label-free-neutrophil-enrichment-from-patient-derived-airway

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

Airway secretions contain a large number of immune-related cells, e.g., neutrophils, macrophages, and lymphocytes, which can be used as a major resource ...

Massachusetts Institute of Technology

Fluorescence resonance energy transfer (FRET)-based biosensors are powerful tools for measuring spatio-temporal signaling dynamics in single living cells with subcellular resolution. There are quite a number of already existing sensors and this technology is increasingly used to obtain quantitative dynamic datasets. In this chapter, we describe the analysis of endogenous extracellular signal-regulated kinase (ERK) activity in living cells using the EKAR2G (ERK activity reporter second generation) probe. We focus on the generation of stable cell lines expressing the EKAR2G sensor as well as data acquisition and analysis.

Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors.

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.

Patient-Derived Airway Secretion Dissociation Technique To Isolate and Concentrate Immune Cells Using Closed-Loop Inertial Microfluidics.

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1-deficient (Thbs1-/-) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1-/- mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger.

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis.

In blood samples from patients with viral infection, it is often important to separate viral particles from human cells, for example, to minimize background in performing viral whole genome sequencing. Here, we present a microfluidic device that uses spiral inertial microfluidics with continuous circulation to separate host cells from viral particles and free nucleic acid. We demonstrate that this device effectively reduces white blood cells, red blood cells, and platelets from both whole blood and plasma samples with excellent recovery of viral nucleic acid. Furthermore, microfluidic separation leads to greater viral genome coverage and depth, highlighting an important application of this device in processing clinical samples for viral genome sequencing.

Article	Year
Measuring ERK Activity Dynamics in Single Living Cells Using FRET Biosensors. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 27924569	2017
Patient-Derived Airway Secretion Dissociation Technique To Isolate and Concentrate Immune Cells Using Closed-Loop Inertial Microfluidics. Analytical chemistry\| PubMed ID: 28402103	2017
Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis. JCI insight\| PubMed ID: 29415890	2018
Negative Selection by Spiral Inertial Microfluidics Improves Viral Recovery and Sequencing from Blood. Analytical chemistry\| PubMed ID: 29536737	2018

Hyunryul Ryu

Biography

JoVE Journal Publications

Other Publications