The isolation and culture of primary fibroblasts derived from keloid tissue are the basis for further studies of keloids. Fibroblasts from keloid tissue can be easily acquired through this protocol, which can provide an abundant and stable source of cells in the laboratory for keloid research. Begin by using sterile tweezers to place the keloid tissue in a 50 milliliter sterile centrifuge tube containing 10 to 25 milliliters of PBS supplemented with 1%PSA.
Meanwhile, add four milliliters of PBS PSA solution to each well of a six-well plate. Now take the tissue out of the tube, and wash twice with PBS PSA solution. Using a pair of sterile forceps, transfer the tissue sequentially from one well to the next.
Then remove the adipose and epidermis layers with surgical scissors, leaving the dermis untouched. Use a pair of scissors to dissect the trimmed dermis into three to five square millimeter pieces and transfer the pieces to the next well with a pair of sterile forceps. Wash the pieces in PBS PSA solution.
Using sterilized forceps, place 10 to 30 dermis tissue pieces spaced less than five millimeters apart in Petri dishes. Place the dishes upside down in an incubator until the pieces dry and stick to the dish. Next, add seven milliliters of DMEM supplemented with 10%FBS and 1%PSA, and incubate the dishes as before.
After three days, replace half the supernatant with complete culture medium. Observe the fibroblasts daily under 40X microscopic magnification. Remove the tissue pieces in the culture medium when the fibroblasts reach 90%confluency.
Wash the fibroblasts in PBS and add two milliliters of sterile trypsin EDTA solution. After incubating the cells in a humidified incubator, gently tap the culture dish and observe it under the microscope. Add two milliliters of complete medium to end to the digestion once most of the cells have detached.
Now transfer the cell suspension to a 15 milliliter sterile centrifuge tube and centrifuge the tube at 300G for three minutes at room temperature. Discard the supernatant carefully and resuspend the cell pellet in complete medium. Seed the fibroblasts into a nine centimeter cell culture dish and incubate in a humidified incubator.
To perform immunofluorescent analysis after culturing the fibroblasts on round coverslips and incubating them in primary and secondary antibodies, add 50 microliters of DAPI solution to the glass slide to stain the cellular nuclei. Place the washed coverslips on glass slides using forceps. Finally, transfer the samples to a wet dark box for fluorescent microscopy.
Fibroblast outgrowths of the tissue were observed at five days after processing. The fibroblasts displayed high proliferation rates and reached confluency after 10 days. The fibroblasts had elongated and spindle-like cell bodies, which were aligned in bundles at high confluency.
Immunofluorescent staining returned positive red immunofluorescence of the fibroblasts and blue immunofluorescence of the cellular nucleus. Flow cytometric assays showed CD90 positivity in almost all fibroblasts. This study has described an optimized method and provided clear instructions to solve existing challenges and increase the chance of success for the isolation and culture of keloid fibroblasts.
