Isolation of Corneal and Limbal Cells From Human Donor Cornea

Öz

Objective: Isolation of corneal cells while preserving their specific phenotypes is crucial for regenerative medicine applications. Here, we aimed to evaluate corneal cell isolation techniques from human corneal tissue. Methods: We isolated corneal cells and limbal stem cells from human donor corneas obtained from the eye bank by employing both enzymatic and explant cell culture approaches and monitored the morphological characteristics of the isolated cells. Results: Explant culture demonstrated suitability for the isolation of corneal endothelial and epithelial cells, while both explant and enzymatic culture processes yielded successful results for limbal and corneal stromal tissues. The optimal conditions for enzymatic culture were determined to be 2 mg/mL enzyme solution for 30 minutes at 37 °C. On the other hand, the feasibility of explant cultures was increased by re-transferring and culturing tissue specimens to isolate an enlarged quantity of cells in an extended culture periods. However, this approach resulted in morphological changes in the isolated cells. The earliest passage at which isolated primary cells (keratocyte and limbal stem cells) could be immortalized was Passage 1, enabling subculturing and cell stock creation. On the other hand, isolated primary limbal stem cells demonstrated the capacity to differentiate into corneal epithelial and keratocyte cells. Conclusion: This study provides a comprehensive and detailed presentation that will serve as an informative resource in the literature for corneal cell culture, corneal tissue engineering, and drug delivery studies.

Anahtar Kelimeler

• cornea
• primary human corneal cells
• limbus
• limbal stem cells
• differentiation
• immortalization

İnsan Donör Korneasından Korneal ve Limbal Hücrelerin İzole Edilmesi

Öz

Amaç: Gözün en dış tabakasında yer alan kornea, ışığın merceğe etkin bir şekilde iletilmesini sağlayan, saydam ve damarsız bir dokudur; bu yönüyle görme keskinliğine önemli ölçüde katkı sağlar. Dıştan içe doğru sırasıyla epitel, Bowman membranı, stroma, Descemet membranı ve endotel olmak üzere beş farklı tabakadan oluşan korneada, her bir tabakada bulunan özelleşmiş hücre tipleri, dokunun bütünlüğü için hayati öneme sahip spesifik işlevler üstlenir. Ayrıca, korneaya komşu bir doku olan limbus bölgesinde, kornea dokusunun yenilenmesini sağlayan kök hücreler bulunmaktadır. Korneal hücrelerin kendi özgün fenotiplerini koruyacak şekilde izole edilmesi, rejeneratif tıp uygulamaları açısından büyük önem taşımaktadır. Bu çalışmada, insan kornea dokusundan korneal hücre izolasyon tekniklerinin değerlendirilmesi amaçlanmıştır. Yöntem: Göz bankasından temin edilen insan donör kornealarından korneal hücreler ve limbal kök hücreler, enzimatik ve eksplant hücre kültürü yaklaşımları kullanılarak izole edildi ve elde edilen hücrelerin morfolojik özellikleri gözlemlendi. Bulgular: Eksplant kültürü yöntemi, korneal endotel ve epitel hücrelerinin izolasyonu için uygunluk göstermiştir. Bununla birlikte, limbal ve korneal stromal dokuların izolasyonunda hem eksplant hem de enzimatik kültür yöntemleri başarılı sonuçlar vermiştir. Enzimatik kültür için en uygun koşul; 2 mg/mL enzim çözeltisinin 37°C’de 30 dakika inkübasyonu olarak belirlenmiştir. Diğer yandan, eksplant kültürlerin verimliliği, doku örneklerinin yeniden transfer edilerek kültüre alınmasıyla artırılmış; bu da daha uzun süreli kültürlerde hücre sayısının artırılmasını sağlamıştır. Ancak bu yaklaşım, izole edilen hücrelerin morfolojisinde değişikliklere yol açmıştır. Keratosit ve limbal kök hücreler gibi izole edilen primer hücrelerin ölümsüzleştirilebildiği en erken pasaj, Pasaj 1 olarak belirlenmiş ve bu da alt kültürleme ile hücre stoğu oluşturulmasına olanak tanımıştır. Öte yandan, izole edilen primer limbal kök hücrelerin korneal epitel ve keratosit hücrelerine farklılaşma kapasitesi gösterdiği tespit edilmiştir. Sonuç: Bu çalışma, korneal hücre kültürü, kornea doku mühendisliği ve ilaç salınımı çalışmaları için literatürde yol gösterici nitelikte kapsamlı ve ayrıntılı bir kaynak sunmaktadır.

Anahtar Kelimeler

• kornea
• insan primer korneal hücre izolasyonu
• limbus
• limbal kök hücreler
• farklılaşma
• ölümsüzleştirme

Kaynakça

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