To start, incubate fertilized chicken eggs in a humidified incubator at 38 degrees Celsius. Now dissect stage 30 to 33 chicken embryo dorsal skins in HBSS. Then incubate the skins in two times calcium and magnesium free saline with 0.25%EDTA for 15 to 20 minutes.
Carefully separate the epithelium and the MESENCHYME using watchmaker's forceps and move the separated material to HBSS, placed on ice. Collect the separated mesenchyme into a 15 milliliter centrifuge tube and add two milliliters of 0.1%collagenase trypsin made in PBS. Then incubate the solution in a water bath.
Next, use a pasteur pipette to disperse the skin mesenchyme into a single cell suspension. Then add eight milliliters of DMEM containing 10%fetal bovine serum to halt digestion. Centrifuge the cells at 233G for five minutes.
Then resuspend the pellet in culture medium. Next place a cell culture insert in a well of a six well dish, drop a 10 microliter droplet of the mesenchymal cells onto the insert. In the well outside of the insert, pipette two milliliters of DMEM with 10%fetal bovine serum.
Incubate the plated cells at 37 degrees in an incubator set with 5%carbon dioxide and 95%air for one hour. Using a one milliliter pipette, transfer the intact epithelium on top of the plated mesenchymal droplet. Flatten the intact epithelium over the mesenchyme to form the reconstituted skin explant.
Leave a thin layer of medium on the insert to keep the skin explant semi wet. Providing an air/liquid interface incubate the reconstituted skin explant at 37 degrees in an incubator with 5%carbon dioxide and 95%air environment. The ex vivo organ cultures appear homogeneous at first short feather buds formed after two to three days in culture and long feather buds formed after four to five days in culture.
