An In Vitro Method to Generate Astrocyte Scaffolds within Hydrogel Micro-columns

1. Development of the Agarose Hydrogel Micro-columns

1. Make an agarose 3% weight/volume (w/v) solution by weighing 3 g of agarose and transferring it to a sterile beaker containing 100 mL of Dulbecco's phosphate buffered saline (DPBS). Add a sterile magnetic bar to the beaker, and place it on the surface of a hot plate/stirrer. Keep the beaker covered to prevent evaporation of its contents in the next step.
2. Heat the agarose and DPBS at a temperature of 100 °C and stir at 60-120 rpm. Adjust these settings as necessary, and constantly monitor the progression of the dissolution process as the solution changes initially from opaque to a clear appearance that signifies that the agarose has been completely dissolved.
   Caution: The hot beaker and the solution are hot!
3. As the agarose solution is heated and stirred, retrieve four empty 10 cm Petri dishes and add 20 mL of DPBS to two of them. Place acupuncture needles (diameter: 300 µm, length: 40 mm), glass microliter capillary tubes (diameter: 701.04 µm, length: 65 mm, capacity: 25.0 µL), and a bulb dispenser inside the biosafety cabinet. When the liquid agarose solution clears out, maintain constant heating at approximately 50 °C and stirring to avoid gelation of the agarose.
4. Introduce an acupuncture needle into the bottom opening of a bulb dispenser. Insert a capillary tube over the needle exposed to the outside. Secure the capillary tube by entering part of it into the rubber section of the bulb dispenser cylinder.
5. Transfer 1 mL of liquid agarose with a micropipette to the surface of an empty Petri dish, and place one end of the capillary tube vertically (with the needle inserted) in contact with agarose, while the rubber cap of the bulb dispenser is being pinched inwards. Slowly release the pressure on the bulb dispenser cap to draw agarose into the capillary tube.
   NOTE: The transfer of liquid agarose into the capillary tubes must be performed rapidly. If the liquid agarose is left to cool on the Petri dish surface for sufficient time (approximately 60 s), it starts to gel, preventing suitable suctioning of the agarose along the capillary tube.
6. Place each bulb-tube-needle assembly in a free Petri dish, and let the agarose gel inside the capillary tubes solidify for 5 min. Carefully pull the capillary tube with your hands out of the rubber stopper in the bulb dispenser cylinder, leaving the needle and the agarose gel in place inside the tube.
7. Manually extract the acupuncture needle by slowly pulling it out of the capillary tube; the newly solidified agarose cylinder also slides out of the tube in this procedure, still surrounding the needle. Gently nudge the micro-column along the acupuncture needle with the tip of sterile forceps to move it to the end. Place the needle over an open, DPBS-containing Petri dish and push the micro-column into the DPBS with forceps.
   NOTE: If the agarose micro-column remains within the glass capillary tube upon removal of the acupuncture needle, slowly push the agarose micro-column out of the capillary tube with a 25-mm gauge needle and into the dish with DPBS.
8. Sterilize microscalpels or forceps using a hot bead sterilizer. Make 4% w/v agarose by weighing 4 g of agarose and transferring it to 100 mL of DPBS. Heat and stir as explained in step 1.2 to obtain a clear 4% liquid agarose solution. Maintain heating and stirring of the solution throughout the following steps.
9. Transfer the Petri dishes containing the micro-columns to a dissection hood, and move a micro-column with fine forceps to an empty Petri dish. Utilize the stereoscope for visual guidance and a microscalpel to cut the micro-columns to the desired length. Trim both ends to form beveled ends at 45^o angles from horizontal to facilitate handling of the micro-columns during extracellular matrix (ECM) and cell addition.
10. Repeat the previous step for three more micro-columns in the same Petri dish and line up the four constructs in parallel with a separation of <3 mm between each of the cylinders. Load 50 µL of 4% agarose solution with a micropipette and pour a streak/line of liquid over the micro-column array to connect and bundle the constructs into groups of four (hereafter called "micro-column boats"). Avoid movement of 4% agarose to the ends of the micro-columns, which may clog the interior, by minimizing the distance between the constructs and adding the agarose quickly in a thin line.
    NOTE: Arranging groups of four micro-columns into "boats" is not required to fabricate the astrocytic scaffolds. Nevertheless, the boats serve to hasten the fabrication process and to offer a safer way to move and handle micro-columns in later steps.
