Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

Hyunryul Ryu; Kyungyong Choi; Yanyan Qu; Taehong Kwon; Janet S. Lee; Jongyoon Han

doi:10.3791/57673

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Method Article

Label-free Neutrophil Enrichment from Patient-derived Airway Secretion Using Closed-loop Inertial Microfluidics

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DOI:

10.3791/57673

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June 7th, 2018

Hyunryul Ryu¹ , Kyungyong Choi¹^,² , Yanyan Qu³ , Taehong Kwon¹^,² , Janet S. Lee³^,⁵ , Jongyoon Han¹^,²^,⁴

¹Research Laboratory of Electronics, Massachusetts Institute of Technology, ²Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, ³Department of Medicine, University of Pittsburgh, ⁴Department of Biological Engineering, Massachusetts Institute of Technology, ⁵Vascular Medicine Institute, University of Pittsburgh

Summary

In this research, we demonstrate a label-free neutrophil separation method from clinical airway secretions using closed-loop operation of spiral inertial microfluidics. The proposed method would expand the clinical in vitro assays for various respiratory diseases.

Abstract

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource to evaluate a variety of pulmonary diseases, both for research and clinical purposes. However, due to the heterogeneous and viscous nature of patient mucus, there is currently no reliable dissociation method that does not damage the host immune cells in the patient airway secretion. In this research, we introduce a sample preparation method that uses inertial microfluidics for the patient's immune assessment. Regardless of the heterogeneous fluidic properties of the clinical samples, the proposed method recovers more than 95% of neutrophils from airway secretion samples that are diluted 1,000-fold with milliliters of clean saline. By recirculating the concentrated output stream to the initial sample reservoir, a high concentration, recovery, and purity of the immune cells are provided; recirculation is considered a trade-off to the single-run syringe-based operation of inertial microfluidics. The closed-loop operation of spiral microfluidics provides leukocytes without physical or chemical disturbance, as demonstrated by the phorbol 12-myristate 13-acetate (PMA)-induced elastase release of sorted neutrophils.

Introduction

Since cells are encapsulated in a large amount of mucus in airway secretions, the functional assessment of leukocytes by an in vitro assay has been hindered. Dithiothreitol (DTT) is the most common lysis buffer to dissociate and homogenize the sputum for cytological analysis and detection of mediators while providing tolerable viability of isolated cells¹^,². However, DTT can interfere with surface-bound antigens of airway neutrophils, resulting in the disruption of neutrophil function such as elastase and myeloperoxidase (MPO) release²^,³.There....