All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.
1. Rat or Mouse Brain Dissection
NOTE: Both mice and rats can be used. Animals older than P8 may also be used (we tested up to P12), but removing the meninges will become increasingly difficult. Further, the cell yield and health will diminish, since brain cells from older animals will have formed intricate processes and connections that may break during tissue dissociation, leading to increased cell death. Cortical preparations are described here, but other brain regions can also be used.
- Place 0.05% trypsin-EDTA in a 37 °C water bath.
- Euthanize animals (pregnant rats for E19 pups, or P0-P8 animals) by slowly raising CO2 concentration, and isolating the brains. The following steps require a sterile technique.
- If working with embryonic tissue, first remove the embryos from the uterus and open the embryonic sacs, then immediately cut off the heads with scissors. When all heads are cut off, transfer them into a pre-cooled dissection medium.
- Once the heads are submerged in the medium, open the skin and skull (both are very soft in young animals) with forceps and pinch off the cerebellum. Carefully lever the rest of the brain up and out of the skull.
NOTE: Two to four E19 embryos yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.
- For newborn or older pups, cut off the heads with scissors, and isolate the brains by medially cutting the skin in a straight line from the back of the head to the tip of the nose. Pull the skin aside and carefully cut the skull left and right, then lift the skull bone up. Cut off the cerebellum, and lever the rest of the brain up and out of the skull.
NOTE: Two to three P0-P8 pups yield enough tissue for at least eight 10 cm dishes of cortical astrocytes plated at 500,000 cells per dish on the day of dissection.
- Separate the cortex from the underlying midbrain structure. Place the forceps within the longitudinal fissure between the hemispheres, then move the forceps in between the cortex and midbrain structures of one hemisphere (perpendicular to the longitudinal fissure) to pinch each hemisphere free.
- Using the forceps, remove all meninges from each hemisphere (and its hippocampus), then separate the hippocampi. If hippocampal astrocytes are required, move each hippocampus into a 15 mL tube filled with 14 mL dissection medium on ice.
- Remove further areas from each hemisphere's cortex if specific regions are required (e.g., frontal cortex). Cut any cortical tissue to be used for generating astrocytes into pieces no larger than 1 mm3. Collect all pieces of cortical tissue in a separate 15-mL tube filled with a dissection medium on ice.
2. Tissue Digestion and Dissociation
- Once all tissue pieces are collected, wait for them to settle to the bottom of the tube, then aspirate as much medium as possible.
- Add 2-3 mL of 0.05% trypsin-EDTA using a serological pipette, and incubate the tubes in a 37 °C water bath for 20 min.
NOTE: Release all trypsin-EDTA from the pipette swiftly to mix the tissue pieces and allow them to settle again.
- Carefully aspirate the trypsin-EDTA, leaving tissue pieces in as little liquid as possible, at the bottom of the tube.
- Add 5 mL of pre-cooled dissection medium to wash off the remaining trypsin-EDTA. Pipette to the side of the upper part of the tube to achieve mixing of the tissue pieces.
- Aspirate the dissection medium, and leave the tissue pieces in as little liquid as possible. Repeat this washing step with pre-cooled dissection medium twice more.
- After the final washing step, add 1 mL of DMEM+ (warmed to room temperature) using a 1,000 µL pipette tip, and triturate the tissue by pipetting up and down without introducing bubbles.
NOTE: It is important to avoid producing bubbles, as this may change the pH of the solution, to which astrocytes are quite sensitive.
- Place a 100 µm cell strainer in the opening of a 50-mL tube and pre-wet the filter with 4.5 mL of pre-cooled DMEM+.
- Pipette the 1 mL of dissociated tissue suspension onto the cell strainer and add another 4.5 mL pre-cooled DMEM+ to wash the cells through the cell strainer.
3. Passaging Cells
- The day before passaging, coat cell culture plates with 0.04% PEI.
- On day in vitro (DIV) 7 after the dissection, place the 10-cm dishes with cells in DMEM+ on a laboratory shaker in the incubator and shake the dishes at 110 rpm for 6 h.
- Wash off the PEI from coated coverslips with deionized, distilled water twice. Add NB+H medium to the plates (500 µL per well for 24-well plates and 2 mL per well for 6-well plates). Pre-equilibrate the plates in the incubator at 37 °C and 5% CO2 while the 10 cm dishes are shaking, or for at least 30 min.
- 20-30 min before the end of the 6 h shaking, pre-warm 1x PBS, DMEM+, NB+H, and 0.25% trypsin-EDTA to 37 °C in the water bath.
- After 6 h of shaking, take the culture dishes off the shaker and immediately replace the medium (that contains shaken-off neurons, oligodendrocytes, and microglia) with 10 mL pre-warmed 1x PBS per dish.
NOTE: It is important to immediately aspirate the old medium containing detached cells to avoid the non-astrocytic cells re-attaching.
- Remove the PBS and add 3 mL pre-warmed trypsin-EDTA per dish. Incubate each dish for 4 min in the incubator at 37 °C and 5% CO2
- Add 5 mL pre-warmed DMEM+ to the 3 mL trypsin-EDTA in each dish, pipette the cells off the dish using a 5 mL serological pipette, and collect the cell suspension in a 50 mL tube.
NOTE: Pipette directly from the bottom of the dish - the floor of the dish should become clearer as the cells detach. Only use serum-containing medium (in place of DMEM+), as serum inactivates trypsin.
- Centrifuge the cell suspension at 3,220 x g at 20 °C for 4 min.
- Remove the supernatant and resuspend the cell pellet in 1 mL pre-warmed NB+H.
4. Cell Plating for Final Astrocyte Cultures
- Count the cells.
- Plate 20,000 cells per well in 6-well plates (in 2 mL NB+H medium per well) or 5,000 cells per well in 24-well plates (in 500 µL NB+H medium per well).
- Keep the cell cultures in the incubator at 37 °C and 5% CO2 until further treatment or analysis.
