Box 2. Isolation of schwann cell colonies

• Sterilize cloning cylinders by autoclaving.
• Identify colonies of SKP-derived Schwann cells morphologically under the tissue culture microscope, and then place a pen mark on the underside of the slide or dish to identify the location(s) of these colonies.
• Remove the medium from the tissue culture dish or slides, and coat one end of the cloning cylinder with sterile vacuum grease. With the aid of a low magnification inverted tissue culture microscope, place the cylinder over an individual Schwann cell colony, and press it down to form a tight seal around the colony. Fill the cylinder with 200–500 l of trypsin versene or trypsin EDTA (undiluted) and incubate at 37 °C for 5 min. Cells should then be easily detached from the substrate with gentle trituration.
• Remove all contents from the cylinder (including the putative Schwann cells), into a conical tissue culture tube and dilute the cell suspension in 10 ml of wash medium containing 10% FBS to inactivate the enzyme. Mix the suspension by repeated inversion and then pellet cells by centrifugation in a tabletop centrifuge at 1,200 r.p.m. for 6–8 min. Cells can then be resuspended in fresh Schwann cell differentiation medium.

Note: Plating these trypsinized cells at high density will improve cell viability and the efficiency of expansion.