¹Ministry of Agriculture and Forestry, Foot and Mouth Disease Institute, Cell Department, TR-06510 Ankara - TURKEY DOI : 10.9775/kvfd.2021.25993 Cell lines provide a useful in vitro model to study in different biotechnological fields. The purity of the cell lines plays a pivotal role in research and production activities. This study aims to present a new method to establish pure cell lines by using bovine tongue tissue. For this purpose, the bovine tongue was obtained from the local slaughterhouse. After establishing primary cell culture, the cells were treated with EDTA (0.02%) for 3-5 min at 37°C. Primarily detached fibroblasts were collected into 5 mL EMEM (with 20% FBS), centrifuged and transferred to a new culture flask with EMEM (10% FCS) medium. The remainder cells in the primary flask were incubated for 8 hours with DMEM (10% FCS). Thereafter, the same process was applied with Na2EDDA (0.01%), and the cells were washed twice with DMEM (with 20% FBS). Reincubation was carried out with decreased FBS concentration (10%) and EGF (10 ng/mL) at 37°C, 5% CO2 in humidified air conditions. The same processes were repeated after 48 h. Conformational studies of pure cultures were done with immunostaining technique with anti-cytokeratin and anti-vimentin monoclonal antibodies where, after purification, fibroblasts displayed vimentin-positive and epithelial cells cytokeratin-positive. In conclusion, this study successfully demonstrated an easy, effective, and efficient method to generate pure novel cell lines from primary tissue culture or mixed cell cultures. Keywords : Primary cell culture, Fibroblast cell, Epithelial cell, Pure culture