Research Laboratory of Electronics, ‡Department of Electrical Engineering and Computer Science, §Department of Biological Engineering, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.
Department of Medicine and the ⊥Vascular Medicine Institute, University of Pittsburgh , NW628 Montefiore University Hospital, 3459 Fifth Avenue, Pittsburgh, Pennsylvania 15213, United States.
Anal Chem. 2017 May 16;89(10):5549-5556. doi: 10.1021/acs.analchem.7b00610. Epub 2017 Apr 21.
Assessment of airway secretion cells, both for research and clinical purposes, is a highly desired goal in patients with acute and chronic pulmonary diseases. However, lack of proper cell isolation and enrichment techniques hinder downstream evaluation and characterization of cells found in airway secretions. Here, we demonstrate a novel enrichment method to capture immune-related cells from clinical airway secretions using closed-loop separation of spiral inertial microfluidics (C-sep). By recirculating the output focusing stream back to the input reservoir and running continuously with a high flow processing rate, one can achieve optimal concentration, recovery and purity of airway immune cells from a large volume of diluent, which was not readily possible in the single-pass operation. Our method reproducibly recovers 94.0% of polymorphonuclear leukocytes (PMNs), with up to 10 PMNs in clear diluted buffer from 50 μL of airway secretions obtained from mechanically ventilated patients. We show that C-sep isolated PMNs show higher neutrophil elastase (NE) release following activation by phorbol 12-myristate 13-acetate (PMA) than cells isolated by conventional mucolytic method. By capturing cells without chemically disrupting their potential function, our method is expected to expand the possibility of clinical in vitro cell based biological assays for various pulmonary diseases such as acute respiratory distress syndrome, pneumonia, cystic fibrosis, and bronchiectasis.
评估气道分泌物细胞，无论是用于研究还是临床目的，都是急性和慢性肺部疾病患者的迫切需求。然而，缺乏适当的细胞分离和富集技术阻碍了对气道分泌物中细胞的下游评估和表征。在这里，我们展示了一种使用螺旋惯性微流控（C-sep）的闭环分离来从临床气道分泌物中捕获免疫相关细胞的新型富集方法。通过将输出聚焦流循环回输入储液器并以高流量处理率连续运行，可以从大量稀释剂中实现气道免疫细胞的最佳浓度、回收率和纯度，而在单次通过操作中则不容易实现。我们的方法可重复地回收 94.0%的多形核白细胞（PMN），从机械通气患者的 50 μL 气道分泌物中，可在清澈的稀释缓冲液中获得多达 10 个 PMN。我们表明，与传统粘液溶解方法分离的细胞相比，C-sep 分离的 PMN 在被佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）激活后释放更高的中性粒细胞弹性蛋白酶（NE）。通过捕获细胞而不破坏其潜在功能，我们的方法有望为各种肺部疾病（如急性呼吸窘迫综合征、肺炎、囊性纤维化和支气管扩张症）的临床体外细胞基础生物检测提供更多可能性。
