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

Summary

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.

Abstract

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' 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' 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% ± 1.33% of cells were viable right after bioprinting. After 1 week of incubation, the viability of astrocytes significantly increased to 83.54% ± 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 reproducible protocol provides a valuable method to biofabricate 3D neural-like tissue rich in astrocytes that resembles cells' native microenvironment, useful to researchers that aim to understand astrocytes' functionality and their relation to the mechanisms involved in neurological diseases.

Introduction

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 synaptogenesis^1,2. 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 astrogliosis^3,4, forming a glial scar that prevents healthy tissue exposition to degenerative agents⁵. This event results in changes in astrocytes' gene expression, morphology, and function^6,7. Therefore, studies involving astrocytes' 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 signaling^12,13. 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 CNS^14,15,16.

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.

Protocol

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 São Paulo (CEUA 2019/ 9292090519).

1. Mice brain dissection

1. 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.
2. 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 µm filter.
3. 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.
4. 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.
5. Using a spatula previously sterilized with ethanol 70%, lift the brain from the cranial cavity and place it in the culture dish containing 10 mL of cold HBSS.
6. Place the culture dish containing the brain under the stereomicroscope, and using two blunt-tip forceps, remove the meninges from the brain (Figure 1).
7. Separate the cortices from the rest of the brain by gently rolling them away from the median line of the brain using a spatula.
8. Collect both cortices and immediately transfer them to the same microtube containing 1 mL of cold HBSS.

2. Astrocytes isolation and culture

1. 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.
2. Remove HBSS and incubate the tissue with 1 mL of 0.05% trypsin at 37 °C for 5 min.
   NOTE: Only trypsin digestion is sufficient at this point.
3. 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.
4. 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 µm to remove non dissociated fragments.
5. 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 °C. After centrifugation, discard the supernatant and suspend the pellet in 1 mL of astrocytes culture medium.
6. 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 °C and 5% CO₂.
7. Ensure that after 24 h, the cells are adherent. Then, replace the medium and change it every 3 days.
8. After 7 days, remove microglia and oligodendrocytes from the culture by washing the cells with 2 mL of 1x PBS.
9. Replace the PBS solution with the astrocytes culture medium and leave the culture flask in an orbital shaker at 180 rpm overnight.
   ​NOTE: Astrocytes form a confluent monolayer in approximately 10-12 days of culture.

3. 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 make sure there are 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 into a container with 2 L of distilled water for dialysis, letting 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 in 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) to the dialyzed gelatin-MA, and let it 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.

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.

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 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.

5. 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 to be used in the experiment, avoiding storage.

6. 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 movement of the needle.
      NOTE: Use one 35 mm culture dish for each bioprinting.
   2. Press the Print button.
   3. Once the bioprinting over, ensure that the syringe moves away from the dish. Then, close the culture dish and prepare for the crosslinking 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 astrocytes culture medium and incubate at 37 °C and 5% CO₂. Change the medium every 3 days.

7. Assessment of astrocytes viability

1. Viability of bioprinted astrocytes
   1. Transfer the bioprinted construct to a 35 mm culture dish using a spatula.
   2. Wash the construct with 1 mL of 1x PBS.
   3. Deposit 100 µL of the Live/Dead reagent over the construct and keep it at 37 °C for 30 min, keeping it protected from light.
   4. Remove the Live/Dead reagent and wash the construct with 1 mL of 1x PBS.
   5. 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.
   6. 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.
   7. Calculate the number of viable (green) and dead (red) using a computational software.
2. Viability of 2D astrocytes culture
   1. Seed 0.5 x 10⁶ astrocytes (passage 1-3) in a 35 mm confocal dish, add astrocytes medium, and incubate them at 37 °C and 5% CO₂.
   2. When cells are confluent, remove the culture medium and wash with 1 mL of 1x PBS.
   3. Deposit 200 µL of the Live/Dead reagent, and keep the dish at 37 °C for 30 min, protected from light.
   4. Remove the Live/Dead reagent and wash the cells with 1 mL of 1x PBS.
   5. Take the dish to a confocal microscope coupled with a digital camera, and use 488 and 570 nm excitation for image acquirement.
   6. Calculate the number of viable (green) and dead (red) using a computational software.

