| Summary: | 博士 === 國立清華大學 === 化學工程學系 === 92 === The development of ex vivo culture systems that facilitate the expansion of hematopoietic stem and progenitor cells is crucial to stem cell research and clinical application. In this study, two serum-free, stroma-free and cytokine-containing culture media (SF-HSC and SF-MNC) for white blood cells (WBC), CD34+ cells, CD34+CD38－ cells, colony-forming unit cells (CFU cells), and long-term culture-initiating cells (LTC-IC) expansion were systematically developed and optimized using the two-level factorial design and steepest ascent methods. The experimental results showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-HSC were BIT2 (1.5 g/L BSA, 4.39 μg/mL insulin, 60 μg/mL transferrin, and 25.94 μM 2-ME), and CC-S6 (8.46 ng/mL TPO, 4.09 ng/mL IL-3, 15 ng/mL SCF, 6.73 ng/mL FL, 0.78 ng/mL IL-6, 3.17 ng/mL G-CSF, and 1.30 ng/mL GM-CSF) in the Iscove’s modified Dulbecco’s medium (IMDM), respectively. After one-week culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38－ cells, CFU cells, and LTC-IC were 64.2-, 27.4-, 72.4-, 22.2-, and 8.1-fold,respectively. The experimental results also showed that the optimal compositions of the serum substitutes and the cytokine cocktail in SF-MNC were BIT (4 g/l BSA, 0.71 μg/ml insulin, and 27.81μg/ml transferring), and CC-S9 (5.53 ng/ml TPO, 2.03 ng/ml IL-3, 16 ng/ml SCF, 4.43 ng/ml FL, 2.36 ng/ml IL-6, 1.91 ng/ml G-CSF, 1.56 ng/ml GM-CSF, 2.64 ng/ml SCGF, and 0.69 ng/ml IL-11) in the IMDM, respectively. After 6-day culture, the increases in the total number of WBC, CD34+ cells, CD34+CD38－ cells, CFU cells, and LTC-IC were 1.4-, 30.4-, 63.9-, 10.7-, and 2.8-fold,respectively.
If SF-HSC or SF-MNC were changed twice per week, WBC and CD34+ cell could grow up continuously for at least 10 weeks. Its expansion ability of CD34+ cells and CFC was superior or comparable to that of X-vivo 20TM, StemlineTM, and StemspanTM commercial media. Furthermore, SF-HSC and SF-MNC has the lowest concentration of cytokine cocktail. These systematic methodologies are helpful in improving the ex vivo expansion system for hematopoietic stem cell and progenitor cells.
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