中国仓鼠卵巢细胞
流式细胞术
单克隆抗体
转染
分子生物学
生物
细胞
重组DNA
细胞培养
抗体
荧光素
化学
荧光
生物化学
免疫学
基因
遗传学
物理
量子力学
作者
Takeshi Okumura,Kenji Masuda,Kazuhiko Watanabe,Kenji Miyadai,Koichi Nonaka,Masayuki Yabuta,Takeshi Ōmasa
标识
DOI:10.1016/j.jbiosc.2015.01.007
摘要
To screen a high-producing recombinant Chinese hamster ovary (CHO) cell from transfected cells is generally laborious and time-consuming. We developed an efficient enrichment strategy for high-producing cell screening using flow cytometry (FCM). A stable pool that had possibly shown a huge variety of monoclonal antibody (mAb) expression levels was prepared by transfection of an expression vector for mAb production to a CHO cell. To enrich high-producing cells derived from a stable pool stained with a fluorescent-labeled antibody that binds to mAb presented on the cell surface, we set the cell size and intracellular density gates based on forward scatter (FSC) and side scatter (SSC), and collected the brightest 5% of fluorescein isothiocyanate (FITC)-positive cells from each group by FCM. The final product concentration in a fed-batch culture of cells sorted without FSC and SSC gates was 1.2-1.3-times higher than that of unsorted cells, whereas that of cells gated by FSC and SSC was 3.4-4.7-fold higher than unsorted cells. Surprisingly, the fraction with the highest final product concentration indicated the smallest value of FSC and SSC, and the middle value of fluorescence intensity among all fractionated cells. Our results showed that our new screening strategy by FCM based on FSC and SSC gates could achieve an efficient enrichment of high-producing cells with the smallest value of FSC and SSC.
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