8. Immunostaining of astrocytes

1. Glial fibrillary acidic protein (GFAP) staining of 3D bioprinted astrocytes
   NOTE: To investigate the presence of other cell markers, change the primary antibody accordingly.
   1. Remove the medium from the well and wash the construct with 1 mL of 3x PBS.
   2. Add 4% paraformaldehyde (PFA) in PBS to the well until the construct is completely covered and leave it for 2 h at 4 °C.
   3. 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 °C in PBS. Make sure the well plate is sealed to avoid PBS evaporation.
   4. Treat the sample with glycine 0.1 mol/L for 5 min.
   5. Wash with 1 mL of 1x PBS for 5 min.
   6. Permeabilize the sample with PBS containing 0.1% Triton X-100 and 10% FBS for 1 h at 25 °C under orbital agitation.
   7. Incubate the construct with chicken anti-GFAP (primary antibody dilution 1:500) at 4 °C overnight.
   8. 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 °C.
   9. Incubate the sample with Alexa fluor 488-conjugated anti-chicken (secondary antibody dilution 1:500) and 1 µg/mL DAPI for 1 h at 25 °C under orbital agitation.
      NOTE: Keep the sample protected from light.
   10. Wash the sample with 1 mL of PBS for 5 min, 3 times.
   11. Transfer the construct to a 35 mm confocal dish.
       NOTE: Make sure the dish is well sealed to prevent the sample from drying out.
2. Glial fibrillary acidic protein (GFAP) staining of 2D astrocytes culture
   1. Seed 0.5 x 10⁶ astrocytes (passage 1-3) in a 35 mm confocal dish, add astrocytes medium, and incubate them at 37 °C and 5% CO₂.
   2. When the cells are confluent, remove the culture medium and wash them with 1 mL of 1x PBS.
   3. Repeat steps 8.1.2-8.1.10.
3. Astrocytes cytoskeleton staining
   1. Repeat steps 8.1.1-8.1.6.
   2. Add 200 µL of 50 µg/mL of fluorescent-conjugated phalloidin solution in PBS and 1 µg/mL of DAPI over the construct.
   3. Incubate for 1 h at 25 °C under orbital agitation, keeping the sample protected from light.
   4. Wash the sample with 1 mL of PBS for 5 min 3 times , and transfer the construct to a confocal dish using a spatula.
      ​NOTE: Make sure the dish is well sealed to prevent the sample from drying out.

9. Confocal imaging

1. Take the dishes to a confocal microscope coupled with a digital camera for imaging (359, 488, and 570 nm excitation).
2. 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.

Representative Results

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 (Supplemental file) as an interconnected frame of square shape (0.6 x 0.6 mm), with pores of 1 mm, aiming to facilitate the diffusion of nutrients and oxygen. The frame was composed of 6 layers placed on top of each other, changing at an angle of 90° in every layer (Figure 2A i). The designed structure possessed approximately 5 mm of high (Figure 2A ii), allowing tissue manipulation. The bioink composition also allowed the fabrication of constructs of different shapes (Figure 2B).

The preparation of the bioink composed of gelatin, GelMA, and fibrinogen comprised two crosslinking steps. First, GelMA was crosslinked under UV light, in which inter and intramolecular covalent bonds are formed, followed by fibrin crosslinking. In this step, thrombin enzymatically cleaves fibrinogen chains, resulting in the formation of fibrin fibers¹⁷, a reaction stabilized by Ca²⁺ ions¹⁸. Then, GelMA and fibrin fibers form a stable interpenetrated polymer network (IPN) supplemented with laminin, suitable support for cell attachment and spread^19,20 (Figure 2C).

Prior to bioprinting, after 12 days of isolation, astrocytes were characterized by the presence of GFAP, a protein constituent of astrocyte intermediate filaments⁴ (Figure 3A). Then, trypsinized astrocytes were mixed with the gelatin/GelMA/fibrinogen solution at a density of 1 x 10⁶ cells/mL, generating an astrocytes-laden bioink. The bioink was transferred to a 5 mL syringe connected to a 22 G blunt needle, composing the bioprinter printhead (Figure 3B i). The needle allowed the bioink extrusion without clogging and preventing high shear stress to the cells.

Due to the viscoelastic properties of gelatin, which behaves as a fluid at higher temperatures and as a gel at lower temperatures²¹, the bioprinted construct retained shape fidelity (Figure 3B ii). After the bioprinting of two consecutive layers of bioink, the formation of a well-defined structure was observed (Figure 3C), with cells entrapped within the biomaterial.

After bioprinting and crosslinking processes, the construct was incubated with astrocyte medium, and after 1 day of bioprinting, most of the cells still presented a round morphology (Figure 3D). Bioprinted scaffolds maintained integrity after 7 days of incubation, and although some round cells were observed, a large number of astrocytes spread throughout the construct, presenting astrocytic morphology and interconnection (Figure 3E).

