Separation of basophils from human blood by a seal-less flow-through centrifuge

通过无密封流通离心机从人体血液中分离嗜碱性粒细胞

• 批准号: 9572273
• 负责人: Yoichiro Ito
• 金额: $ 0.44万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9572273
• 关键词: Allergic inflammation; Alpha Cell; Antibodies; Basophils; Biological Assay; Blood; Blood specimen; Cell Count; Cell Density; Cell Separation; Cells; Chronic; Classification; Cultured Cells; Dendritic Cells; Erythrocytes; Goals; Harvest; Hematopoietic stem cells; Hemolysis; Human; Hypersensitivity; Immunity; Leukocytes; Liquid substance; Methods; Mus; Percoll; Phosphate Buffer; Population; Preparation; Red Blood Cell Count; Reporting; Research; Role; Running; Saline; Sampling; Stem cells; Sterility; Suspensions; System; Time; Tissues; Tube; White Blood Cell Count procedure; Whole Blood; base; cell injury; density; improved; neutrophil; novel; operation; peripheral blood; seal

We have developed a novel flow-through density gradient cell separation method for continuously harvesting human hematopoietic progenitor cells (1, 2), leukocytes and mouse dendritic cells. This system continuously separates a large number of cells into five fractions according to their densities. As the blood contains a huge number of red blood cells (over 1,000 times of the number of leukocytes), the pretreatment with dilution and hemolysis is usually essential for harvesting leukocytes. As the result, the cell separation time was prolonged due to the increased sample volume by the dilution which might cause cell damage and hemolysis. Therefore, we modified the present method for improving the above two points. Before the separation, Percoll density medium with the density of 1.050, which was similar to that of blood, was flowed through the sample channel instead of phosphate buffered saline before introducing the whole blood sample without diluton. And, separated cells were separated again to improve the purity. We separated basophils, which had the fewest number among leukocyte population and reported having the novel role on chronic allergic inflammation (3), under these modified conditions. A set of isosmotic Percoll media with the densities of 1.050, 1.074, 1.079, 1.090, 1.095 and 1.104 g/ml was prepared, and introduced into the channel to form a density gradient. Then the anti-coagulated whole blood was continuously fed into inlet 1, through which Percoll medium with the density of 1.050 g/ml was flowing. Harvested fractions with the density of 1.079 g/ml and 1.090 g/ml were washed, and the cell pellets were re-suspended into just 1 ml of the density medium with 1.050 g/ml. This cell suspension was fed through inlet 1 and separated under the same condition, again. Leukocyte classification of basophils in the density of 1.079 g/ml was approximately 72% and that of neutrophils in 1.090 g/ml was approximately 98% after the second run, respectively. The red blood cell counts were about 5% in each fraction. Without diluting the blood sample, the time required for cell separation was shortened and the repeating operation made possible to remove most of red blood cells. The present method might be useful for purifying some functional cells by FACS, as the preparative separation not using any antibodies and hemolytic agents. References 1. Y. Ito, K. Shinomiya, A new continuous-flow cell separation method based on cell density: principle, apparatus, and preliminary application to separation of human buffy coat, J Clin Apher. 16(2001)186-191. 2. H. Shiono, H. M. Chen, T. Okada, Y. Ito, Colony-forming assay for human hematopoietic progenitor cells harvested by a novel continuous-flow cell separation method, J. Chromatogr. A 1151(2007) 153-157. 3. H. Karasuyama, K. Obata, T. Wada, Y. Tsujimura, K. Mukai, Newly appreciated roles for basophils in allergy and protective immunity, Allergy 66 (2011) 1133-1141.

我们已经开发了一种新型的流通密度梯度细胞分离方法，用于连续收集人造血祖细胞（1、2），白细胞和小鼠树突状细胞。该系统根据其密度不断将大量细胞分为五个部分。由于血液中含有大量的红细胞（白细胞数量超过1000次），因此稀释和溶血的预处理通常对于收集白细胞至关重要。结果，由于样品体积增加，细胞分离时间延长了，这可能会导致细胞损伤和溶血。 因此，我们修改了当前改善上述两个点的方法。在分离之前，将密度为1.050的percoll密度培养基（类似于血液）流过样品通道，而不是磷酸盐缓冲盐水，然后再引入没有稀释剂的整个血液样品。并且，将分离的细胞再次分离以提高纯度。我们分离了嗜碱性粒细胞，在这些修饰的条件下，在白细胞种群中数量最少，并报告说在慢性过敏性炎症中具有新作用（3）。 制备了一组密度为1.050、1.074、1.079、1.090、1.095和1.104 g/mL的同质percoll培养基，并引入了通道中以形成密度梯度。然后将抗凝的全血连续喂入入口1，通过该血液，密度为1.050 g/ml的percoll培养基流动。洗涤1.079 g/mL的密度和1.090 g/ml的收获级分，并将细胞颗粒重新悬浮为1.050 g/ml的1 mL密度​​培养基。该细胞悬浮液通过入口1馈送并在相同条件下分离。嗜碱性粒细胞的白细胞分类在1.079 g/mL的密度下约为72％，中性粒细胞为1.090 g/ml的白细胞分别在第二次运行后约98％。每个部分的红细胞计数约为5％。 在不稀释血液样本的情况下，缩短了细胞分离所需的时间，重复操作使消除大部分红细胞的重复操作成为可能。目前的方法可能对通过FACS纯化某些功能性细胞有用，因为制剂分离不使用任何抗体和溶血剂。 参考 1。Y.Ito，K。Shinomiya，一种基于细胞密度的新连续流细胞分离方法：原理，设备和初步应用人类Budy Coat的分离J Clin Apher。 16（2001）186-191。 2. H. Shiono，H。M. Chen，T。Okada，Y。ITO，通过一种新型的连续流细胞分离方法收获的人类造血祖细胞的菌落形成分析，J。Chromatogr。 A 1151（2007）153-157。 3。H.Karasuyama，K。Obata，T。Wada，Y。Tsujimura，K。Mukai，新近赞赏的嗜碱性粒细胞在过敏和保护性免疫中的作用，过敏66（2011）1133-1141。