Sığır Dil Dokusu Primer Kültürü Kullanılarak Yeni Bir Epitel veFibroblastik Hücre İzolasyonu ve Saflaştırma Yöntemi

Hücre hatları, farklı biyoteknolojik alanlarda çalışmak için elverişli in vitro modeller olup, saflıkları araştırma ve üretimlerde çok önemli bir rol oynar. Bu çalışmanın amacı, sığır dil dokusunu kullanarak saf hücre hatlarının oluşturulması için yeni bir yöntem oluşturmaktır. Mezbahadan alınan sığır dil dokusundan primer hücre kültürü hazırlandıktan sonra, 37°C’de 3-5 dak EDTA (%0.02) ile muamele edilen hücreler, fibroblastlar yüzeyden ayrılmaya başladığında, %20 FBS içeren 5 mL EMEM ortamında toplandı, santrifüjlendi ve %10 serum içeren EMEM ortamında yeni bir kültür kabına aktarıldı. Eski kültür kabında kalan hücreler 8 saat 5 mL DMEM (%10 FBS’li) ile inkübasyonu takiben, aynı işlem Na2EDDA (%0.01) ile uygulandıktan sonra hücreler, %20 FBS’li DMEM ile iki kez yıkandı ve bu defa %10 FBS ve 10 ng/ml EGF içeren DMEM ile inkübe edildi. 48 saat sonra aynı işlemler tekrarlandı. Saf kültürlerde sitokeratin ve vimentin için spesifik monoklonal antikorlar kullanılarak doku orijini doğrulaması amacıyla immün boyama protokolü uygulandı. Sonuçlarımız, immünositokimyasal analize göre, saflaştırılmış fibroblast hücrelerinin vimentin pozitif olduğunu ve saflaştırılmış epitel hücrelerinin de sitokeratin pozitif gösterdiğini gösterdi. Sonuç olarak, bu çalışma, primer kültürler ya da karışık hücre kültürlerinden saf yeni hücre hatları oluşturmak için kolay, etkili ve verimli bir yöntem ortaya koymuştur.

A Novel Epithelial and Fibroblastic Cell Isolation and Purification Method Using Primary Culture of Bovine Tongue Tissue

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 antivimentin 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.

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