Because the parameters of bioprinting, such as speed, could directly affect cell viability, different bioprinting speeds (400, 600, and 800 mm/min) were tested and astrocytes survival evaluated using the Live/Dead assay with calcein-AM (live cells, green fluorescence) and ethidium homodimer-III (EthD-II) (dead cells, red fluorescence). The percentage of viable cells was quantified using a computational software, by calculating the number of living and dead cells. Cell viability was evaluated at time 0 (right after bioprinting), and results showed that at the lower speed, 400 mm/min, viable cells represented 74.08% ± 1.33% of total cells, being significantly higher than cells bioprinted at 600 and 800 mm/min (60.25% ± 1.93% and 62.94 ± 6.18%, respectively) (Figure 4A). Therefore, the speed of 400 mm/min was used in this work.

Prior to bioprinting, 2D cultured astrocytes were characterized as the percentage of viable cells, and viability of bioprinted astrocytes was normalized to this condition. Results showed that 2D culture presented 90.98% ± 0.94% of viable cells. Viability of bioprinted astrocytes (day 7) was 83.54% ± 3.00%, representing 0.92 ± 0.03 of the 2D value, which was significantly higher than that of day 0 (0.81 ± 0.01) (Figure 4B). Images of astrocytes stained with the live/dead reagent are presented in Figure 4C, and show that after bioprinting, cells possessed a round morphology (Figure 4C i). After 1 week of incubation, astrocytes spread throughout the construct (Figure 4C ii), showing a distinct morphology of cells from 2D culture (Figure 4C iii).

Bioprinted astrocytes were stained to show cell density and cell morphology within the construct. Figure 5A shows a bioprinted construct after 7 days of incubation, with high density of astrocytes stained with phalloidin, an F-actin cytoskeleton dye. Although few round cells were observed, astrocytes presented mainly a star-like morphology. Bioprinted astrocytes showed to be GFAP positive when stained after 7 days of bioprinting, indicating that cells maintained their astrocytic phenotype (Figure 5B i). Figure 5B ii and 5B iii show the Z-stacked images of the bioprinted GFAP+ astrocytes within the construct. These results indicate that the bioink composition provided a biocompatible microenvironment to promote astrocytes' adhesion, spread, and growth.

Figure 1: Extraction of brain and cortex separation for primary astrocytes culture. Primary astrocytes were isolated from the cortex of C57Bl/6 mice pups' (post-natal day 1) brain. After extracting the brain from the animal, the meninges were removed under a microscope, and the cortex separated followed by tissue digestion and astrocytes culture. Please click here to view a larger version of this figure.

Figure 2: Schematic illustration of 3D bioprinting process. (A) CAD file designed for the 3D bioprinting of neural tissue showing (i) 2 layers, top view, and (ii) 6 layers, side view, of the construct. (B) Images showing the capacity of the bioink to print structures of different shapes (i) square, (ii) capillary, and (iii) star. (C) Scheme of biomaterials crosslinking after 3D bioprinting showing, where GelMA is crosslinked under UV light followed by fibrin crosslinking in a thrombin:Ca²⁺ bath. Please click here to view a larger version of this figure.

Figure 3: 3D boprinting of astrocytes-laden gelatin/GelMA/fibrinogen bioink. (A) Characterization of astrocytes after 12 days of isolation and culture, stained for GFAP, green and DAPI, blue. (B) (i) Printhead setup and (ii) 3D bioprinted construct right after bioprinting. (B) Two-layered construct showing the bioprinted frame. Magnification of 4x. (C) Image of bioprinted astrocytes 1 day after bioprinting, showing cells in a round morphology. (D) Image of bioprinted astrocytes 7 days after the bioprinting process, showing cells with astrocytic morphology with few round cells, indicating their affinity with the mimetic tissue. Magnification of 10x. Please click here to view a larger version of this figure.

Figure 4: Assessment of bioprinted astrocytes viability. (A) Viability of astrocytes bioprinted at different speeds. (B) Viability of bioprinted astrocytes on (i) day 0 (right after bioprinting) and (ii) after 7 days of bioprinting, normalized to viability of astrocytes in 2D culture. Statistical analysis by means of One-Way Anova with Tukey's test, n = 3, *p < 0.05. (C) Fluorescent images of astrocytes stained with Live/Dead reagent. Bioprinted astrocytes on (i) day 0 (right after bioprinting), (ii) day 7, and (iii) 2D culture of astrocytes. Magnification of 10x. Please click here to view a larger version of this figure.

