Isolation of Schwann Cells from Mouse Spinal Roots

Take a nerve root and chop it into small pieces.

Transfer to a digestive enzyme-containing tube and incubate.

The enzyme digests the extracellular matrix.

Add media containing serum to stop digestion.

Pipette the content repeatedly, then centrifuge and discard the supernatant.

Resuspend the pellet.

Pass the suspension through a strainer to remove undigested tissue. Incubate the collected cells in a plate.

Fibroblast and Schwann cells or SCs attach to the plate and divide.

Remove the unattached cells. Wash the attached cells with a phosphate buffer.

Add digestive enzymes to loosen the digestion-sensitive SCs. Stop digestion with media.

Completely detach SCs by adding media.

Transfer the cells to a tube and centrifuge. Discard the supernatant and resuspend the cells in SC-specific media.

Transfer the cells to an uncoated plate and incubate.

Any remaining fibroblasts will adhere, leaving SCs in suspension.

Transfer the suspension to a matrix-coated plate.

Incubate for Schwann cell attachment and division.

Slice the nerves into 3- to 5-millimeter pieces with scissors. Add 1 milliliter of 0.25% trypsin, and transfer the nerve pieces to 5-milliliter centrifuge tubes. Incubate at 37 degrees Celsius for 18 to 20 minutes. Next, to stop the digestion, add 3 to 4 milliliters of DMEM containing 10% fetal bovine serum. Pipette the mixture up and down gently, about 10 times. Centrifuge at 800 times g for 5 minutes.

After centrifugation, discard the supernatant, and resuspend the precipitate in 2 to 3 milliliters of DMEM supplemented with 10% FBS. Filter the cell suspension through a 400-mesh filter, and then use the suspension to inoculate a 60-millimeter Petri dish. Incubate the Petri dishes for 4 to 5 days at 37 degrees Celsius in the presence of 5% carbon dioxide.

When the culture has reached 90% confluence, wash the cells once using 1X PBS. Then, to digest the Schwann cells, add 1 milliliter of 0.25% trypsin at 37 degrees Celsius per 60-millimeter dish. After 8 to 10 seconds at room temperature, stop the digestion by adding 3 milliliters of DMEM supplemented with 10% FBS. To detach the Schwann cells, gently blow on the cells with a pipette.

Verify by microscope that the cells are detached. Then, collect the medium, which contains the Schwann cells, and centrifuge at 800 x g for 5 minutes. Discard the supernatant, and resuspend the precipitate in 3 milliliters of medium. Use the suspension to inoculate uncoded 60-millimeter Petri dishes, and incubate the dishes at 37 degrees Celsius for 30 to 45 minutes. Transfer the medium, which will contain the Schwann cells to a poly-L-lysine-coated medium dish, and incubate the dish at 37 degrees Celsius for 2 days.