample from drying out.</li>
	</ol>
	</li>
	<li>Glial fibrillary acidic protein (GFAP) staining of 2D astrocytes culture
	<ol>
		<li>Seed 0.5 x 106 astrocytes (passage 1-3) in a 35 mm confocal dish, add astrocytes medium, and incubate them at 37 &#176;C and 5% CO2.</li>
		<li>When the cells are confluent, remove the culture medium and wash them with 1 mL of 1x PBS.</li>
		<li>Repeat steps 8.1.2-8.1.10.</li>
	</ol>
	</li>
	<li>Astrocytes cytoskeleton staining
	<ol>
		<li>Repeat steps 8.1.1-8.1.6.</li>
		<li>Add 200 &#181;L of 50 &#181;g/mL of fluorescent-conjugated phalloidin solution in PBS and 1 &#181;g/mL of DAPI over the construct.</li>
		<li>Incubate for 1 h at 25 &#176;C under orbital agitation, keeping the sample protected from light.</li>
		<li>Wash the sample with 1 mL of PBS for 5 min 3 times , and transfer the construct to a confocal dish using a spatula. 
		&#8203;NOTE: Make sure the dish is well sealed to prevent the sample from drying out.</li>
	</ol>
	</li>
</ol>
9. Confocal imaging
<ol>
	<li>Take the dishes to a confocal microscope coupled with a digital camera for imaging (359, 488, and 570 nm excitation).</li>
	<li>Use magnification of 10x for an overall visualization and 40 or 63x for zoomed images of cells. 
	NOTE: Make sure the sample is sitting flat. If necessary, place a coverslip over the sample to increase the flatness.</li>
</ol>

The authors have no conflicts to disclose.

The 3D bioprinting technology has emerged as a biofabrication alternative that allows the engineering of refined constructs that structurally and physiologically resemble native tissues22, including the brain23. The biofabrication of neural-like tissues allows for in vitro native microenvironment modeling, being an important tool for understanding the cellular and molecular mechanisms associated with the development and treatment of many diseases that affect the CN...

