Limbal doku türevli hücrelerin ın vitro çoğaltılmasında doku iskelesi olarak fonksiyonelleştirilmiş kollajen film kullanımının değerlendirilmesi
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Dosyalar
Tarih
2020
Yazarlar
Dergi Başlığı
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Cilt Başlığı
Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Gözümüzün ön yüzeyini oluşturan korneanın iyi bir görme işlevi için orijinal bütünlüğünün yaşam boyu korunması gerekmektedir. Korneanın işlevsel bütünlüğü, limbal bölgede yer alan limbal epitelyal kök hücreler tarafından rejenerasyon yoluyla korunarak yaşam boyu sürdürülür. Herhangi bir nedenle bu kök hücrelerin zarar görmesi, korneanın işlevini yitirmesine ve sonuç olarak ciddi görme kayıplarına neden olmaktadır. Bu patolojinin tedavisi için uygulanan geleneksel (otogreft, allogreft) yaklaşımlar kliniklerde arzu edilen güvenilirlikte başarılı olma konusunda yetersiz kalmaktadır. Bu nedenle ileri biyomühendislik yöntemlerinden olan doku mühendisliği alanında geliştirilen yöntemlere ihtiyaç duyulmaktadır. Günümüzde, in vitro çoğaltılan limbal epitelyal kök hücrelerin, riski düşük ve güvenilirliği yüksek bir tedavi seçeneği olarak hastaya sunulması en güncel tedavi yaklaşımıdır. Limbal epitelyal kök hücrelerin in vitro çoğaltılmasında kullanılan en yaygın destek materyali insan amniyotik membranıdır (iAM). Olumlu sonuçlarına rağmen, iAM'nin kliniklerde kullanımı ciddi standardizasyon problemleri ve bulaşıcı hastalık riskleri gibi olumsuzluklar nedeniyle kısıtlı kalmaktadır. Bu nedenle, iAM yerine geçebilecek alternatif bir destek materyali geliştirilmesine gereksinim duyulmaktadır. Geliştirilecek alternatif destek materyalinin başarısı, in vitro ortamda, hücrelerin doğal mikroçevresini olabildiğince taklit edebilmesine doğrudan bağlıdır. Bu yaklaşıma bağlı olarak bu doktora tez çalışmasında, destek materyali olarak hyaluronik asit (HA) ile zenginleştirilen ve 4-arm PEG süksinimidil glütarat (4SP) ile stabilize edilerek fonksiyonelleştirilen kollajen filmlerin ve hücre kültürü ortamının makromoleküler yönden kalabalıklaştırılmasının limbal doku türevli hücrelerin in vitro çoğaltılması üzerine etkisi araştırıldı. HA ile zenginleştirilerek ve 4SP ile stabilize edilerek üretilen kollajen bazlı filmlerin kimyasal karakterizasyonu 2,4,6-trinitrobenzen sülfonik asit (TNBSA) ve enzimatik degredasyon analizleri ile yapıldı. Üretilen filmlerin yapısal karakterizasyonları ise morfolojik ve mekanik yönden incelendi. Makromoleküler kalabalıklaştırıcı optimizasyonu primer insan korneal fibroblastları (HCFs) ile yapıldı ve belirlenen optimal kalabalıklaştırıcı limbal doku türevli hücrelere uyarlandı. Üretilen filmlerin ve makromoleküler kalabalıklaştırma yönteminin üretilen hücrelere etkisi öncelikle hücre morfolojisi, canlılığı, metabolik aktivitesi ve proliferasyon oranı açısından incelenerek değerlendirildi. Hücrelerin ekstrasellüler matriks (ECM) depolama düzeyleri sodyum dodesil sülfat-poliakrilamid jel elektroforezi (SDS-PAGE) ve immunositokimyasal yöntemlerle incelendi. Son olarak, üretilen hücrelerin immunositokimyasal yöntemle fenotipik analizi de yapıldı. HA ile zenginleştirilmiş kollajen filmlerdeki kollajen moleküllerinin kimyasal olarak en etkin çapraz bağlanması minimal 5 mM 4SP konsantrasyonunda sağlanmıştır. Ficoll™ kokteyl makromoleküler kalabalıklaştırıcı olarak HCFs kültüründe korneal stromaya özgü ECM (Kollajen I, III, IV, V, VI) birikimini en yüksek düzeyde sağlarken; aynı zamanda biriken ECM bileşenleri, doğal kornea stromasına benzer şekilde, homojen ve fibriller bir dağılım da sergilemektedir. HA ile zenginleştirilmiş ve 4SP ile stabilize edilerek fonksiyonelleştirilmiş kollajen filmler ve makromoleküler kalabalıklaştırıcı Ficoll™ kokteyl limbal doku türevli hücrelerin morfolojisini, metabolik aktivitesini ve canlılığını korurken, aynı zamanda proliferasyon oranlarını da arttırmaktadır. Ayrıca hücrelerin limbal epitelyal kök hücre mikroçevresine özgü ECM (Kollajen IV, fibronektin, laminin) birikimi arttırılırken fenotipik devamlılığı, incelenen işaretleyiciler (PAX6, vimentin, CK12) yönünden de korunmaktadır. Bu tezin bulgularına göre, makromoleküler olarak (Ficoll™ koktely ile) kalabalıklaştırılmış büyütme ortamı, HA (%0.5 w/w) ile zenginleştirilmiş ve 4SP (5 mM) ile stabilize edilmiş kollajen bazlı doku iskelesi üzerinde in vitro çoğaltılan limbal doku türevli hücrelerin canlılığını, metabolik aktivitesini ve proliferasyonunu destekleyip limbal epitelyal kök hücre mikroçevresine özgü ECM birikimini hızlandırırken, aynı zamanda hücrelerin fenotipik özelliklerinin de devamlılığını sağlamaktadır.
