İpek Mikrotaşıyıcı Destekli Biyoreaktörlerde Göbek Kordonu Mezenkimal Kök Hücre Kültürasyonu

Abstract

Mesenchymal stem cells (MSCs) play a significant role in tissue engineering and regenerative medicine due to their ability to differentiate into specialized cell types such as osteoblasts, chondrocytes, myocytes, and adipocytes under specific environmental conditions. MSCs can be isolated from many tissues in the body, primarily bone marrow, adipose tissue, and umbilical cord. Depending on their source of isolation, MSCs exhibit characteristic differences and are mainly used in clinical studies related to inflammation, wound healing, infection, and various degenerative diseases affecting organs and tissues. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) are increasingly used in the field of tissue engineering due to their ability to maintain in vitro differentiation potential over extended periods compared to other adult MSCs, their low immunogenic response, and non-tumorigenic nature. Large-scale cell production is required for the clinical use of UC-MSCs. For this purpose, microcarrier-supported bioreactor systems have been developed. In these systems, microcarriers produced using polymers with appropriate properties based on the needs of the culture are utilized. Within the scope of this thesis, silk fibroin (SF) polymer was selected for microcarrier production due to its biocompatibility, biodegradability, and favorable mechanical properties. The emulsified phase separation method was used for the production of microcarriers. Using this method, four different SF concentrations—2.5%, 3.5%, 4.5%, and 5.5% (w/v) were used to fabricate the microcarriers. As part of the characterization, size measurements showed that the diameters of 2.5%, 3.5%, 4.5%, and 5.5% SF microcarriers were 277 µm, 213 µm, 226 µm, and 234 µm, respectively. In terms of surface roughness, no surface irregularities were observed in the 2.5% SF microcarriers, while the 3.5% SF group exhibited significantly rough surfaces. Static culture was performed using the produced microcarriers. MTT analysis showed that the absorbance values on day 7 for 2.5%, 3.5%, and 4.5% SF microcarriers were similar, whereas the lowest absorbance value was recorded for the 5.5% SF group. To evaluate the effect of surface topography on UC-MSC attachment and proliferation, dynamic culture studies were conducted using 2.5% and 3.5% SF microcarriers. The 3.5% SF microcarriers demonstrated more homogeneous cell distribution under dynamic conditions. MTT analysis revealed that absorbance values obtained with 3.5% SF microcarriers on days 3 and 7 were approximately six times higher than those of 2.5% SF microcarriers. Flow cytometry analyses conducted following static and dynamic cultures confirmed that the cells proliferating on the surface of the microcarriers retained their stem cell characteristics after being removed from the surface. This study is the first to investigate the effect of entirely natural silk fibroin-based microcarriers, produced without the use of any coating or additional materials such as collagen or gelatin, on the proliferation of UC-MSCs under dynamic culture conditions.