Isolation and Culture of Primary Schwann Cells from a Human Dermis
DEŞİFRE METNİ
Precoat T25 flasks with poly-L-lysine or PLL, using 5 milliliters of DPBS, and place the precoated flasks at 37 degrees Celsius for three hours. Later, wash the PLL coating matrix twice with DPBS. After washing the isolated pieces of the dermis with 5 milliliters of DMEM basal medium, mince the dermis into small pieces with scissors.
Digest the minced dermis with 5 milliliters of collagenase at 37 degrees Celsius for 2 and 1/2 hours, with gentle trituration using a pipette tip every 30 minutes until the dermis dissociates completely. Once digested, filter the cell suspension with a 70-micrometer cell strainer, followed by dilution of the filtered cell suspension with 5 milliliters of complete DMEM medium to stop the enzymatic activity of collagenase.
Next, centrifuge the cell suspension at 870 times g for 5 minutes at room temperature, and resuspend the cell pellet in 2 milliliters of DMEM complete medium to divide the cells. After repeating the centrifugation process as described, aspirate the supernatant to use the cell pellet to isolate the Schwann cells or fibroblasts.
To isolate the Schwann cells, resuspend the cell pellet in 5 milliliters of fresh DMEM complete medium, and quantify the cell number and viability before seeding 5 milliliters of the resuspended cells in precoated T25 flasks at a density of 4.0 times 10 to the 3 cells per milliliter. Incubate the flask at 37 degrees Celsius and 5% carbon dioxide. After 16 hours, confirm cell adhesion by microscopy at 10x magnification. Then, remove the non-adherent cells, and wash the flask thrice with 5 milliliters of DPBS.
Next, incubate the cells with 5 milliliters of 10 micromolar cytosine arabinoside in a DMEM complete medium. After 24 hours, aspirate the cytosine arabinoside-containing medium before rinsing the flask. Refill the culture flask with 5 milliliters of Schwann cells complete culture medium to incubate for 48 hours while refreshing the medium every other day until Schwann cells reach 80% confluency.
