Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves

1. Schwann cell culture

1. Coating for Schwann cell culture
   1. Coat the cell culture dishes under sterile conditions. Apply 2 mL of 0.01% poly-L-lysine (PLL) to two 60 mm tissue culture (TC) dishes each and incubate overnight at 4 °C.
   2. Remove the PLL, wash the TC dishes 2x with distilled water, and incubate with 2 mL of 1 µg/cm² laminin overnight at 4 °C. Wash the TC dishes 2x with aqua dest, and let the plates air-dry.
2. Medium preparation for Schwann cell culture
   1. Prepare 50 mL of Schwann cell medium by adding 10% heat-inactivated fetal calf serum (FCS), 2 µM forskolin, 10 nM neuregulin, and 50 µg/mL gentamycin to Dulbecco′s Modified Eagle′s Medium (DMEM)/F-12 (high glucose) under sterile conditions.
   2. Prepare 70 mL of Leibovitz's L-15 medium with 50 µg/mL gentamycin under sterile conditions.
3. Enzymatic digestion of the sciatic nerve
   ​NOTE: The next steps are performed under sterile conditions.
   1. Prepare the enzymatic digestion solution containing 0.25% dispase II, 0.05% type I collagenase, and 50 µg/mL gentamycin in 10 mL of DMEM (high glucose).
   2. Centrifuge the tube at 188 x g for 5 min at 4 °C, remove the supernatant with a 25 mL serological pipette, and transfer the pellet with the remaining Leibovitz's L-15 medium into a 60 mm tissue culture dish using a 1,000 mL pipette.
   3. Rinse the 50 mL tube with 10 mL of the enzymatic digestion solution and add it to the dish containing the nerve fibers. Distribute the tissue in the dish carefully with the tip of a pipette to maximize the accessible surface for digestion.
   4. Incubate at 37 °C and 5% CO₂ for 18 h, and stop the digestion by adding 10 mL of 40% FCS in Hanks' balanced salt solution, without Ca²⁺ and Mg²⁺ (HBSS).
4. Cell separation
   1. Transfer the digested nerves into a 50 mL tube using a serological pipette and centrifuge at 188 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 10 mL of DMEM containing 10% FCS and 50 µg/mL gentamycin. Resuspend the pellet 20 times subsequently, using a 10 mL, 5 mL, 2 mL, 1 mL, and 200 µL pipette tip.
   2. Filter the cell suspension through a 100 µm cell strainer and centrifuge at 188 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet with 4 mL of DMEM containing 10% FCS and 50 µg/mL gentamycin.
   3. Add 2 mL of the cell suspension to each of the two PLL- and laminin-coated 60 mm TC dishes, and incubate at 37 °C and 5% CO₂. Leave the plates untouched for 2 days in the incubator to protect the cells from mechanical stress and to support adherence.
5. Schwann cell differentiation
   1. After 2 days, remove the medium and carefully rinse the plates 2x with DMEM (high glucose), 10% FCS, and 50 µg/mL gentamycin. Afterward, add 2 mL of Schwann cell medium. Replace the Schwann cell medium every 2nd day, and observe the cell appearance and confluency using a microscope.
6. Cell trypsinization and magnetic separation
   1. When the cells reach a confluency of about 80% (6-12 days of culture), carefully wash the plates 2x with 3 mL of DPBS and incubate with 2 mL of 0.05% Trypsin/EDTA (prewarmed to 37 °C) for 3 min. When the cells detach from the plate bottom, inactivate digestion by the addition of 2 mL of DMEM with 10% FCS and 50 µg/mL gentamycin.
      NOTE: Stick to a trypsinization time of strictly 3 min, and proceed rapidly afterward.
   2. Resuspend the cell pellet in 2 mL of magnetic cell separation buffer containing DPBS with 0.5% bovine serum albumin (BSA) and 2 nM EDTA. Combine 10 µL of the cell suspension with 10 µL of trypan blue and count the cells using a staining chamber.
   3. Centrifuge the cell suspension at 188 x g for 10 min at 4 °C and resuspend the cell pellet in 90 µL of magnetic cell separation buffer per 1 x 10⁷ cells. Add 10 µL of Thy-1 microbeads per 1 x 10⁷ cells. Resuspend the solution a few times and incubate for 15 min in the dark at 8 °C.
   4. Add 2 mL of the magnetic cell separation buffer to the cell suspension and centrifuge at 300 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 500 µL of magnetic cell separation buffer.
   5. Moisten the magnetic cell separation column with 1 mL of the magnetic cell separation buffer. Place the magnetic cell separation column in the magnetic cell separator. Apply the cells to the magnetic cell separation column. Collect the flow through and centrifuge at 300 x g at 4 °C for 10 min.
      ​NOTE: Fibroblasts are positively selected and remain in the column, while Schwann cells pass the column. Fibroblasts can be collected with a stamp (e.g., as a negative control for Schwann cell staining protocols).
   6. Discard the supernatant and resuspend the pellet in 1 mL of the coculture medium. Count the cells after staining with trypan blue and a staining chamber.