Derivation of Leptomeninges Explant Cultures from Postmortem Human Brain Donors

The brain donation registration includes documentation by the registrant of their intent to donate. The autopsy permission for tissue retrieval is provided by the next of kin as permitted by law. Research studies using collected autopsy specimens are reviewed by the institutional review board (IRB) to ensure compliance with Health Insurance Portability and Accountability Act (HIPAA) regulations.

NOTE: Leptomeninges samples are collected by the brain dissector or neuropathologist during a brain dissection and are stored in 50 mL conical tubes containing 25-30 mL meninges growth media. The sample is stored at 4 °C until sample preparation. The processing should be performed as soon as possible as the viability of cells decreases with time post mortem.

1. Set-up before Starting the Leptomeninges Dissection

NOTE: Steps 1.1 – 1.3 are to be performed inside a biosafety cabinet.

1. Prepare meninges growth media by combining 375 mL of Dulbecco's Modified Eagle Media, 100 mL of fetal bovine serum, 5 mL 10 mM non-essential amino acids, 5 mL 200 mM L-alanyl-L-glutamine, 5 mL 100 mM sodium pyruvate, 5 mL 10,000 U/mL penicillin/streptomycin and 5 mL 250 µg/mL Amphotericin B in 500 mL filter unit. Filter and mix. Use media for up to four weeks.
2. Place all materials in the biosafety cabinet before starting the dissection: 4x Petri dishes, 2x 6-well plates, 2x scalpels, 15-20 sterilized 15-mm cover slips, phosphate buffer saline (PBS), serological pipettes, aspirating pipettes.
3. Add 1 mL of sterile-autoclaved 0.1% gelatin solution to each well of a 6-well plate. Set the plate aside for 30-60 min. Prepare 2x 6-well plates.

2. Preparation of Leptomeninges Sample

NOTE: Steps 2.1 – 2.3 are to be performed inside a biosafety cabinet.

1. Add approximately 10 mL of PBS to a sterile 10-cm tissue culture dish and place the meninges sample in the dish. Using forceps, gently wash leptomeninges with PBS to remove blood.
2. Transfer the meninges into a new 10-cm tissue culture dish containing 10 mL of fresh PBS and continue washing. Repeat as necessary to remove as much blood as possible.
3. Using two scalpels, cut leptomeninges tissue into an approximately 6 cm x 6 cm large piece.
4. Place lid on a culture dish and transfer to a dissecting microscope in a horizontal laminar flow hood.

3. Dissection of Leptomeninges Tissue

NOTE: Steps 3.1 – 3.3 are to be performed inside a horizontal laminar flow hood using a dissecting microscope.

1. Remove blood vessels from a region on the leptomeninges piece (step 2.3) large enough to create at least 20-25 3 mm x 3 mm pieces. Hold the leptomeninges in place with one scalpel and with the other, use a gentle and shallow scraping motion to separate the vessels.
2. Cut small squares of tissue from the prepared region. Cut in a stepwise process by first making larger pieces and then slicing those into smaller sections.
3. Use one scalpel to hold the tissue in place and another scalpel to cut the tissue with a rocking motion. Due to the consistency of the tissue, it is difficult to slide the scalpels against each other. This will only tear the tissue apart and results in ragged edges.
4. Continue cutting the pieces in half until there are about 20-25 3 mm x 3 mm pieces.

4. Transfer of Dissected Meninges Pieces onto Tissue Culture Plates

NOTE: Step 4 is to be performed inside a biosafety cabinet.

1. Aspirate gelatin from 6-well plate (from step 1.3). Add 1 mL of meninges growth media to each well. Using sterile forceps, place 3-4 biopsy pieces into each well.

5. Placement of Cover Slips over Meninges Pieces

NOTE: Steps 5.1 – 5.2 are to be performed inside a horizontal laminar flow hood using a dissecting microscope.

1. Cover the pieces with 1-2 sterile 15-mm circular coverslips per well.
2. Press down firmly with forceps to ensure the pieces are touching the bottom of the plate. This allows attachment of tissue pieces to the bottom of the well.
3. Place the 6-well plate into a 37 °C, 5% CO₂ incubator.