Astrocytes are the most abundant cell type in the Central Nervous System (CNS) and play a key role in brain homeostasis. In addition to enduring neuronal support, astrocytes are responsible for modulating neurotransmitters uptake, maintaining the blood-brain barrier integrity, and regulating neuronal synaptogenesis1,2. Astrocytes also have an essential role in CNS inflammation, responding to injuries to the brain in a process that leads to astrocitary reactivity or reactive astrogliosis3,4, forming a glial scar that prevents healthy tissue exposition to degenerative agents5. This event results in changes in astrocytes&#39; gene expression, morphology, and function6,7. Therefore, studies involving astrocytes&#39; functionality are helpful for the development of therapies to treat neurologic disorders.
In vitro models are crucial for studying mechanisms related to neurological injuries, and although successful isolation and two-dimensional (2D) culture of cortical astrocytes have been established8, this model fails to provide a realistic environment that mimics native cell behavior and to reproduce the complexity of the brain9. In 2D condition, the poor mechanical and biochemical support, low cell-cell and cell-matrix interactions, and cell flattening leading to the absence of basal-apical polarity, affect cell signaling dynamics and experimental outcomes leading to altered cell morphology and gene expression, which compromise response to treatments10. Therefore, it is crucial to develop alternatives that provide a more realistic neural environment, aiming to translate the results to the clinic.
Three-dimensional (3D) cell culture represents a more advanced model that recapitulates with increased fidelity features of organs and tissues, including the CNS11. Regarding glial culture, 3D models contribute to the maintenance of astrocytes morphology, cell basal-apical polarity, and cell signaling12,13. The 3D bioprinting technology emerged as a powerful tool to biofabricate 3D living tissues in a controlled manner by using cells and biomaterials to recreate the structure and properties of native tissues. The use of this technology has led to a substantial improvement of results prediction and has contributed to regenerative medicine applied to the CNS14,15,16.
The protocol described here details the isolation and culture of cortical astrocytes. The protocol also details a reproducible method to bioprint astrocytes embedded in gelatin/gelatin methacryloyl (GelMA)/fibrinogen, supplemented with laminin. In this work, an extrusion-based bioprinter was used to print the biomaterial composition containing cortical astrocytes at a density of 1 x 106 cells/mL. Bioprinting shear stress was minimized by controlling the printing speed, and astrocytes showed high viability after the process. Bioprinted constructs were cultured for 1 week, and astrocytes were able to spread, attach, and survive within the hydrogel, maintaining the astrocytic morphology and expressing a specific marker glial fibrillary acidic protein (GFAP)4.
This procedure is compatible with piston-driven extrusion-based bioprinters and can be used to bioprint astrocytes derived from different sources. The 3D bioprinted model proposed here is suitable for a wide range of neural engineering applications, such as studies of the mechanisms involved in astrocytes functionality in healthy tissues and understanding the progression of neurological pathologies and treatment development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3D Bioprinter</td>
			<td>3D Biotechnology Solutions</td>
			<td></td>
			<td>Extrusion-based bioprinter</td>
		</tr>
		<tr>
			<td>Blunt-tip forceps</td>
			<td>Integra Miltex</td>
			<td>6--30</td>
			<td>Forceps for brain dissection previously sterilized</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich</td>
			<td>9048-46-8</td>
			<td>Protease free, fatty acid free, essentially globulin free</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>10043-52-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask</td>
			<td>Fisher Scientific</td>
			<td>156340</td>
			<td>Culture flask T25</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Corning Incorporated</td>
			<td>352340</td>
			<td>Cell strainer 40 &#181;m</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td>Confocal TCS SP8 microscopy coupled with an Olympus FluoView 300 confocal system</td>
		</tr>
		<tr>
			<td>Conical tubes</td>
			<td>Thermo Scientific</td>
			<td>339651, 339652</td>
			<td>Sterile tubes of 15 mL and 50 mL</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Abcam</td>
			<td>ab224589</td>
			<td>DAPI staining solution</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>12500062</td>
			<td>DMEM/F-12 50/50, 1X (Dulbecco&#39;s Mod. Of Eagle&#39;s Medium/Ham&#39;s F12 50/50 Mix) with L-glutamine</td>
		</tr>
		<tr>
			<td>Dyalisis tubing</td>
			<td>Sigma-Aldrich</td>
			<td>D9527</td>
			<td>Molecular weight cut-off = 14 kDa</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher Scientific</td>
			<td>64-15-5</td>
			<td>Reagent grade</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>12657011</td>
			<td>Research Grade</td>
		</tr>
		<tr>
			<td>Fibrinogen</td>
			<td>Sigma-Aldrich</td>
			<td>9001-32-5</td>
			<td>Fibrinogen cristalline powder from bovine plasma</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Sigma-Aldrich</td>
			<td>9000-70-8</td>
			<td>Gelatin powder from porcine skin</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>56-40-6</td>
			<td>Glycine powder</td>
		</tr>
		<tr>
			<td>Hanks Buffered Salt Solution (HBSS)</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>14175095</td>
			<td>No calcium, no magnesium, no phenol red</td>
		</tr>
		<tr>
			<td>L-Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>56-85-9</td>
			<td>L-Glutamine crystalline powder</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma-Aldrich</td>
			<td>114956-81-9</td>
			<td>Laminin 1-2 mg/mL L in 50 mM Tris-HCl</td>
		</tr>
		<tr>
			<td>Live dead kit cell imaging kit</td>
			<td>Thermo Scientific</td>
			<td>R37601</td>
			<td>Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm)</td>
		</tr>
		<tr>
			<td>Methacrylic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>760-93-0</td>
			<td>For GelMA preparation</td>
		</tr>
		<tr>
			<td>Microtubes</td>
			<td>Corning Incorporated</td>
			<td>MCT-150-C</td>
			<td>Microtubes of 1,5 mL</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 22G</td>
			<td>Fisher Scientific</td>
			<td>NC1362045</td>
			<td>Sterile blunt needle</td>
		</tr>
		<tr>
			<td>Operating scissor</td>
			<td>Integra Miltex</td>
			<td>05--02</td>
			<td>Sharp scissor for brain dissection previously sterilized</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>30525-89-4</td>
			<td>Paraformaldehyde powder</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>15070063</td>
			<td>Pen Strep (5,000 Units/ mL Penicillin; 5,000 ug/mL Streptomycin)</td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning Incorporated</td>
			<td>430591, 430588</td>
			<td>Sterile petri dishes of 35 and 100 mm</td>
		</tr>
		<tr>
			<td>Phalloidin</td>
			<td>Abcam</td>
			<td>ab176753</td>
			<td>iFluor 488 reagent</td>
		</tr>
		<tr>
			<td>Photoinitiator</td>
			<td>Sigma-Aldrich</td>
			<td>106797-53-9</td>
			<td>2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone</td>
		</tr>
		<tr>
			<td>Phosphate buffer saline (PBS)</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>10010023</td>
			<td>PBS 1 x, culture grade, no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>25988-63-0</td>
			<td>Poly-L-lysine hydrobromide mol wt 30,000-70,000</td>
		</tr>
		<tr>
			<td>Primary antobody</td>
			<td>Abcam</td>
			<td>ab4674</td>
			<td>Chicken polyclonal to GFAP</td>
		</tr>
		<tr>
			<td>Secondary antibody</td>
			<td>Abcam</td>
			<td>ab150176</td>
			<td>Alexa fluor 594 anti-chicken</td>
		</tr>
		<tr>
			<td>Spatula</td>
			<td>Miltex</td>
			<td>V973-70</td>
			<td>Number 24 cement spatula previously sterilized</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Fisherbrand</td>
			<td>3000038</td>
			<td>Microscope for brain dissection</td>
		</tr>
		<tr>
			<td>Syringe 5 mL</td>
			<td>BD</td>
			<td>1222C84</td>
			<td>Sterile syringe</td>
		</tr>
		<tr>
			<td>Syringe filter 2 &#181;m</td>
			<td>Fisher Scientific</td>
			<td>09-740-105</td>
			<td>Polypropylene filter for sterilization</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>Sigma-Aldrich</td>
			<td>9002--04-4</td>
			<td>Thrombin cristalline powder from bovine plasma</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>9002-93-1</td>
			<td>Laboratory grade</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>15400054</td>
			<td>Trypsin no phenol red 1 x diluted in PBS</td>
		</tr>
		<tr>
			<td>Versene solution</td>
			<td>Gibco; Life Technologies Corporation</td>
			<td>15040066</td>
			<td>Versene Solution (0.48 mM) formulated as 0.2 g EDTA(Na4) per liter of PBS</td>
		</tr>
		<tr>
			<td>Well plate</td>
			<td>Thermo Scientific</td>
			<td>144530</td>
			<td>Sterile 24-well plate</td>
		</tr>
	</tbody>
</table>