The cornea is the outermost surface of our eyes. For a good visual function, original integrity of the cornea must be maintained during the whole life span. The functional integrity of the cornea is maintained during the whole life span through regeneration by limbal epithelial stem cells, located in the limbal region. Any reason causing to a damage of these stem cells can lead to loss of corneal function and consequently to occurrence of severe visual loss. Traditional approaches (i.e. autograft, allograft) are inadequate in providing the desired success and safety level for the treatment of this pathology in the ophthalmology clinics. Therefore, developing new methodologies using the advanced bioengineering methods in the field of tissue engineering are essential. At the present time, as an advanced cellular therapy approach, transplantation of in vitro expanded limbal epithelial stem cells is the safest treatment option with the highest success level. The most common used scaffold for in vitro expansion of limbal epithelial stem cells is the human amniotic membrane (hAM). Despite of the positive outcomes, the applications of hAM both in the clinics and in the laboratories remains limited due to the serious standardization problems and transmission risk of infectious diseases. Therefore, development of an alternative scaffold which is able to replace hAM is an urgent need. The success of the alternative scaffold depends on the ability of the mimicking the natural microenvironment of the cells. In this doctoral thesis, we investigated the effect of Hyaluronic acid (HA)- enriched and 4-arm PEG succinimidyl glutarate (4SP)-stabilized collagen films in combination of macromolecular crowding method of the cell culture medium on in vitro expansion of limbal tissue-derived cells. Chemical characterization of collagen-based films fabricated by enriching with HA and stabilizing with 4SP was done by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) and enzymatic (collagenase) degradation analyses. Structural characterizations of the fabricated films were examined morphologically and mechanically. Optimization of macromolecular crowding was performed with primary human corneal fibroblasts (HCFs) and the determined optimal crowder was adapted to the limbal-tissue derived cells. The effects of the fabricated films and macromolecular crowding methodology on in vitro expanded cells were primarily examined in terms of cellular morphology, cell viability, metabolic activity and proliferation rate of the cells. Deposition of extracellular matrix (ECM) levels of the cells was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunocytochemical methods. Finally, phenotypic analysis of the in vitro expanded cells was done by immunocytochemical method. The most effective minimal concentration of 4SP for cross-linking chemically to the collagen molecules in the HA-enriched collagen films was determined as 5 mM. FicollTM cocktail provides the highest corneal stroma-specific ECM (Collagen I, III, IV, V, VI) deposition in the culture of HCFs for macromolecular crowding, and the deposited ECM components exhibit homogeneous distribution and fibrillary morphology similar to the native corneal stroma. HA-enriched and 4SP-stabilized collagen films and macromolecular crowding method via using FicollTM cocktail causes to an increase in cellular proliferation rate while maintaining the cellular morphology, metabolic activity and viability of limbal tissue-derived cells. In addition, while the deposition of limbal epithelial stem cell microenvironment specific ECM (Collagen IV, fibronectin, laminin) is increased, cellular phenotypic maintenance is found to be protected in terms of the examined markers (PAX6, vimentin, CK12). According to the results of this thesis study, HA (0.5% w/w)-enriched 4SP (5 mM)- stabilized collagen-based tissue scaffold in combination with the macromolecularly crowded (via FicollTM cocktail) growth medium maintains the viability and metabolic activity of the in vitro expanded limbal tissue-derived cells and support their proliferation and also accelerates the limbal epithelial stem cell microenvironment-specific ECM (collagen IV, fibronectin, laminin) deposition while maintaining the phenotype of the cells.