Figure 5: Characterization of 3D bioprinted astrocytes. Immunofluorescence of 3D bioprinted astrocytes after 7 days of incubation stained for (A) F-actin (phalloidin, red) and nuclei (DAPI, blue) with magnification of 10x and 40x and for (B) GFAP, green (i) with magnification of 10x, (ii) Z-stacked showing the X-Y-Z axis of the bioprinted construct, and (iii) image showing the X-Z axis of GFAP+ astrocytes. Please click here to view a larger version of this figure.

Supplemental File. Please click here to download this File.

Discussion

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 CNS¹¹. Due to the important role of glial cells in neural functionality, cortical astrocytes have been used in many studies, such as brain development²⁴, biomolecules transport²⁵, neurite outgrowth²⁶, and in brain-like tissue biofabrication¹⁴.

Methods for engineering 3D culture of astrocytes were reported previously, using different biomaterials and scaffolding techniques^27,28,29. Similarly, a method for 3D bioprinting human induced pluripotent stem cells (hiPSCs)-derived neural aggregates was also reported, and showed the capacity of these bioprinted cells to differentiate and mature in vitro³⁰. However, there are no reports of methods for biofabrication of 3D brioprinted astrocytes constructs in the literature. Then, this protocol aimed to describe a reproducible method for 3D bioprinting cortical astrocytes.

In this protocol, astrocytes were isolated from the cerebral cortices of C57Bl/6 mice pups, an animal model widely used in research^31,32, which can be replaced by astrocytes derived from other sources, such as hiPSCs and spinal cord. After isolation, culture, and subculture, astrocytes remain in a proliferative state up to passage 3, which is the maximal number of splitting recommended due to their intrinsic limitation of proliferation⁸. It was verified that proliferation of astrocytes from passage 4 was limited, being difficult to achieve the desired amount of cells for 3D bioprinting.

In the described method, a concentration of 1.0 x 10⁶ cells/mL of biomaterial solution was used, as a proof of concept to evaluate the capacity of cells to survive the bioprinting process and to maintain their viability and intrinsic properties during culture within the hydrogel. In the previous work, a 3D bioprinted neural-like tissue was biofabricated by co-culturing astrocytes and neurons, and to increase cellular interaction, astrocytes concentration was 8.0 x 10⁶ cells/mL¹⁴. Then, concentration of cells may be optimized for specific studies.

A bioink for 3D bioprinting is composed of cells and a biomaterial or a combination of biomaterials³³. In this protocol, a combination of gelatin/GelMA/fibrinogen was used, which showed to be favorable for both bioprinting and maintainance of astrocytes in culture. The production of a bioprintable bioink is challenging when using extrusion-based bioprinting technique. The biomaterial composition must possess viscoelastic properties that, at the same time, allow the extrusion of the bioink, while retaining the 3D shape after the printing process³⁴. In addition, it should be able to maintain cell viability during the process, which depending on the bioprinting conditions, can cause shear stress to the cells leading to death³⁵.

Bioprintability was assured by gelatin, a biomaterial that possesses optimal viscoelastic properties due to its sol-gel transition capacity³⁶. This allows gelatin macromolecules to rearrange and behave as a fluid during extrusion, and as a gel after bioprinting, maintaining the 3D structure. In addition, as a collagen derivate, gelatin is composed of glycine-amino acid peptide triplet repetitions, which assure cell-specificity²¹. However, intra and intermolecular bonds of gelatin are weak, and due to its thermoreversibility, gelatin has no stability at 37 °C, being released from the construct during cell culture. Therefore, GelMA became an alternative as a stable hydrogel, due to the covalent bonds formed after UV light exposition, maintaining cell-specificity properties¹⁹. The physical properties of GelMA, such as porosity, degradation, and elastic modulus can be tuned to suit different tissue engineering applications¹⁹. The stiffness of GelMA scaffolds can be controlled by varying the methacryloyl substitution³⁷, allowing to achieve a stiffness similar to that of the tissue to be modeled. That is, by decreasing the degree of functionalization, a lower stiffness can be achieved³⁷. Therefore, due to the softness of the mouse brain^38,39 and the direct effect of extracellular matrix (ECM) stiffness on cell behavior⁴⁰, in this protocol a low degree of gelatin functionalization was proposed. In the previous works, the capability of GelMA hydrogels to mimic CNS physical features, showing high suitability for culturing astrocytes, neurons, and neural stem cells^14,41,42 was reported.

