O-17: Improved Protocol for Isolating Dermal Fibroblasts (Pages: 0-0)


Mostafazadeh A *, Pandamooz S , Akhavan-Niaki H , Mousavi Kani N , Abedian Z , Motevallizadeh Ardekani A , Hadipour ,

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Objective: Fibroblasts are the main component of connective tissue which have been used for a wide range of investigative purposes such as tissue engineering because of their outstanding mitotic potential. Isolating dermal fibroblasts is an appropriate way to expand these fast growing cells in vitro. This study was performed to increase the number of viable cells after enzymatic digestion of specimen with collagenase. Materials and Methods: Dermal fibroblasts were isolated from fresh human foreskin of donors aged from 1 to 3 months. After was hing and epidermal layer was removed by adding 0.5% Dispase II at 37°C for 2 hours and dermal parts were digested with 0.1% Collagenase at 37°C. The released cells were collected every 20 minutes from upper aqueous phase, centrifuged and resuspend in complete growth medium (DMEM+ 10% FBS +1% penicillin/streptomycin), then seeded on 6-well tissue culture plates at 106 cells/ml after determining the viability with trypan blue staining. The adherent cells reached to confluency within 7 days and were subcultured to 25-ml cell culture flask and stained with Giemsa after another 7 days. Results: Staining the cells with trypan blue has determined > 90% cell viability of resulting suspension which had been pooled earlier. Moreover morphology of cultured fibroblasts was confirmed with Giemsa staining. Conclusion: Although using dissociated fibroblast culture method is more convenient than skin explant culture, its enzymatic digestion is critical, as a large number of cells can be lost over prolonged exposure to collagenase. By cutting the specimens into small sizes and collecting released cells every 20 minutes. Cell yield can be maximized while their viability is maintained