Isolation and Culture of Primary Schwann Cells from a Human Dermis

All procedures involving sample collection have been performed in accordance with the institute's IRB guidelines.

1. Isolation of Schwann cells (SCs) and fibroblasts from the dermis

1. Pre-coat T25 flasks with poly-L-lysine (PLL, 0.01 µg/µL) using 5 mL of 1x DPBS. Place the pre-coated flasks at 37 °C for 3 h. Wash the PLL coating matrix twice using 1x DPBS (5 mL).
   NOTE: T25 flasks can be prepared ahead of time.
2. Rinse the isolated pieces of the dermis with 5 mL of DMEM basal medium.
3. Mince the dermis into small pieces with scissors or a scalpel.
4. Digest the minced dermis with 5 mL of collagenase (2 mg/mL in DMEM basal medium) at 37 °C for 2.5 h. Gently triturate the minced dermis with a pipette tip every 30 min until the dermis completely dissociates.
5. Filter the cell suspension with a 70 µM cell strainer. It is necessary to apply mechanical force using a 1 mL syringe to fully filter the suspension.
6. Dilute the filtered cell suspension with 5 mL of complete DMEM medium (DMEM basal medium + 5% FBS + 1x of antibiotic) to stop the enzymatic activity of collagenase.
7. Centrifuge the cell suspension at 870 x g for 5 min at RT to pellet the cells.
8. From this cell pellet, both fibroblasts and SCs will be obtained. Resuspend the cell pellet in 2 mL of DMEM complete medium to divide the cells (1 mL cell suspension/tube) and centrifuge at 870 x g for 5 min at RT.
9. Aspirate the supernatant. Resuspend the cell pellet in 5 mL of fresh DMEM complete medium.
10. Quantify the cell number and viability.
    NOTE: Here, a specialized cell sampling and staining device (Table of Materials) was used to quantify the cell number and viability following the manufacturer's instructions.
11. Seed the pre-coated T25 flasks with 5 mL of resuspended cells at a density 4 x 10³ cells/mL. Incubate the flask at 37 °C in the presence of 5% CO₂ overnight (~12-16 h incubation).
12. After 16 h, confirm cell adhesion by microscopy (Table of Materials) at 10x magnification.
13. Remove the non-adherent cells and wash the flask 3x with 5 mL of 1x DPBS.
14. Add 5 mL of 10 µM cytosine arabinoside (antimitotic agent, kills fibroblasts) in DMEM complete medium. Incubate the flask at 37 °C in the presence of 5% CO₂ for 24 h.
15. Aspirate the cytosine arabinoside-containing medium from the flask. Rinse the flask 3x with 5 mL of 1x DPBS.
16. Refill the culture flask with 5 mL of SCs complete culture medium (Table of Materials) and incubate at 37 °C in the presence of 5% CO₂ for 48 h. Refresh SC complete culture medium every other day until SCs reach 80% confluence.