3d bioprinting of murine cortical astrocytes for engineering neural-like tissue

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also respond to neural injuries and act to protect the tissue from degenerative events. In vitro studies of astrocytes&#39; functionality are important to elucidate the mechanisms involved in such events and contribute to developing therapies to treat neurological disorders. This protocol describes a method to biofabricate a neural-like tissue structure rich in astrocytes by 3D bioprinting astrocytes-laden bioink. An extrusion-based 3D bioprinter was used in this work, and astrocytes were extracted from C57Bl/6 mice pups&#39; brain cortices. The bioink was prepared by mixing cortical astrocytes from up to passage 3 to a biomaterial solution composed of gelatin, gelatin-methacryloyl (GelMA), and fibrinogen, supplemented with laminin, which presented optimal bioprinting conditions. The 3D bioprinting conditions minimized cell stress, contributing to the high viability of the astrocytes during the process, in which 74.08% &#177; 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% &#177; 3.00%, indicating that the 3D construct represents a suitable microenvironment for cell growth. The biomaterial composition allowed cell attachment and stimulated astrocytic behavior, with cells expressing the specific astrocytes marker glial fibrillary acidic protein (GFAP) and possessing typical astrocytic morphology. This&#160;reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells&#39; native microenvironment, useful to researchers that aim to understand astrocytes&#39; functionality and their relation to the mechanisms involved in neurological diseases.

This work aimed to develop a neural-like tissue using the 3D bioprinting technology to deposit layer-by-layer primary astrocytes-laden gelatin/GelMA/fibrinogen bioink. Astrocytes were extracted and isolated from the cerebral cortex of mice pups (Figure 1), added to a biomaterial composition, allowing the biofabrication of a living 3D construct.
The computer-aided-design (CAD) was developed using the G-code&#160;(Supplemental file) as an interconne...

Watch this Scientific Journal Video about 3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue at JoVE.com

3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Astrocytes are glial cells with an essential role in the central nervous system (CNS), including neuronal support and functionality. These cells also ...

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Bioengineering

6.3K Views.  Universidade Federal de São Paulo. Here we report a method of 3D bioprinting murine cortical astrocytes for biofabricating neural-like tissues to study the functionality of astrocytes in the central nervous system and the mechanisms involving glial cells in neurological diseases and treatments.