6. Cell Culture Maintenance

NOTE: Steps 6.1 – 6.3 are to be performed inside a biosafety cabinet. Handle culture dishes with care as coverslips should not move and dislodge the meninges pieces.

1. After 2 days in culture, add an additional 1 mL of fresh meninges growth media to each well.
2. At Day 7, carefully aspirate the media and add 2 mL of fresh media to each well.
3. After Day 9, continue to change growth media every other day until culture becomes confluent.

7. Passaging

NOTE: All steps are to be performed inside a biosafety cabinet. Once meningeal fibroblasts have migrated out of the meningeal tissue pieces and start to grow towards the edges of the culture vessel, expand meningeal fibroblasts into larger culture dish.

1. Before starting the passaging, coat the culture dish with 1% gelatin for 15-30 min.
2. Aspirate media from 6-well dish and wash cells with 2 mL PBS.
3. Add 1 mL of trypsin/EDTA to each well and incubate at 37 °C for 5-10 min. It is not necessary to remove the cover slips or the meningeal tissue for this step.
4. When cells are rounded and start to detach, add 2 mL of culture media.
5. Pipette cells up and down to completely disrupt all clumps and transfer the cell solution to larger culture dish.
6. Use a ratio of 1:3 to 1:4 to combine cells from three 6-well dishes into one T75 culture dish.
7. Once meningeal fibroblasts cells are confluent in T75 culture flasks, trypsinize the cells, and cryopreserve in cryopreservation media at 1×10⁶ cells/mL per vial. One T75 culture flask with confluent meningeal fibroblasts contains about 5-7 million cells.

8. Characterization of the Meningeal Fibroblasts by Immunostaining

NOTE: Before starting the procedure, prepare 4% paraformaldehyde solution, 5% normal goat serum in PBS (blocking buffer), 0.3% Triton X-100 in PBS (permeabilization buffer), primary and secondary antibody solutions, and 1 µg/mL of Hoechst 33342 diluted in PBS from 10 mg/mL stock.

1. Seed 15,000 to 30,000 meningeal fibroblasts in 8-well chamber slide coated with 0.1% gelatin.
2. After 24 h when the cells are attached, fix cells in 100 µL 4% paraformaldehyde per well for 10 min at room temperature. Wash the wells 3 times with 1x PBS.
3. Permeabilize the cells with 150 µL of 0.3% Triton X-100 for 5 min at room temperature. Wash the wells 3 times with 1x PBS.
4. Add 200 µL blocking solution (PBS + 5% normal goat serum) for 1 h at room temperature.
5. Aspirate the blocking solution, and add 150 µL of desired primary antibodies diluted in blocking solution. For double-staining, prepare working dilutions of primary antibodies in separate centrifuge tubes on ice as follows:
   1. Prepare 1:300 dilution of anti-Fibronectin + 1:200 dilution of anti-nestin in blocking solution. Prepare 1:250 dilution of anti-SERPINH1 + 1:1,000 dilution of anti-TUJ1 in blocking solution. Prepare 1:250 dilution of anti-SERPINH1 + 1:100 dilution of anti-SOX2 in blocking solution
6. Incubate at 4 °C overnight.
7. Wash the wells 3 times with 1x PBS.
8. Prepare a working 1:400 dilution of fluorescently labeled secondary antibodies goat anti-rabbit (green; Ex/Em² 495/519 nm) and goat anti-mouse (red; Ex/Em² 590/617 nm) in blocking solution and add 100 µL to each well. Incubate for 1 h, in the dark, at room temperature. Then wash wells once with 1x PBS.
9. Add 100 µL of 1 µg/mL Hoechst 33342 (blue; Ex/Em² 358/461 nm) solution to each well and incubate in the dark for 3 min. Wash wells 3 times with 1x PBS, leaving in the final 1x PBS wash in the well, and cover the slide with aluminum foil.
10. Analyze cells under fluorescent microscope.