Generating a Primary Culture of Astrocytes from a Rat Pup Brain

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Primary cell culture preparation

1. Brain tissue isolation
   NOTE: Isolation should be performed on ice, using ice cold solutions.
   1. Use newborn pups 1-3 days old for primary neonatal cell culture.
      ​NOTE: For the study presented here, Sprague Dawley hSOD1^G93A and non-transgenic rats were used. For genotyping, rat tails were used for later DNA extraction and PCR.
   2. Sprinkle the pup's head with 70% ethanol and immediately decapitate it fast using scissors.
   3. Cut open the skin using small-angled scissors to expose the skull. Open the skull by making a cut from the foramen magnum toward the orbits. Next, make a perpendicular midline cut. Remove the brain from the skull and place it in a Petri dish containing phosphate-buffered saline (PBS).
      NOTE: Clean the tools between pups to prevent cross-contamination between transgenic and non-transgenic pups. Isolation of the cortices from the rest of the brain should be done under a stereomicroscope.
   4. Using the tip of the forceps, tear the connections between both hemispheres. Then, separate the hemispheres with curved forceps by gently pushing a hemisphere from the center to the side.
   5. Remove the meninges by carefully tearing them with straight and curved forceps.
   6. Remove the hippocampus by pinching it with a curved forceps. Discard the hippocampus or use it for another cell culture preparation.
      NOTE: Further steps should be performed under the laminar flow hood to ensure sterile conditions.
2. Tissue homogenization and dissociation
   1. Transfer one cortex into a 15 mL tube filled with 3 mL of cold PBS. Pipet the suspension up and down with a 1 mL tip, completing 10-15 strokes until the suspension becomes homogeneous.​NOTE: Be very cautious not to produce bubbles in this process.
   2. Centrifuge at 500 × g for 5 min. Remove the supernatant and resuspend the pellet in 3 mL of cold PBS by pipetting it up and down with a 1 mL tip. Repeat the centrifugation step.
      NOTE: All solutions from this point should be prewarmed to 37 °C.
   3. Discard the supernatant and resuspend the pellet in 2 mL of complete Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin). Transfer the homogenate to a 2 mL tube.
   4. Pass the homogenate through 21 G and 23 G needles (three times each) to make a suspension of single cells.
      NOTE: Be very cautious not to produce bubbles in this process.
3. Pour the cell suspension prepared from one cortex into a 60 mm diameter Petri dish or a T25 flask (with the surface pretreated with a polypeptide coating for the growth of adherent cells) containing 3 mL of complete DMEM. Lightly shake the Petri dish so that the suspension is uniformly distributed.
4. Grow the cells in the incubator in a humidified atmosphere of 5% CO₂/95% air at 37 °C.
5. Change the medium 48 h after isolation and replace the medium every 3 days.
6. To promote astrocyte growth, when cells reach 70%-80% confluency, wash them with prewarmed PBS to remove the loosely attached glial cells and traces of FBS in the medium.
   1. Trypsinize the underlying layer of astrocytes by adding 1 mL of prewarmed trypsin solution (0.25% trypsin, 0.02% EDTA in PBS, sterile-filtered).
   2. Place the dish in the incubator at 37 °C for 2-5 min.
   3. Check the cells under the microscope. When they start to detach, add 4 mL of complete DMEM.
   4. Collect the cell suspension and transfer it to a 15 mL tube. Centrifuge at 500 × g for 5 min.
   5. Discard the supernatant and resuspend the pellet in 1 mL of complete medium. Count the cells using a hemocytometer.
   6. Replate the cells at a density of 10⁴ cells/cm² in 5 mL of fresh complete DMEM.
7. Change the medium every 3 days. To minimize the presence of other glial cell types, after astrocytes reach 50% confluence and prior to each media replacement, wash the cells with complete DMEM.
   1. Aspirate the supernatant medium with a 1 mL pipette and gently dispense it onto the layer of cells several times. Ensure that the whole surface of the cell layer is covered during this washing step.
8. Once the cells reach 80% confluence (usually after 14 days), repeat the trypsinization and the collection of astrocytes as described in step 1.6.
9. Seed 5 × 10³ of astrocytes on a 7 mm circular glass coverslip coated with poly-L-lysine (50 µg/mL). Use them in experiments after 48 h.
10. To promote microglia, after step 1.7, allow the glial cells to reach a confluent layer. When microglial cells appear on top of the astrocyte layer (after 10-15 days, recognized by their smaller, more oval bodies and shorter processes), shake the Petri dish on an orbital shaker for 2 h at 220 rpm.
11. Dispersing and seeding microglial cells
    1. Lightly wash the detached and loosely attached cells by aspirating the supernatant medium with a 1 mL pipette tip and gently dispense it onto the layer of cells several times. Be sure to cover the whole surface of the cell layer during this washing step, collect the medium with the detached cells, and transfer it to a 15 mL tube.
    2. Centrifuge at 500 × g for 5 min. Discard the supernatant and resuspend the pellet in 1 mL of medium.
    3. Pass the cell suspension through a 21 G needle to obtain a single-cell suspension.
       NOTE: Most published methods use trypsin or papain to dissociate the tissue; however, 21 G needles have the advantage of preventing the overdigestion of cells, allowing for a gentler dissociation.
    4. Count the cells using a hemocytometer. Seed 5 × 10³ microglial cells on a 7 mm circular glass coverslip coated with poly-L-lysine (50 µg/mL). Use them in experiments after 48 h.
       ​NOTE: It is difficult to obtain a pure microglial culture by shaking, since the oligodendrocyte precursor cells and astrocytes may still be present in a variable number depending on the experimenter's experience.