Wharton Gel-Derived Exosome Isolatıon: A Method Comparative Study

Öz

Exosomes are nano-sized biomolecules that originate from the endosome, have an important role in immune modulation, and provide intercellular interaction. Exosomes have a wide variety of membrane proteins such as tetraspanins, proteins, Annexin and Rab proteins, and they are candidates for use in the treatment and diagnosis of infections, nervous system, cancer and neurodegenerative diseases. It is important for diagnosis and treatment that exosomes released from almost all body fluids can be isolated both homogeneously and in large numbers. Therefore, the purification of exosomes and obtaining specific exosomes for diagnosis is an important step in their production for therapeutic purposes. This study aimed to isolate the most and purest exosomes by using different isolation methods, ultracentrifugation, differential centrifugation-filtration and precipitation from commercial mesenchymal stem cell culture medium derived from wharton gel. Imaging, purity and size analysis of exosome characterization was determined for methods. As a result, it was observed that as a result of the isolation with the ultracentrifuge method, a much larger number of exosomes were isolated compared to both precipitation and differential centrifugationfiltration methods. According to the differential centrifugation-filtration method, it was observed that the number of exosomes was much less, but the exosomes were of the same purity as the ultracentrifuge method. Since exosomes play an active role in treating almost every disease, they provide ease of use in diagnosing diseases, and their good biodistribution, biocompatibility and low immunogenicity will also provide an advantage in the treatment phase.

Anahtar Kelimeler

• Wharton Gel
• Exosome
• Isolation Methods

Wharton Jel Kaynaklı Eksozom İzolasyonu: Metot Karşılaştırma Çalışması

Öz

Eksozomlar endozomdan köken almış, immün modülasyonda önemli role sahip, hücrelerarası etkileşimi sağlayan, nano boyutta biyomoleküllerdir. Eksozomlar tetraspanninler, proteinler, Annexin ve Rab proteinleri gibi çok çeşitli zar proteinlerine sahiptirler ve bu içeriklere sahip olmaları ile enfeksiyon, sinir sistemi, kanser ve nörodejeneratif hastalıkların tedavi ve teşhisinde kullanılmaya adaydır. Hemen hemen bütün vücut sıvılarından salınan eksozomların hem homojen hem de çok sayıda izole edilebilmesi teşhis ve tedavi için önemlidir. Bu nedenle eksozomların saflaştırılması ve tanıya yönelik spesifik eksozom izolasyonu tedavi amaçlı üretimi açısından önemli bir basamaktır. Bu çalışmada wharton jel kaynaklı ticari mezenkimal kök hücre kültür besiyerinden (medyum) farklı izolasyon metotları olan ultrasantrifüj, diferansiyel ultrasantrifüj-filtrasyon ve çökeltme (presipitasyon) metodu kullanılarak en çok sayıda ve en saf eksozom izolasyonu yapılması amaçlanmıştır. Eksozomların karakterizasyonu için; görüntülenmesi, saflığı ve boyut analizi her üç metot için belirlenmiştir. Sonuç olarak ultrasantrifüj metodunda, hem diferansiyel ultrasantrifüjfiltrasyon, hem de presipitasyon metoduna göre çok daha fazla sayıda çok daha küçük boyutta eksozom izolasyonu olduğu görülmüştür. Diferansiyel ultrasantrifüj-filtrasyon metodu sonucunda ise eksozom sayısının çok daha az olduğu ancak ultrasantrifüj yöntemi ile elde edilen eksozomlarla aynı saflıkta olduğu görülmüştür. Eksozomların hemen hemen her hastalığın tedavisinde etkin rol almalarından dolayı hastalıkların teşhisinde kullanım kolaylığı sağlamakla birlikte iyi biyo-dağılım, biyouyumluluk ve düşük immünojeniteye sahip olmaları da tedavi aşamasında avantaj sağlayacaktır.

Anahtar Kelimeler

• Wharton Jel
• Eksozom
• İzolasyon Metotları

Teşekkür

Bu çalışmanın yapılmasında; laboratuvar olanak ve elverişli çalışma ortamı sunan, Erciyes Üniversitesi, Genom ve Kök Hücre Merkezi’ne teşekkürlerimi sunarım.

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