11. Let the micro-column boat cool for 1-2 min to permit gelation of the added 4% agarose.Pick up the micro-column boat with fine forceps by the connecting 4% agarose, and move the micro-columns to the other DPBS-containing Petri dish prepared in step 1.1.3. Fabricate more boats with the remaining micro-columns as desired.
12. Move the Petri dishes containing the micro-columns and/or boats to a biosafety cabinet and sterilize with exposure to ultraviolet (UV) light for 30 min.
    Caution: Wear appropriate protection to prevent exposure to UV.
13. Store the dishes with the micro-column boats in DPBS at 4 °C, if ECM addition and cell plating will not occur immediately afterwards, up until 1 week. Repeat the micro-column fabrication steps if the constructs are not used during this timeframe.

2. Primary Cell Culture and Isolation

1. Cortical astrocyte isolation from rat pups
   1. In preparation for the following steps, add 20 mL of Hank's balanced salt solution (HBSS) to four 10 cm Petri dishes that will serve as reservoirs for the dissected tissues. Keep all the dishes on ice. Sterilize clean surgical scissors, forceps, micro-spatula, and micro-scalpels in a hot bead sterilizer.
   2. Prewarm 0.25% trypsin with 1 mM ethylenediaminetetraacetic acid (EDTA) and 0.15 mg/mL deoxyribonuclease (DNase) I in HBSS at 37 °C. In addition, prewarm the astrocyte culture medium at 37 °C, which consists of Dulbecco's Modified Eagle Medium (DMEM) with Ham's F-12 Nutrient Mixture and 10% fetal bovine serum (FBS).
   3. Anesthetize postnatal day 0 or day 1 Sprague-Dawley rat pups by exposing them to hypothermic conditions and euthanize by decapitation. Pin the head into a stage using a 19 mm gauge needle placed in the snout anterior to the eyes.
   4. Make an incision with a scalpel down the middle posterior to anterior, from the base of the neck up to just behind the eyes. Carry out another incision going laterally from directly behind the eyes downwards, forming a T-shape. Use fine forceps to pull the cranial skin/skull off to the side.
   5. Hold the head by the snout (facing up) by placing one prong of the forceps in the mouth of the animal and the other on the outside surface. Remove the brain with a sterile micro-spatula and place it in one of the Petri dishes filled with chilled HBSS. Place this Petri dish on ice immediately afterwards and at all times except when in use.
   6. Situate a granite block previously stored at -20 °C below a stereoscope inside a dissection hood. Place the Petri dish with the brain tissue on the surface of the granite block to preserve its low temperature during the dissection procedure. Use the stereoscope as visual guidance throughout the following steps.
   7. If the olfactory bulbs remain intact, extract them by cutting with forceps or microscalpels. In addition, use a microscalpel to remove the cerebellum and to make a midline incision separating the two cerebral hemispheres. Transfer the hemispheres with forceps to a Petri dish containing fresh, chilled HBSS.
   8. Dissect the midbrain structures from the inside of the hemispheres with a microscalpel to obtain only the separated cortices. Use fine forceps to peel the meninges, a sheet-like structure, off from the cortical tissue and to transfer the isolated cortices to a new Petri dish with cold HBSS. Use the microscalpel to mince the tissue in order to increase the surface area for trypsin action in the next step.
   9. Use a Pasteur pipette to transfer the cortices to a 15 mL centrifuge tube containing 4 mL of prewarmed trypsin-EDTA (8 cortices in each tube). Expose the cortical tissue to trypsin-EDTA for 5-7 min at 37 °C.
   10. With a Pasteur pipette, carefully remove the trypsin-EDTA and add 400 µl of 0.15 mg/mL DNAse I solution to the centrifuge tube. Agitate the tube or vortex until all the tissue is dissociated and there are no remaining tissue pieces in the liquid. If it is not possible to completely dissolve the tissue, remove the remaining fragments with the tip of a Pasteur pipette.
   11. Centrifuge the tube containing the dissociated cell solution at 200 x g for 3 min. Remove the supernatant with a Pasteur pipette without disturbing the cells. Add 1 mL of astrocyte culture medium (defined in step 2.1.2) to the tube with a micropipette and agitate to resuspend and create a homogeneous solution.
