Dolgulu Yatak Biyoreaktörde Mezenkimal Kök Hücre Üretiminin Araştırılması
Karaaslan, Mustafa Caner
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In the thesis study, it was aimed to investigate mesenchymal stem cell production in packed bed bioreactor where non-woven polyester fiber matrices (NWPF) are used as carrier. In the preliminary studies, MC3T3-E1 mouse pre-osteoblastic cells were used. Surface modification was carried out with 3M sulfuric acid in order to allow the cells to adhere to non-woven polyester fabric discs. The hydrophilicity of the discs was determined by water contact angle analysis. For this purpose, a cell culture study was performed with NWPF discs in static conditions, and the cells were adhered to the discs and evaluated by Scanning Electron Microscope (SEM). In the next stage of the study, preliminary dynamic cell culture studies were performed in the packed bed bioreactor (PBB) with MC3T3-E1 cells with a working volume of 0.5L, using Fibra-Cel® discs. Cells removed from discs on Day 1, 3, 5, and 7 of the culture were counted, and at the end of the culture, it was observed that the number of cells increased approximately 5-fold in the bioreactor. After carrying out bioreactor optimization, studies were performed with rat adipose mesenchymal stem cell (rAdMSC). The effects of different operating parameters such as packed bed height, mixing speed and cell seeding density in the bioreactor on the production efficiency of the cells were investigated. In order to increase the cell production efficiency in the bioreactor and reduce the production of material and make the production more economical, the scale of the bioreactor was reduced and a PBB design with a volume of 100mL was designed. In the last part of the thesis study, the viability of rat adipose mesenchymal stem cells on NWPF discs were investigated by MTT 3- [4,5-dimethylthiazol-2-yl] - 2,5-diphenyltetrazolium bromide) analysis, and their morphologies were F-actin / DAPI and crystal violet staining. As a result of the dynamic culture, the number of cells increased 4 times. Cell removal was carried out to examine the morphology of cells after removal from NWPF discs. It has been observed that 85% of cells have been successfully removed from disks. For this purpose, the culture was successfully continued by adding the cells removed to 75 cm2 flask. Glucose, lactate and urea analysis were performed to examine the metabolic activities of the cells. Although 50% of the medium was changed on certain days during the culture period, the number of cells increased continuously and the glucose concentration decreased 2.5-fold compared to the beginning of the culture. Due to the decrease in glucose concentration, lactate and urea concentrations in the medium increased 2-fold and 5-fold, respectively. From the obtained results, it was determined that there was no inhibition due to the lactate and urea concentrations accumulated in the dynamic cell culture. As a result, the dynamic cell culture made in PBB within the scope of the presented thesis showed that rat AdMKHs are attached to the fibers of NWPF disks and spread to all regions and have the potential to reach the number of cells needed in clinical applications.
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