Besides GelMA, fibrinogen, a native biopolymer that forms fibrin fibers through enzymatic reaction, has also been widely used to biofabricate neural-like tissue, showing high specificity and offering a suitable microenvironment for neural cells to attach and grow^30,43,44. Other biomaterials, such as alginate and chitosan have been used to bioprint neural-like tissues, mixed to gelatin, GelMA, and/or fibrinogen, aiming to improve printability and physical properties of the 3D scaffolds^14,30. In the present method, an optimal bioprintability, physical stability, and cell-specificity was achieved using gelatin, GelMA, and fibrinogen as components of the bioink. In order to increase astrocytes' recognition to the microenvironment, laminin-a component of the brain ECM-was used to supplement the bioink.

In this protocol, gelatin was used at a concentration of 4% (w/v), which allowed the sol-gel transition of the bioink at 25 °C within approximately 10 min. A faster gelation can be achieved by increasing the concentration of gelatin. However, sterilization by filtration using 0.2 µm filter can be compromised. The bioink filtration is a critical step, verifying that higher concentrations of gelatin (>5% w/v) may prevent filtration. An alternative to achieve a faster gelation point during bioprinting is to leave the syringe containing the bioink at 4 °C for 2 min before connecting it at the bioprinter printhead. After bioprinting, it is crucial to maintain the construct at 25 °C to avoid destabilizing the 3D structure and maintain a gelled condition prior to UV exposition. Notably, the construct should be solid when the fibrinogen crosslinker solution is added to the plate. For GelMA crosslinking, it is important to flip the sample (2 x 60 s each side) to ensure UV exposition throughout the construct. For fibrinogen crosslinking, the crosslinker solution should cover the construct completely. Therefore, if using a larger dish or plate, the volume should be adjusted. After crosslinking, the hydrogel should look homogeneous under phase contrast microscopy and cells should be homogeneously distributed throughout the construct, possessing a round morphology.

This protocol allowed the gelatin/GelMA/fibrinogen bioink to print structures of different shapes, maintaining the integrity and the 3D shape of the constructs. Due to the limitations of the temperature to reach 25 °C inside the laminar flow, the bioprinting was performed in the culture room outside the laminar flow. Bioprinting time was approximately 1 min for each sample, and no contamination was observed during cell culture.

Cell integrity can be affected by bioprinting, due to the shear stress caused in the process³⁵. This can be controlled by optimizing printing parameters, such as printing speed. In this work, different bioprinting speeds were tested, 400, 600, and 800 mm/min, observing a significant decrease in cell viability when speed was increased from 400 to 600 mm/min. At 400 mm/min, around 74% of the cells remained viable after bioprinting, and this value increased significantly after 7 days of incubation (>80%). Therefore, lower speeds were not used in order to avoid the excessive exposition of cells to the environment. The 2D culture of astrocytes showed higher viability (~90%) as compared to the bioprinted cells. However, as shown by fluorescent images, morphology is affected by the type of culture. While 2D cells were flat, astrocytes in 3D environment possessed a star-like shape, interconnecting with each other by cellular processes.

Notably, the mimetic microenvironment was favorable for cortical astrocytes culture, as cell viability significantly increased after 1 week, suggesting cell proliferation within the construct. Laminin, a glycoprotein present in the brain ECM, was added to the bioink aiming to achieve a higher adherence of the astrocytes to the hydrogel¹⁴. Cytoskeleton F-actin staining showed that bioprinted astrocytes were in high density within the construct, indicating that the cell concentration used in this protocol allowed astrocytes interconnection. Immunohistochemistry for GFAP localization, a marker correlated with astrocytes' extensive arborization and cell hypertrophy⁴⁵, corroborated by F-actin staining, showed that cells presented typical astrocytic morphology, indicating that the mimetic 3D system was favorable for cells to attach and behave as in their native environment.

The protocol presented here describes an efficient and reproducible procedure for 3D bioprinting cortical astrocytes. Due to the importance of astrocytes in neuroinflammation response to injury, as well as in regulating neuronal functionality, studies involving this glial cell could contribute to many aspects in the understanding of diseases that affect the CNS. Therefore, the 3D in vitro model presented here is useful in future applications that aim to study astrocyte-neuron interactions, the functionality of astrocytes in brain pathologies, and the potential of astrocytes as therapeutic targets.

Materials

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