The Investıgatıon Of Surface Modıfıcatıons On Polyester Based Fıbrous Matrıces For Cell Expansıon In Packed Bed Bıoreactors

Abstract

This study was financially supported by Hacettepe University Scientific Research Projects Coordination Unit (BAP) with the project entitled “The Investigation of The Effects of Surface Modifications on Fiber Based Polyester Matrices for Mesenchymal Stem Cell Production in Packed Bed Bioreactors” (project no: FYL-2018-17359). This study aims to investigate the effects of surface modifications on polyester matrices for mesenchymal stem cell (MSC) production in a packed bed bioreactor. Firstly, sulfuric acid and sodium hydroxide treatments were applied on PET with different parameters in order to increase hydrophilicity and all surface-treated groups of PET disks were examined through water contact angle measurements, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR), Scanning Electron Microscope (SEM) and Energy Dispersive X-Ray Spectroscopy (EDS) analyses. After characterization studies, 3M H2SO4 and 0.05M NaOH treated PET disks were further studied in vitro with MC3T3-E1 cells and cell activity was found to be higher in sodium hydroxide treated PET disks than sulfuric acid-treated PET disks. Secondly, collagen type-1 (30 µg/disk) and vitronectin (0.15 and 0.60 µg/disk) were coated on the PET surfaces via physical and chemical immobilization methods. In the characterization studies, hydroxyproline analysis and SEM analysis demonstrated that the physical immersive coating technique was more efficient and resulted in evenly distributed collagen type-1 on the PET disk surface. Meanwhile, the results of SEM and ATR-FTIR analyses of collagen type-1 and vitronectin crosslinking on PET disks were similar to that of the control group. Also, the results of static cell culture conducted with rAdMSCs demonstrated no significant differences between collagen type-1 and vitronectin coated PET disks with plain sodium hydroxide treated PET disks. Thus, sodium hydroxide treated PET disks were selected for dynamic cell culture studies. In the last part of the thesis, rAdMSC expansion was investigated in our custom made packed-bed bioreactor using sodium hydroxide treated PET as packing material. In dynamic studies, two different cell seeding densities were used: 30 x 106 cells/ 1 g disks and 10 x 106 cells/0.5 g disks. The specific growth rate and doubling time of rAdMSCs with higher seeding density were calculated as 0.06 h-1 and 65 h and it was determined that glucose concentration in the culture medium was insufficient. And the specific growth rate and doubling time of rAdMSCs harvested from the bioreactor were calculated as 0.2516 h-1 and 42 h, which were comparable to the characteristic values given in the literature. In the differentiation studies of rAdMSCs harvested from the bioreactor, ALP-von Kossa staining was done in the osteogenic differentiation studies, whereas oil red o staining was done for adipogenic differentiation studies. In addition, it was determined that harvested cells had similar SOX2, Nanog and OCT 4 gene expressions in comparison to the control group. However, in the dynamic studies growth rate of cells in the bioreactor exhibited a trend independent of cell seeding density, which should be further investigated in a more comprehensive cell expansion study.