   12. Transfer 10 mL of astrocyte culture medium with a serological pipette to a T-75 flask. Add 250 µl of the 1 mL cell solution (prepared in step 2.1.11) with a micropipette to the flask to plate one pup brain worth of cells per flask. Distribute the discharged cell solution evenly across the culture medium and gently agitate the flask to further promote an even distribution.
   13. Culture the plated flasks in a humidified incubator at 37 °C and 5% CO₂. After reaching 24 and 72 h in culture, mechanically agitate the flask to detach non-adherent cell types, such as neurons and oligodendrocytes.
   14. Afterwards, at each of these timepoints, perform a media change. Hold the flask vertically so the culture media lies on the bottom of the flask, not covering the adhered cells. Aspirate the culture medium with a Pasteur pipette, pressing the tip of the pipette against the bottom corner of the flask to avoid extracting the cells. Place the flask in the original horizontal position and gently add 10 mL of astrocyte medium over the cells with a serological pipette.
   15. Return the flasks to the incubator after each media change. After 90% confluency is achieved, mechanically agitate the flask once more to remove any remaining non-adhering cells.
   16. Passage the astrocytes by taking out the culture medium with a vacuum and a Pasteur pipette. Add 5 mL of 0.25% trypsin-EDTA with a serological pipette over the adhered cells. Gently agitate the flask to ensure the trypsin covers all the cells, and incubate the flask for 5-7 min at 37 °C and 5% CO₂.
   17. Quench trypsin by adding 1 mL of astrocyte medium to the cells with a serological pipette. Extract the cell solution from the flask with a serological pipette and transfer it to a sterile 15 mL centrifuge tube. Centrifuge the tube at 200 x g for 3 min.
   18. Remove the supernatant with a serological pipette and resuspend it in 1 mL of astrocyte culture medium. Agitate the tube to ensure the cell solution is homogeneous. Count the number of cells in the solution with a hemocytometer or an automatic cell counter.
       NOTE: A flask that is 90% confluent typically yields approximately 3 million astrocytes.
   19. Add 10 mL of astrocyte culture medium to a T-75 flask. Perform a 1:4 dilution by transferring 250 µl of the cell solution (step 2.1.18) with a micropipette to the T-75 flask already containing culture medium. Gently agitate to ensure a homogeneous cell distribution throughout the surface of the flask.
   20. Repeat steps 2.1.16-2.1.19 each time 90% confluency is achieved to passage the cells.
2. Cortical Neuron Isolation from Rat Fetuses
   1. ​Follow similar preparations as steps 2.1.1 and 2.1.2, with the exception that the prewarmed media is co-culture media, consisting of Neurobasal medium + 2% B-27 supplement (for neurons) + 1% G-5 serum-free supplement (for astrocytes) + 0.25% L-glutamine.
   2. Euthanize timed-pregnant embryonic day 18 Sprague-Dawley rats with carbon dioxide asphyxiation and confirm death by decapitation.
   3. Extract the rat fetuses and dissect the cortices from the rest of the cerebral tissue in Petri dishes containing HBSS on the surface of the chilled granite block, using a stereoscope for visual guidance and sterilized scissors, microscalpel, and forceps. After the successive dissection of the heads, brains, cerebral hemispheres, and cortices, transfer each tissue to a new HBSS-filled Petri dish.
   4. Mince the cortical tissue into smaller fragments to increase the surface area for trypsin. Transfer 4-6 cortices with a Pasteur pipette to a tube with 5 mL of prewarmed trypsin-EDTA and maintain at 37 °C to dissociate the tissue. At 5-7 min manually agitate the tube to mix and prevent clumping of the tissue.
   5. Remove the tube from 37 °C after 10 min. As explained previously in step 2.1, extract the trypsin-EDTA and substitute with 1.8 mL of 0.15 mg/mL DNAse solution. Afterwards, vortex the tissue until the solution appears homogeneous, with no tissue fragments floating in the liquid.
   6. Centrifuge the dissociated tissue solution at 200 x g for 3 min. After removing the supernatant, resuspend in 2 mL of co-culture medium. Count the number of cells in this solution with a hemocytometer or an automatic cell counter.
      NOTE: The usual yield is 3.0-5.0 x 10⁶ cells/cortical hemisphere.
   7. Prepare a new cell solution with a density of 2.0-4.0 x 10⁵ cells/mL in the co-culture media defined above.

3. Development of the Astrocytic Cables Inside the Micro-columns

1. ECM core fabrication
   NOTE: The ECM has to be added to the interior of the hydrogel micro-columns on the same day in which cell seeding will be performed.
