Magnetic Separation of Schwann Cells from Mouse Sciatic Nerves

Protocolo

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.

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Materiais

Name	Company	Catalog Number	Comments
Bovine serum albumin	Carl Roth, Karlsruhe, Germany	8076.4
Cell strainer, 100 µM	BD Bioscience, Heidelberg, Germany	352360
Centrifuge 5810-R	Eppendorf AG, Hamburg, Germany	5811000015
CO2 Incubator Heracell	Heraeus Instruments, Hanau, Germany	51017865
Dispase II	Sigma Aldrich GmbH, Steinheim, Germany	4942078001
Distilled water (Water Purification System)	Millipore, Molsheim, France	ZLXS5010Y
DMEM/F-12, GlutaMAX	Thermo Fisher Scientific, Schwerte, Germany	31331093
DPBS (no calcium ions and no magnesium ions)	Sigma Aldrich GmbH, Steinheim, Germany	D8537-6X500ML
FCS	Sigma Aldrich GmbH, Steinheim, Germany	F7524	FCS must be tested for Schwann cell culture
Gentamycin	Thermo Fisher Scientific, Schwerte, Germany	5710064
HBSS (no calcium ions and no magnesium ions)	Thermo Fisher Scientific, Schwerte, Germany	14170138
Laminin	Sigma Aldrich GmbH, Steinheim, Germany	L2020-1MG
MACS Multistand	Miltenyi Biotec, Bergisch Gladbach, Germany	130042303
Microscissors	Fine Science Tools GmbH, Heidelberg, Germany	15000-08
Microscope	Motic, Wetzlar, Germany	Motic BA 400
Microscope slide	VWR, Radnor, USA	630-1985
MiniMACS separator	Miltenyi Biotec, Bergisch Gladbach, Germany	130091632
MS columns	Miltenyi Biotec, Bergisch Gladbach, Germany	130-042-201
Neubauer counting chamber	Assistant, Erlangen, Germany	40441
Pipettes	Eppendorf AG, Hamburg, Germany	2231300004
Poly-L-Lysin	Sigma Aldrich GmbH, Steinheim, Germany	P4707-50ML
Serological pipette, 10 mL	Sarstedt, Nümbrecht, Germany	861254025
Serological pipette, 25 mL	Sarstedt, Nümbrecht, Germany	861685001
Serological pipette, 5 mL	Sarstedt, Nümbrecht, Germany	861253001
TC dish 60, cell +	Sarstedt, Nümbrecht, Germany	833901300
Thy-1 Microbeads (MACS Kit)	Miltenyi Biotec, Bergisch Gladbach, Germany	130-094-523
Trypsin-EDTA (0.05%), phenol red	Thermo Fisher Scientific, Schwerte, Germany	25300-054
Type I Collagenase	Sigma Aldrich GmbH, Steinheim, Germany	C1639
Water bath type 1008	GFL, Burgwedel, Germany	4285