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

Yıl 2016, Cilt: 8 Sayı: 3, 1 - 5, 01.12.2016

Hatice İsan Kübra Tüfekçi Meltem Elif Göklü Ranan Gülhan Aktaş

Öz

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.

Anahtar Kelimeler

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

Öz

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.

Anahtar Kelimeler

HepG2, freeze-thaw, centrifuge, dimethylsulfoxide

Kaynakça

• Auwera Van Der I, Cornillie F. Cryopreservation of pro- nucleate mouse ova: slow versus ultrarapid freezing. Hum Reprod 1990; 5 (5): 619-21.
• Anchordoguy TJ, Cechini CA, Crowee JH, Crowee LM. Insights into the cryoprotective mechanism of dimethyl sulfoxide for phospholipid bilayer. Cryobiology 1991; 28: 467-73.
• McGann LE. Optimal temparature ranges for control of coding rate. Cryobiology 1991; 28: 467-73.
• Rinkes IH, Toner M, Ezzel RM, Tompkins RG, Yarmush ML. Effect of dimethyl sulfoxide on cultured rat hepato- cytes in sandwich configuration. Cryobiology 1992; 29 (4): 443-53.
• Akiyama Y, Terada R, Hashimoto M, et al. Rod-shaped Tissue Engineered Skeletal Muscle with Artificial An- chors to Utilize as a Bio-Actuator. Journal of Biomechan- ical Science and Engineering 2010; 5 (3): 236-244.
• Nakagawa K, Morishima N, Matsumoto T. Effect of Three-Dimensional Culture and Cyclic Stretch Stimula- tion on Expression of Contractile Protein in Freshly Iso- lated Rat Aortic Smooth Muscle Cells. Journal of Biome- chanical Science and Engineering 2009; 4 (2): 286-297.
• Terracio L, Miller B, Borg T. Effects of Cyclic Mechanical Stimulation of the Cellular Components of the Heart: in Vitro. In Vitro Cellular & Developmental Biology 1988; 24 (1): 53-58.
• Wang J.H.-C, Yang G, Li Z, Shen W. Fibroblast Respons- es to Cyclic Mechanical Stretching Depend on Cell Ori- entation to the Stretching Direction. Journal of Biome- chanics 2004; 37: 573-576.
• Hashimoto S, Hino H, Iwagawa T. Effect of Excess Gravi- tational Force on Cultured Myotubes in Vitro. Journal of Systemics, Cybernetics and Informatics 2013; 11(3):50- 57.
• Hashimoto S, Okada M. Orientation of Cells Cultured in Vortex Flow with Swinging Plate in Vitro. Journal of Systemics Cybernetics and Informatics 2011; 9 (3): 1-7.
• Hashimoto S, Sato F, Hino, et al. Responses of Cells to Flow in Vitro. Journal of Systemics Cybernetics and In- formatics 2013; 11(5): 20-27.
• Sugaya Y, Sakamoto N, Ohashi T, et al. Elongation and Random Orientation of Bovine Endothelial Cells in Re- sponse to Hydrostatic Pressure: Comparison with Re- sponse to Shear Stress. JSME International Journal, Se- ries C, 2003; 46( 4):1248-1255.
• Uttayarat P, Chen M, Li M, et al. Microtopography and Flow Modulate the Direction of Endothelial Cell Mi- gration. Am. J. Physiol. Heart Circ. Physiol 2008; 294: H1027-H1035.
• Azuma N, Duzgun S.A, Ikeda M, et al. Endothelial Cell Response to Different Mechanical Forces. Journal of Vas- cular Surgery 2000; 32(4): 789-794.
• Lee DH, Park JC, Suh H. Effect of centrifugal force on cellular activity of osteoblastic MC3T3-E1 cells in vitro. Yonsei medical journal 2001; 42 (4): 405-10.
• Coleman SR. Structural fat grafts - The ideal filler? Clin Plast Surg 2001; 28 (1):111.
• Boschert MT, Beckert BW, Puckett CL, et al. Analysis of lipocyte viability afterliposuction. Plast Reconstr Surg 2002; 109: 761Y765; discussion 766Y767.
• Rohrich RJ, Sorokin ES, Brown SA. In search of improved fat transfer viability: A quantitative analysis of the role of centrifugation and harvest site. Plast Reconstr Surg 2004; 113 (1): 391-5.
• Kurita M, Matsumoto D, Shigeura T, et al. Influences of centrifugation on cells and tissues in liposuction aspi- rates: Optimized centrifugation for lipotransfer and cell isolation. Plast Reconstr Surg 2008; 121(3): 1033-41.
• Alvarez JG, Lasso JL, Blasco L, et al. Centrifugation of Hu- man Spermatozoa Induces Sublethal Damage - Separa- tion of Human Spermatozoa from Seminal Plasma by a Dextran Swim-up Procedure without Centrifugation Ex- tends Their Motile Lifetime. Human reproduction 1993; 8 (7) :1087-92.
• Aitken RJ, Finnie JM, Muscio L, et al. Potential importance of transition metals in the induction of DNA damage by sperm preparation media. Human reproduction 2014; 29 (10): 2136-47.
• Zini A, Mak V, Phang D, et al. Potential adverse effect of semen processing on human sperm deoxyribonucleic acid integrity. Fertility and sterility 1999;72 (3): 496-9.
• Katkov II & Mazur P. Influence of centrifugation regimes on motility, yield, and cell associations of mouse sperma- tozoa. J Androl 1988; 19: 232–241.
• Kim S, Agca C, Agca Y. Effects of various physical stress factors on mitochondrial function and reactive oxygen species in rat spermatozoa. Reprod Fert Develop 2013; 25 (7): 1051-64.