Evaluation of Functionalised Collagen Film as a Scaffold for In Vitro Expansion of Limbal Tissue-Derived Cells
Project no. 20031 · Status: Kapanmış (Yayın bekleniyor) · 2018
Summary
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. Ficoll™ 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 Ficoll™ 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 Ficoll™ 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.