An arrayed high-content chemotaxis assay for patient diagnosis

Abstract

Chemotaxis assays are essential tools for the study of gradient sensing and directed cell migration, and have the potential to aid in the diagnosis and characterization of patients with immune disorders. Current methods are limited in their ability to meet the more demanding requirements for clinical applications. Because patient samples have a short lifespan and sometimes a limited volume (e.g. pediatrics), the operational requirements for an efficient chemotaxis assay are increased in the clinical setting. Here we describe a microscale assay platform for gradient generation that overcomes these limitations. Passive fluidic methods are leveraged to provide a reliable microfluidic gradient generation device, operable in only three pipetting steps. In addition, arrayed imaging and advanced cell tracking algorithms enabled a 50-fold increase in throughput over current methods. These methods were employed to aid in the diagnostic evaluation of an infant who presented with severe, recurrent bacterial infections. Analysis of the infant's neutrophils revealed impaired cell polarization and chemotaxis in a gradient of the chemoattractant fMLP. The patient was subsequently diagnosed with an inhibitory mutation in the Rho GTPase, Rac2. The approach also enabled a microenvironmental screen of human primary neutrophil chemotaxis on fibronectin, fibrinogen and laminin with results suggesting that fibronectin, although commonly used, may not be the most appropriate matrix protein for chemotaxis assays. Together, these findings demonstrate the use of arrayed micro-devices to aid in the diagnosis of a primary immunodeficiency disorder, and illustrate the capability for increased throughput microenvironmental studies and screening targeted to specific human diseases.

Microfluidic 3D models of cancer

“…One useful advantage to 3D compartmentalization is that it can be used to investigate signaling mechanisms involved in chemotaxis[44]. The types of molecular transport such as diffusion are more controllable in microsystems due to the scale of the system and the ability to control the shape, length, and material of channels[45]. Traditionally, cell migration/invasion studies have been conducted using transwells, which is a vertical arrangement of compartments.…”

The Actin Regulatory Protein HS1 Interacts with Arp2/3 and Mediates Efficient Neutrophil Chemotaxis

“…Microfluidic Chemotaxis Assays and ImmunofluorescenceMicrofluidic devices were fabricated as previously described (33). In brief, the devices enable the generation of a static gradient of chemoattractant between a source chamber containing fMLP and a sink chamber containing the PLB-985 cells, which were connected by a 1-mm-long, 400-m-wide channel in which the fMLP gradient is generated.…”

Microfluidic kit-on-a-lid: a versatile platform for neutrophil chemotaxis assays

“…[15][16][17][18] In some microfluidic applications, the measurement of neutrophils has been used for clinical diagnostics, 19,20 demonstrating the potential for translation of microfluidic methods into a clinical setting. However, few examples exist of microfluidic techniques being used in hematology research.…”