Martkamchan Sirimarn, Onlamoon Nattawat, Wang Siyu, Pattanapanyasat Kovit, Ammaranond Palanee
Graduate Program in Clinical Biochemistry and Molecular Medicine, Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand.
Center for Emerging and Neglected Infectious Diseases, Mahidol University, Nakhon Pathom, Thailand.
Adv Clin Exp Med. 2016 Sep-Oct;25(5):821-828. doi: 10.17219/acem/35771.
The activation of peripheral blood mononucleated cells (PBMCs) with anti-CD3/CD28-coated magnetic beads promotes intrinsic resistance to HIV as well as cell expansion.
The aim of this study was to define an optimal cell isolation protocol for the expansion of PBMCs using anti-CD3/CD28-coated bead stimulation, with the ultimate goal of using these cells for adoptive therapy.
PBMCs were isolated from healthy donor blood samples. To determine the effect of varying the bead-to-cell ratios on the expansion rate and phenotypic characterization of the expanded cells, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at bead-to-cell ratios of 0.1 : 1, 0.5 : 1 and 1.0 : 1 in the presence of 100 U/mL exogenous IL-2; also, one million PBMCs were stimulated by anti-CD3/CD28-coated beads at a bead-to-cell ratio of 0.5 : 1 in the presence of varying concentrations of IL-2 (20, 100 and 1000 U/mL). Cell expansion was carried out for three weeks. The cell numbers, cell viability and phenotypic characterization were determined by trypan blue exclusion and flow cytometry.
The initial experiments showed no difference in the expansion rate from cells grown with the three different bead-to-cell ratios. PBMCs can be expanded up to 1000-fold at a 0.5 : 1 bead-to-cell ratio after three weeks of cell expansion with a high viability (90%). Furthermore, in the presence of 100 U/mL IL-2, the percentages of CD3-CD16+CD56+ cells showed marked increases.
The results demonstrate that PBMCs were stimulated with anti-CD3/CD28-coated beads. This method may provide an alternative for driving T cell expansion, which may be very useful in adoptive immunotherapy.
用抗CD3/CD28包被的磁珠激活外周血单个核细胞（PBMC）可促进其对HIV的内在抗性以及细胞扩增。
本研究的目的是确定一种最佳的细胞分离方案，用于使用抗CD3/CD28包被的磁珠刺激来扩增PBMC，最终目标是将这些细胞用于过继性治疗。
从健康供体血样中分离PBMC。为了确定改变磁珠与细胞比例对扩增细胞的扩增率和表型特征的影响，在存在100 U/mL外源性白细胞介素-2（IL-2）的情况下，用抗CD3/CD28包被的磁珠以0.1∶1、0.5∶1和1.0∶1的磁珠与细胞比例刺激100万个PBMC；此外，在存在不同浓度IL-2（20、100和1000 U/mL）的情况下，用抗CD3/CD28包被的磁珠以0.5∶1的磁珠与细胞比例刺激100万个PBMC。细胞扩增进行三周。通过台盼蓝排斥法和流式细胞术测定细胞数量、细胞活力和表型特征。
初始实验显示，用三种不同磁珠与细胞比例培养的细胞在扩增率上没有差异。细胞扩增三周后，在磁珠与细胞比例为0.5∶1时，PBMC可扩增至1000倍，且活力较高（90%）。此外，在存在100 U/mL IL-2的情况下，CD3-CD16+CD56+细胞的百分比显著增加。
结果表明，用抗CD3/CD28包被的磁珠刺激PBMC。该方法可能为驱动T细胞扩增提供一种替代方法，这在过继性免疫治疗中可能非常有用。