Video: 3D Bioprinting of Murine Cortical Astrocytes for Engineering Neural-Like Tissue

Tissue Engineering of the Intestine in a Murine Model

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to preoperative weights within 40 days.1 In humans, massive small bowel resection can result in short bowel syndrome, a functional malabsorptive state that confers significant morbidity, mortality, and healthcare costs including parenteral nutrition dependence, liver failure and cirrhosis, and the need for multivisceral organ transplantation.2 In this paper, we describe and document our protocol for creating tissue-engineered intestine in a mouse model with a multicellular organoid units-on-scaffold approach. Organoid units are multicellular aggregates derived from the intestine that contain both mucosal and mesenchymal elements,3 the relationship between which preserves the intestinal stem cell niche.4 In ongoing and future research, the transition of our technique into the mouse will allow for investigation of the processes involved during TESI formation by utilizing the transgenic tools available in this species.5The availability of immunocompromised mouse strains will also permit us to apply the technique to human intestinal tissue and optimize the formation of human TESI as a mouse xenograft before its transition into humans. Our method employs good manufacturing practice (GMP) reagents and materials that have already been approved for use in human patients, and therefore offers a significant advantage over approaches that rely upon decellularized animal tissues. The ultimate goal of this method is its translation to humans as a regenerative medicine therapeutic strategy for short bowel syndrome.

This article and the accompanying video present our protocol for generating tissue-engineered intestine in the mouse, using an organoid units-on-scaffold approach.

Tissue-engineered small intestine (TESI) has successfully been used to rescue Lewis rats after massive small bowel resection, resulting in return to ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and fibroblasts are first isolated, counted and mixed at desired cell ratios. They are co-cultured in individual wells in ultra-low attachment 96-well plates. Within 3 days, beating spheroids form. These spheroids are then picked up by a nozzle using vacuum suction and assembled on a needle array using a 3D bioprinter. The spheroids are then allowed to fuse on the needle array. Three days after 3D bioprinting, the spheroids are removed as an intact patch, which is already spontaneously beating. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials. 3D bioprinted cardiac patches exhibit mechanical integration of component spheroids and are highly promising in cardiac tissue regeneration and as 3D models of heart disease.

This protocol describes 3D bioprinting of cardiac tissue without the use of biomaterials, using only cells. Cardiomyocytes, endothelial cells and ...

Isolation of Murine Adipose Tissue-derived Microvascular Fragments as Vascularization Units for Tissue Engineering

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several angiogenic and prevascularization strategies have been established. However, most cell-based approaches include time-consuming in vitro steps for the formation of a microvascular network. Hence, they are not suitable for intraoperative one-step procedures. Adipose tissue-derived microvascular fragments (ad-MVF) represent promising vascularization units. They can be easily isolated from fat tissue and exhibit a functional microvessel morphology. Moreover, they rapidly reassemble into new microvascular networks after in vivo implantation. In addition, ad-MVF have been shown to induce lymphangiogenesis. Finally, they are a rich source of mesenchymal stem cells, which may further contribute to their high vascularization potential. In previous studies we have demonstrated the remarkable vascularization capacity of ad-MVF in engineered bone and skin substitutes. In the present study, we report on a standardized protocol for the enzymatic isolation of ad-MVF from murine fat tissue.

We present a protocol to isolate adipose tissue-derived microvascular fragments that represent promising vascularization units. They can be rapidly isolated, do not require in vitro processing and, thus, may be used for one-step prevascularization in different fields of tissue engineering.

A functional microvascular network is of pivotal importance for the survival and integration of engineered tissue constructs. For this purpose, several ...

Microfluidic Bioprinting for Engineering Vascularized Tissues and Organoids

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic bioprinting to generate a scaffold with multilayer interlacing hydrogel microfibers. To achieve smooth bioprinting, a core-sheath microfluidic printhead containing a composite bioink formulation extruded from the core flow and the crosslinking solution carried by the sheath flow, was designed and fitted onto the bioprinter. By blending gelatin methacryloyl (GelMA) with alginate, a polysaccharide that undergoes instantaneous ionic crosslinking in the presence of select divalent ions, followed by a secondary photocrosslinking of the GelMA component to achieve permanent stabilization, a microfibrous scaffold could be obtained using this bioprinting strategy. Importantly, the endothelial cells encapsulated inside the bioprinted microfibers can form the lumen-like structures resembling the vasculature over the course of culture for 16 days. The endothelialized microfibrous scaffold may be further used as a vascular bed to construct a vascularized tissue through subsequent seeding of the secondary cell type into the interstitial space of the microfibers. Microfluidic bioprinting provides a generalized strategy in convenient engineering of vascularized tissues at high fidelity.

We provide a generalized protocol based on a microfluidic bioprinting strategy for engineering a microfibrous vascular bed, where a secondary cell type could be further seeded into the interstitial space of this microfibrous structure to generate vascularized tissues and organoids.

Engineering vascularized tissue constructs and organoids has been historically challenging. Here we describe a novel method based on microfluidic ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.