A simple method for the establishment of tissue culture melanocytes from regenerating fowl feathers.

A quick and simple method for the establishment of tissue cultures of nonembryonic domestic fowl melanocytes was desired. The selected source of these cells was the 14-d-old regenerating feather. Three procedures were compared on the basis of the yield and purity of melanocytes. For the first method, 2 mm of the proximal end of the feather was cut off under sterile conditions and placed immediately in Hanks' balanced salt solution (BSS) containing antibiotics. The feather was split longitudinally and the pulp removed. The tissue was placed pulp side down in several drops of Ham's F12 medium containing 2.5 micrograms/ml Fungizone, 50 micrograms/ml gentamicin, 100 micrograms/ml streptomycin, 100 micrograms/ml penicillin, and 10% fetal bovine serum. After 2 h at 37 degrees C, the tissue was attached to the dish and new medium was added and changed every 3 d thereafter. Cells migrated from the tissue starting on Day 2 and the tissue was removed on Day 5. Large dendritic peripheral cells and small round central cells were seen. Approximately 6.5 X 10(4) cells were present on Day 10 and 8 X 10(4) cells were counted on Day 20. By Day 30, the pigmented melanocytes were large, flat, dendritic cells. Electron microscopy and the use of the dopa reaction indicated that the population of cells was almost entirely melanocytes. The second method used was similar to the first, the only difference being that the feather sheath was also removed and thus only the collar of cells remained. The third method tried was similar to the second with the difference that the collar of cells was gently agitated with 0.25% trypsin for 5, 10, and 20-min intervals at 37 degrees C. The trypsin supernatant fluid was removed by gentle centrifugation and medium plus fetal bovine serum was added to stop tryptic action. The second method showed no advantage over the first. The purity and yield of melanocytes in the third method were lower than in either of the previous two methods. The number of cells desired can be controlled by varying the number of the feather pieces used per culture.