The cornea is the outermost surface of our eyes. For a good visual function, original integrity of the cornea must be maintained during the whole life span. The functional integrity of the cornea is maintained during the whole life span through regeneration by limbal epithelial stem cells, located in the limbal region. Any reason causing to a damage of these stem cells can lead to loss of corneal function and consequently to occurrence of severe visual loss. Traditional approaches (i.e. autograft, allograft) are inadequate in providing the desired success and safety level for the treatment of this pathology in the ophthalmology clinics. Therefore, developing new methodologies using the advanced bioengineering methods in the field of tissue engineering are essential. At the present time, as an advanced cellular therapy approach, transplantation of in vitro expanded limbal epithelial stem cells is the safest treatment option with the highest success level. The most common used scaffold for in vitro expansion of limbal epithelial stem cells is the human amniotic membrane (hAM). Despite of the positive outcomes, the applications of hAM both in the clinics and in the laboratories remains limited due to the serious standardization problems and transmission risk of infectious diseases. Therefore, development of an alternative scaffold which is able to replace hAM is an urgent need. The success of the alternative scaffold depends on the ability of the mimicking the natural microenvironment of the cells. In this doctoral thesis, we investigated the effect of Hyaluronic acid (HA)- enriched and 4-arm PEG succinimidyl glutarate (4SP)-stabilized collagen films in combination of macromolecular crowding method of the cell culture medium on in vitro expansion of limbal tissue-derived cells. Chemical characterization of collagen-based films fabricated by enriching with HA and stabilizing with 4SP was done by 2,4,6-trinitrobenzenesulfonic acid (TNBSA) and enzymatic (collagenase) degradation analyses. Structural characterizations of the fabricated films were examined morphologically and mechanically. Optimization of macromolecular crowding was performed with primary human corneal fibroblasts (HCFs) and the determined optimal crowder was adapted to the limbal-tissue derived cells. The effects of the fabricated films and macromolecular crowding methodology on in vitro expanded cells were primarily examined in terms of cellular morphology, cell viability, metabolic activity and proliferation rate of the cells. Deposition of extracellular matrix (ECM) levels of the cells was analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunocytochemical methods. Finally, phenotypic analysis of the in vitro expanded cells was done by immunocytochemical method. The most effective minimal concentration of 4SP for cross-linking chemically to the collagen molecules in the HA-enriched collagen films was determined as 5 mM. FicollTM cocktail provides the highest corneal stroma-specific ECM (Collagen I, III, IV, V, VI) deposition in the culture of HCFs for macromolecular crowding, and the deposited ECM components exhibit homogeneous distribution and fibrillary morphology similar to the native corneal stroma. HA-enriched and 4SP-stabilized collagen films and macromolecular crowding method via using FicollTM cocktail causes to an increase in cellular proliferation rate while maintaining the cellular morphology, metabolic activity and viability of limbal tissue-derived cells. In addition, while the deposition of limbal epithelial stem cell microenvironment specific ECM (Collagen IV, fibronectin, laminin) is increased, cellular phenotypic maintenance is found to be protected in terms of the examined markers (PAX6, vimentin, CK12). According to the results of this thesis study, HA (0.5% w/w)-enriched 4SP (5 mM)- stabilized collagen-based tissue scaffold in combination with the macromolecularly crowded (via FicollTM cocktail) growth medium maintains the viability and metabolic activity of the in vitro expanded limbal tissue-derived cells and support their proliferation and also accelerates the limbal epithelial stem cell microenvironment-specific ECM (collagen IV, fibronectin, laminin) deposition while maintaining the phenotype of the cells.
Açıklama
Anahtar Kelimeler
Kornea, Limbal Doku, Doku Mühendisliği, Biyomalzeme, Kollajen
