Development of a Serum-Free Induction Medium for Genration of Human Neutrophils

• Main Authors: Chia-Hsuan Mu, 穆家萱
• Other Authors: Chao-Ling Yao
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/r74f6d

碩士 === 元智大學 === 化學工程與材料科學學系 === 102 === In recent years, due to the rising rates of cancer, the number of people receiving chemotherapy has also increased. After chemotherapy, cancer patients generally need hematopoietic stem cells (HSCs) transplantation for treatment, but the treatment is often faced with the problem of the patient’s neutrophils and platelets recovering too slowly. Previous research has shown that the number of fully functional neutrophils can be increased by a serum containing medium in vitro. However, using fetal bovine serum (FBS) has many problems, such as unknown infectious pathogens and composition. The purpose of our study is to increase the number of neutrophils by developing a serum-free medium in vitro, and to overcome limitations on clinical transplantation that it can accelerate the recovery of patients’ innate immune system after surgery in the future. We selected 22 serum substitutes from references and used factorial design and path steepest ascent method to identify the optimal factor and concentration. Then, we used cell surface antigen (CD66+ and MPO+) to make sure that the induced cells were neutrophils. In addition, growth kinetics assay, phagocytosis assay, chemotaxis assay, cellular reactive oxygen species (ROS), enzyme-linked immunosorbent assay (ELISA), quantitative real time polymerase chain reaction (Q-PCR), in vitro phagocytosis assay and animal experiment were used to check the functions, we confirmed the neutrophils induced from HSCs that have abilities of phagocytic, migration, generation of ROS and its related gene expression. In addition, the total cell number and neutrophil number after 7-day culture in our developed serum-free neutrophil medium can increase 10- and 250-time, and increase 150- 3500-time after 21-day, respectively. Presently, our research has shown the ability to develop a neutrophil-induced serum-free medium, and to induce and differentiate HSCs into neutrophils in a serum-free system, which has normal neutrophil function. In the future work, we will do the Q-PCR assay, ELISA and animal experiments which will provide a reliable and stable source of neutrophils for basic research and clinical treatments.