Dondurma-Çözme İşleminin Ardından Uygulanan Santrifüjün Ortaya Çıkardığı Mekanik Stresin Karaciğer Kanser Hücreleri İn Vitro Kültürü Üzerine Etkileri

Year 2016, Volume: 8 Issue: 3, 1 - 5, 01.12.2016

Hatice İsan Kübra Tüfekçi Meltem Elif Göklü Ranan Gülhan Aktaş

Abstract

Amaç: Çalışmamızda; dondurma-çözme işleminin ardından direkt kültürü yapılmış ve santrifüj işlemi uygulandıktan sonra kültüre edilmiş HepG2 hücrelerinin in vitro ortamda karşılaştırmalı değerlendirilerek hücre çözme işlemi için en uygun yöntemin belirlenmesi amaçlanmıştır. Gereç ve Yöntem: %10 DMSO Dimethylsulfoxide ve %1 FBS Fetal Bovine Serum içeren DMEM Dulbecco's Modified Eagle's Medium den oluşan dondurma medyumu içerisinde dondurulmuş HepG2 hücreleri, 37°C su banyosu içerisinde çözüldükten sonra iki gruba ayrıldı. I. Gruptaki hücreler direkt kültür metodu uygulanarak; II. Grup hücreler ise 3000 rpm de 3 dak. santrifüj edilmelerinin ardından hücre pelleti ayrıştırılarak eşit oranda besi yeri içerisinde kültüre edildi. Tüm örnekler; Zeiss PrimoVert invert mikroskobunda hergün karşılaştırmalı değerlendirildi. Ardından 7. Gün fikse edilerek Hematoksilen-Eozin ve Tripan Mavisi ile boyandı. Bulgular: İki grup hücrenin morfolojik özellikleri arasında belirgin bir farklılık gözlenmedi. Ancak santrifüj uygulanan hücrelerin bulunduğu kültür kaplarında kültürün her döneminde ölü hücre sayısının daha yüksek olduğu dikkati çekti. Sonuçlar: Çalışmamız; karaciğer kanser hücrelerinin morfolojisi üzerinde dondurma-çözme işlemleri sonrasında uygulanan santrifüj işleminin belirgin bir farklılığa yol açmadığını göstermektedir. Ancak santrifüj sonrası yapılan kültürlerde, ölü hücre sayısının daha fazla olduğu gözlemlenmiştir. Özellikle hücre sayısının az olduğu çalışmalarda çözme sonrası direkt kültürün yapılmasının daha uygun olacağı kanaatine varılmıştır.

Keywords

HepG2, dondurma-çözme, santrifüj, direkt kültür, dimetilsülfoksit

The Effects of Mechanic Stress, Due to Centrifuge After Freezing–Thawing Process, on In Vitro Culture of Liver Cancer Cells

Abstract

Aim: The study aims to examine the effects of centrifuging of HepG2 cells after freezing-thawing process and to clarify the best way for thawing he cells for in vitro culture. Materials And Methods: HepG2 cells were frozen in DMEM Dulbecco's Modified Eagle's Medium containing 10 %DMSO Dimethylsulfoxide and 1% FBS Fetal Bovine Serum . They were thawed in 37°C water-bath immediately and then seperated into two experimental groups: Group I: Cells were cultured directly. Group II: The thawed cell pellet were cultured in the same conditions after centrifuging in 3000 rpm for 3 minutes. All specimens were comparatively examined under Zeiss PrimoVert invert microscope. Subsequently; the cells were fixed on 7th day and stained with Hematoxylene& Eosin and Trypan Blue. Results: There were no significant morphological difference between two groups. However; the dead cell number on daily examinations were strikingly more in the culture of the thawed / centrifuged group. Conclusions: The study demonstrates that centrifuge after thawing has no significant effect on the morphology of liver cancer cells. However, cells undergoesdying process increase after centrifuge. Those results show that direct culture method should be preferred if the cell number is limited in a study.

Keywords

HepG2, freeze-thaw, centrifuge, dimethylsulfoxide

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