   1. Inside a biosafety cabinet, prepare a 1 mg/mL solution of rat tail type I collagen in co-culture medium in a sterile microcentrifuge tube. Maintain the microcentrifuge tube with the ECM solution on ice at all times except when in use.
   2. Transfer 1-2 µL of the ECM solution with a micropipette onto a strip of litmus paper to estimate its pH. Add 1 µL of 1 N sodium hydroxide (NaOH) or hydrochloric acid (HCl) with a micropipette to increase or decrease the pH of the ECM solution, respectively, and pipette up and down to homogenize. Verify the new pH with a litmus paper strip and add more acid or base, as needed, until the pH is stable in the 7.2-7.4 range.
   3. Move the Petri dishes from step 1.2 to a dissection hood, and transfer 4-5 micro-columns or a boat with sterile fine forceps to an empty, sterile 35 or 60 mm Petri dish. Using the stereoscope for guidance, situate the 10 µL tip of a micropipette at one end of each micro-column and suction to empty the lumen of DPBS and air bubbles. Confirm the absence of air bubbles with the stereoscope to ensure that the ECM added in the next step can flow freely across the lumen.
   4. Charge a P10 micropipette with ECM solution, place the 10 µL tip against one end of the hydrogel micro-columns, and deliver enough solution to fill the lumen (approximately 3-5 µL), observing the entry of ECM with the stereoscope. Pipette a small reservoir (2-4 µL) of ECM solution on either end of the micro-column.
      NOTE: Always manage 4-5 micro-columns or one boat at a time to prevent prolonged drying of the constructs, as this may result in the crumpling of the micro-column structure. Completely dried micro-columns firmly attach to the surface of the Petri dishes, which prevents their utilization for cell seeding. Excessive amounts of co-culture media (as a hydration measure) cannot be added to the micro-columns because this may cause the ECM to flow out during the incubation period described below.
   5. Pipette co-culture media in a ring around the Petri dish to provide a humidity sink to prevent the columns from drying out during incubation. Incubate the Petri dish containing the ECM-containing micro-columns at 37 °C and 5% CO₂ for 1 h to promote polymerization of collagen before adding the neurons and/or astrocytes.
      NOTE: The ECM should form a layer coating the inner surface of the hollow lumen, rather than a solid ECM core encompassing the interior, both of which can be observed using the stereoscope. If the ECM forms a core, continue the incubation period until only the layer is left. With this layer formed, the astrocyte cell solution can fill the interior of the micro-column in the plating steps.
   6. During the incubation period, prepare the astrocyte cell solution (as described below).
2. Astrocyte and Neuron Seeding in the Micro-Columns
   1. Passage the plated astrocytes (between the fourth and twelfth passage) as explained in steps 2.1.16-2.1.19. After counting the number of cells in the flask, prepare cell solution at a density of 9.0-12.0 x 10⁵ cells/mL solution suspended in cell-culture media.
   2. Using a stereoscope, place the tip of a P10 micropipette at one end of the micro-columns, and transfer sufficient cell solution (~5 µL) into the lumen to completely fill it. As done above with the ECM, add small reservoirs of cell solution to both ends of each micro-column.
   3. Incubate the plated micro-columns on the Petri dishes at 37 °C and 5% CO₂ for 1 h to promote the attachment of astrocytes to the ECM. If neurons will not be added to micro-columns, proceed to step 3.2.5.
   4. Following the initial incubation period, add 1-2 µL of the cortical neuron solution obtained in step 2.2.7 into both ends of the micro-columns with a micropipette, while observing under the stereoscope. Ensure that sufficient media is present in the dishes to avoid drying, and incubate again for 40 min at 37 °C and 5% CO₂ to allow for the adhesion of neurons.
      NOTE: Cortical neuron solution can also be added 1-2 days after bundle formation, performing step 3.2.4 after carefully removing the culture media from the Petri dish with a micropipette.
   5. After the incubation period, carefully fill the Petri dishes containing the plated micro-columns with 3 or 6 mL of co-culture media for 35 or 60 mm Petri dishes, respectively. Maintain the plated micro-columns in culture at 37 °C and 5% CO₂ to promote the self-assembly of the aligned astrocytic bundles, which should form a bundled, cable-like structure after 6-10 h.