Bekir Uğur ERGÜR, Kemal BAYSAL, Tuğba ŞAN, Özge SAYIN, Pınar AKAN, Deniz ÖZTEKİN, Uğur BORA, Mehmet Emin GÜNEŞ

İnsan Göbek Kordonu Wharton Jölesinden Mezenkimal Kök Hücrelerin Eldesinde İzolasyon Yöntemlerinin Ve Antioksidatif Kültür Koşullarının Etkisi

GİRİŞ ve AMAÇ: Günümüzde, insan kök hücrelerin eldesi ve kültür ortamında çoğaltılması rejeneratif tıp uygulamalarının ilk aşamalarından biridir. Ancak terapötik amaçlar için in vitro hücre çoğaltmada, MKH'lerin hızla muhtemelen oksidatif stresi içeren erken yaşlanmaya girmeleri önemli bir sorundur. Günümüzde üzerinde en fazla çalışılmış ve klinikte tedavide kullanılan hücreler kemik iliği mezenkimal kök hücreleri (Kİ-MKH) olmakla birlikte, insan perinatal dokularında tespit edilmiş mezankimal kök hücreler giderek önem kazanmaktadır. Göbek kordonu çevresinde yer alan Wharton jölesi mezenkimal kök hücrelerinin (WJ-MKH) invaziv olmayan şekilde elde edilmeleri, doku reddine yol açan proteinleri eksprese etmemeleri ve immünosupresif özellikleri nedeni ile Kİ-MKH’lerinden daha kullanışlı olabilecekleri ileri sürülmektedir. Çalışmamız ile WJ-MKH’inin eldesi ve çoğaltılmasında kullanılan izolasyon yöntemlerinin etkinliğinin karşılaştırılması ve anti-oksidatif kültür koşullarının hücre canlılığı ve kök hücrelere özgü yüzey antijen ekspresyonlarına etkisinin belirlenmesi amaçlandı. YÖNTEM ve GEREÇLER: Etik Kurul onayı alındıktan sonra gönüllü onam alınmış, çalışmaya dahil etme kriterlerine uyan toplam 17 sağlıklı gebeden doğum sonrası kordon örnekleri alındı. Wharton jölelesinden enzimatik ve eksplant yöntemi ile kök hücreler elde edilerek, hücre canlılıkları ve proliferasyon kapasiteleri karşılaştırıldı. Kök hücrelerin osteoblast, kondrosit ve adipositlere farklılaşma kapasiteleri immunohistokimyasal olarak gösterildi. BULGULAR: Hücrelerin çoğaltılması sürecinde CD44, CD73, CD90, CD105 yüzey antijen ifadelerinin dördüncü pasaja kadar anlamlı değişmeden eksprese edildiği akış sitometrik olarak belirlendi. Anti oksidan moleküller N-asetil sistein ve askorbik asit ilavesi ile sağlanan kültür koşullarının hücre canlılığı ve hücre yüzey antijen ekspresyonları üzerine etkisi gösterildi. TARTIŞMA ve SONUÇ: Sonuç olarak wharton jölesinden MKH’lerin izolasyonunda eksplant yönteminin yüzey antijen ifadeleri anlamlı değişmeksizin, daha kısa zaman diliminde elde edilen toplam hücre sayısı nedeni ile enzimatik yönteme göre daha avantajlı olduğu düşünüldü.

Anahtar Kelimeler:

wharton jeli, izolasyon yöntemleri, antioksidanlar, mezenkimal kök hücre, hücre canlılığı

The Effect of Antioxidant Culture Conditions and Isolation Methods in Obtaining of Mesenchymal Stem Cells from The Wharton's Jelly of Human Umbilical Cord

INTRODUCTION: Obtaining and growing human stem cells are the first stages of regenerative medicine applications.During the cell proliferation for therapeutic purposes, MSCs rapidly undergo premature aging, possibly involving oxidative stress.Bone marrow mesenchymal stem cells(BM-MSCs) are the most clinically used cells.It's suggested that Wharton-jelly mesenchymal stem cells(WJ-MSC) which are located around the umbilical cord may be more useful than BM-MSCs due to their non-invasive acquisition,not expressing proteins cause tissue rejection,and their immunosuppressive properties.It was aimed to compare the efficiency of the isolation methods used in the reproduction of WJ-MSCs and to determine the effect of anti-oxidative culture conditions on the cell viability and expression of cell-surface antigens. METHODS: Postnatal cord samples were taken from 17 healthy pregnant women who met the inclusion criteria. Stem cells were obtained from Wharton jelly using enzymatic and explant methods, and cell viability and proliferation capacities were compared. RESULTS: The differentiation capacity of stem cells into osteoblasts, chondrocytes and adipocytes was demonstrated immunohistochemically. During the proliferation process, it was determined by flow cytometry that CD44, CD73, CD90, CD105 surface antigen expressions were expressed and unchanged until the fourth passage.The effect of culture conditions provided by addition of anti-oxidant molecules N-acetyl cysteine and ascorbic acid on cell viability and cell surface antigen expressions were demonstrated. DISCUSSION AND CONCLUSION: In conclusion, it was thought that the explant method in the isolation of MSCs from wharton jelly was more advantageous than the enzymatic method due to the total number of cells obtained in a shorter time period, without significantly changing the surface antigen expressions.

Keywords:

Wharton jelly, isolation methods, antioxidants, mesenchymal stem cells, cell viability,

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