Hücrelerinden Arındırılmış Miyokardiyal Doku İle Hücre Tabakası Destekli Kalp Yaması Geliştirilmesi

Abstract

The aim of this thesis is to produce a heart patch by combining cardiovascular cell sheet and decellularized myocardial extracellular matrix (ECM). In the study, two innovative approaches of tissue engineering, ‘‘cell sheet technology’’ and ‘‘decellularization approach’’ were combined. Thus, in vitro heart tissue model, which can be used for the treatment of heart diseases, has been developed.

In the first step of the thesis study, characterization studies of H9C2 rat cardiomyoblast cell line and primary rat cardiac microvascular endothelial cells (CMEC), used in the production of cardiovascular cell sheets, were performed. Then, 3 different methods, temperature responsive culture plates, high cell seeding density and serum concentration and ascorbic acid (AA) treatment, were used to produce cell sheet using only H9C2 cell line. In general, it has been determined that the viability of all cell sheets is highly conserved and the cell sheets were successfully removed from the culture surface on days 7, 9 and 5 of the culture, respectively. In addition, it was observed that the gene expression levels of the cells were influenced by the nature of the cell culture dish surface, cell seeding density and AA and serum concentration in the culture medium. Real-time polymerase chain reaction (RT-PCR) analysis results demonstrated the poly(isopropylacrylamide) (PIPAAm)-coated culture surface enhanced gene expression of skeletal muscle specific L-type voltage-dependent Ca+ channel (Alpha 1s, Cacna1s), while low cell seeding density decreased the expression of Cacna1s and cardiac troponin T (Tnnt2), but increased collagen I (Col1a1) gene expression. It has been shown that Col1a1 gene expression of the cells increased with the use of high serum concentration and AA in culture medium. When the overall effect of ascorbic acid was evaluated, it was determined that myogenic character of H9C2 cells increased in normal serum containing medium, but, cell sheets having high cardiomyogenic character were produced in case of high serum with 100 µg/mL AA.

In order to produce cardiovascular cell sheets, at first H9C2 cells were cultured in retinoic acid (RA; 10 nM, 50 nM and 100 nM) containing medium at different time periods (5 and 7 days) and differentiated into cardiomyocyte cells. As a result of the analyzes (immunofluorescence staining and RT-PCR), it was determined that 100 nM RA treatment for 5 days was the most appropriate condition for differentiation. Subsequently, differentiated H9C2 cells and CMECs (6:1) were co-cultured in media containing different AA (20, 50, 100 µg/mL) and serum (10.5% and 15.0% v/v) contents. Although high levels of viable cell sheets were obtained in all groups, it was determined that cardiomyogenic properties were highest in 10.5% (v/v) FBS and 100 µg/mL AA concentration. Therefore, cell sheets obtained in these conditions were used for the production of heart patches.

Two different methods (SDS and OGP) were used for the production of decellularized myocardial ECM scaffolds, which is the second component of the heart patch. Scanning electron microscope (SEM), immunofluorescence staining, histological staining and DNA analysis showed that decellularization was performed successfully with SDS method and compared to native tissue there was ~89% reduction in DNA content. Otherwise, hydroxyproline assay, GAG analysis and cytotoxicity test showed that the SDS method did not damage the ECM structure and did not contain toxic chemical residues to the cells.

A 2-week in vitro co-culture study was performed on the decellularized ECM scaffold using adipose-derived mesenchymal stem cells (ADMSCs) and CMECs. 5-Azacitidine (5-Aza) was applied for 24 h to stimulate cardiomyogenic differentiation. It has been determined that the cells maintain high viability during culture and form vascular-like lumen structures in the scaffold. However, the analyzes (immunofluorescence staining and RT-PCR) showed that 5-Aza alone was not sufficient for the cardiomyogenic differentiation of ADMSCs on the heart ECM niche.

ECM scaffold seeded with ADMSCs and CMECs was combined with cardiovascular cell sheet to produce the cardiovascular cell sheet supported myocardial patch. To support cardiomyogenic differentiation, 5-Aza treatment for 24 h was applied. It has been observed that, 5-Aza supports the cardiomyogenic differentiation of ADMSCs on the ECM niche with the help of direct co-culture with cardiomyocytes. RT-PCR results showed an increase in Tnnt2 and Gata4 gene expression of the cells. Furthermore, in the absence of a chemical stimulant agent, heart ECM niche and direct co-culture with cardiomyocytes were also found to support the cardiomyogenic differentiation of ADMSCs.

It was determined that cell sheets could be produced successfully without the use of temperature responsive culture plates. Besides, it has been observed that ADMSCs could be differentiated cardiomyogenic with the triple synergistic effect of cardiac ECM niche, direct co-culture with cardiomyocytes and 5-Aza. In conclusion, an in vitro heart tissue model with high vascularization potential has been demonstrated. It has been shown that the produced heart patch is suitable for use in in vivo experiments on rats. If successful results were obtained in these studies, it was determined that the patch could be considered as an alternative approach in heart damage by adapting the clinical studies.