Akciğer Havayolu Modellemesine Yönelik Oluşturulan Deselülerize Doku İskeleleri ile 3b Hücre Kültür Sistemlerinin Kurulması

Abstract

The lungs have the task of providing the necessary oxygen for the cells and carrying out the carbon dioxide formed as a result of metabolic events. During this process, which we call breathing, the lungs encounter many harmful substances such as allergens with protease properties, viruses, bacteria, particles that cause air pollution, cigarette smoke, and toxic chemicals. These harmful substances entering the airway have a destructive effect on the tight junction molecules that provide epithelial cell integrity, leading to the deterioration of its structural integrity and cause epithelial barrier damage. Environmental factors and genetic predispositions cause chronic airway diseases such as asthma and COPD and play a role in increasing or decreasing the severity of the disease. Thousands of people die every year due to chronic airway diseases. Various experimental models are needed to understand these disease mechanisms and to develop treatment methods. Regenerative medicine and tissue engineering applications, which have been developed rapidly in recent years, have an essential place in the understanding and development of disease mechanisms. The tissue and organ decellularization method, which is one of the tissue engineering applications, has been used successfully in both pre-clinical studies and clinical applications in experimental animals for a long time. Decellularized scaffolds obtained by preserving the natural extracellular matrix component of the tissue and removing possible immune response cells are essential in that they can be used in vitro to develop cell culture systems and in vivo studies to meet donor organ deficiency. Since healthy donor lungs are always used for lung transplantation, alternative lung sources on which various cell populations can attach and grow are needed for tissue engineering studies. Within the scope of this thesis, sheep lung has been proposed as a xenogenic alternative to human lungs. In the study, it was aimed to produce tissue scaffolds by applying physical and chemical decellularization methods on sheep lung tissue. In the first stage of the study, it was aimed to create an optimum decellularization method for lung tissue by using detergents that used in lung decellularization such as SDS, Triton X-100 and CHAPS, as well as detergents that were not used in lung decellularization, such as Sodium cholat, NP-40 and ASB-14, by the mixing/shaking technique. sCO2 was used as the physical decellularization method. By comparing the results obtained by physical and chemical methods, the effect of both methods on sheep lungs was revealed. In the second stage of the study, the characterization processes of the obtained decellularized lung tissue scaffolds and hydrogel synthesis from these tissues were carried out. For characterization, it was quantitatively determined how much the DNA contained in the decellularized extracellular matrix was removed from the structure compared to the natural tissue. Then, the tissues were evaluated qualitatively by the histological staining method. Efficacy was investigated by quantitative analyzes of the collagen, glucosaminoglycan (GAG), and elastin ratios remaining in the tissue scaffolds. To analyze the surface characterization of the scaffolds obtained, SEM imaging were performed. After these analyses, hydrogel synthesis and characterization was performed from the tissues of the effective condition and cell culture studies were carried out. As a result of the characterization process, methods with ASB-14, sodium cholate hydrate, and SDS detergents, effective on lung decellularization, were selected. Among these methods, hydrogel synthesis was performed from the decellularized tissue obtained with ASB-14, BEAS-2B cells were cultivated on it, and cell proliferation and differentiation were evaluated on ALI culture.