Joseph M. Martel

Microfluidic, marker-free isolation of circulating tumor cells from blood samples

The ability to isolate and analyze rare circulating tumor cells (CTCs) has the potential to further our understanding of cancer metastasis and enhance the care of cancer patients. In this protocol, we describe the procedure for isolating rare CTCs from blood samples by using tumor antigen–independent microfluidic CTC-iChip technology. The CTC-iChip uses deterministic lateral displacement, inertial focusing and magnetophoresis to sort up to 107 cells/s. By using two-stage magnetophoresis and depletion antibodies against leukocytes, we achieve 3.8-log depletion of white blood cells and a 97% yield of rare cells with a sample processing rate of 8 ml of whole blood/h. The CTC-iChip is compatible with standard cytopathological and RNA-based characterization methods. This protocol describes device production, assembly, blood sample preparation, system setup and the CTC isolation process. Sorting 8 ml of blood sample requires 2 h including setup time, and chip production requires 2–5 d.

Inertial focusing in microfluidics.

When Segré and Silberberg in 1961 witnessed particles in a laminar pipe flow congregating at an annulus in the pipe, scientists were perplexed and spent decades learning why such behavior occurred, finally understanding that it was caused by previously unknown forces on particles in an inertial flow. The advent of microfluidics opened a new realm of possibilities for inertial focusing in the processing of biological fluids and cellular suspensions and created a field that is now rapidly expanding. Over the past five years, inertial focusing has enabled high-throughput, simple, and precise manipulation of bodily fluids for a myriad of applications in point-of-care and clinical diagnostics. This review describes the theoretical developments that have made the field of inertial focusing what it is today and presents the key applications that will make inertial focusing a mainstream technology in the future.

Inertial focusing dynamics in spiral microchannels

This report details a comprehensive study of inertial focusing dynamics and particle behavior in low aspect ratio (h/w ∼ 1/1 to 1/8) spiral microchannels. A continuum of particle streak behavior is shown with longitudinal, cross-sectional, and velocity resolution, yielding a large analyzed parameter space. The dataset is then summarized and compared to prior results from both straight microchannels and other low aspect ratio spiral microchannel designs. Breakdown of focusing into a primary and secondary fluorescent streak is observed in the lowest aspect ratio channels at high average downstream velocities. Streak movement away from the theoretically predicted near inner wall equilibrium position towards the center of the channel at high average downstream velocities is also detailed as a precursor to breakdown. State diagrams detail the overall performance of each device including values of the required channel lengths and the range of velocities over which quality focusing can be achieved.

Tunable Nanostructured Coating for the Capture and Selective Release of Viable Circulating Tumor Cells

A layer-by-layer gelatin nanocoating is presented for use as a tunable, dual response biomaterial for the capture and release of circulating tumor cells (CTCs) from cancer patient blood. The entire nanocoating can be dissolved from the surface of microfluidic devices through biologically compatible temperature shifts. Alternatively, individual CTCs can be released through locally applied mechanical stress.

Continuous Flow Microfluidic Bioparticle Concentrator

Innovative microfluidic technology has enabled massively parallelized and extremely efficient biological and clinical assays. Many biological applications developed and executed with traditional bulk processing techniques have been translated and streamlined through microfluidic processing with the notable exception of sample volume reduction or centrifugation, one of the most widely utilized processes in the biological sciences. We utilize the high-speed phenomenon known as inertial focusing combined with hydraulic resistance controlled multiplexed micro-siphoning allowing for the continuous concentration of suspended cells into pre-determined volumes up to more than 400 times smaller than the input with a yield routinely above 95% at a throughput of 240 ml/hour. Highlighted applications are presented for how the technology can be successfully used for live animal imaging studies, in a system to increase the efficient use of small clinical samples, and finally, as a means of macro-to-micro interfacing allowing large samples to be directly coupled to a variety of powerful microfluidic technologies.

Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood.

Neutrophil chemotaxis is critical for defense against infections and its alterations could lead to chronic inflammation and tissue injury. The central role that transient alterations of neutrophil chemotaxis could have on patient outcomes calls for its quantification in the clinic. However, current methods for measuring neutrophil chemotaxis require large volumes of blood and are time consuming. To address the need for rapid and robust assays, we designed a microfluidic device that measures neutrophil chemotaxis directly from a single droplet of blood. We validated the assay by comparing neutrophil chemotaxis from finger prick, venous blood and purified neutrophil samples. We found consistent average velocity of (19 ± 6 μm/min) and directionality (91.1%) between the three sources. We quantified the variability in neutrophil chemotaxis between healthy donors and found no significant changes over time. We also validated the device in the clinic and documented temporary chemotaxis deficiencies after burn injuries.

A Worldwide Competition to Compare the Speed and Chemotactic Accuracy of Neutrophil-Like Cells

Chemotaxis is the ability to migrate towards the source of chemical gradients. It underlies the ability of neutrophils and other immune cells to hone in on their targets and defend against invading pathogens. Given the importance of neutrophil migration to health and disease, it is crucial to understand the basic mechanisms controlling chemotaxis so that strategies can be developed to modulate cell migration in clinical settings. Because of the complexity of human genetics, Dictyostelium and HL60 cells have long served as models system for studying chemotaxis. Since many of our current insights into chemotaxis have been gained from these two model systems, we decided to compare them side by side in a set of winner-take-all races, the Dicty World Races. These worldwide competitions challenge researchers to genetically engineer and pharmacologically enhance the model systems to compete in microfluidic racecourses. These races bring together technological innovations in genetic engineering and precision measurement of cell motility. Fourteen teams participated in the inaugural Dicty World Race 2014 and contributed cell lines, which they tuned for enhanced speed and chemotactic accuracy. The race enabled large-scale analyses of chemotaxis in complex environments and revealed an intriguing balance of speed and accuracy of the model cell lines. The successes of the first race validated the concept of using fun-spirited competition to gain insights into the complex mechanisms controlling chemotaxis, while the challenges of the first race will guide further technological development and planning of future events.

Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets

Animal models of human disease differ in innate immune responses to stress, pathogens, or injury. Precise neutrophil phenotype measurements could facilitate interspecies comparisons. However, such phenotype comparisons could not be performed accurately with the use of current assays, as they require the separation of neutrophils from blood using species‐specific protocols, and they introduce distinct artifacts. Here, we report a microfluidic technology that enables robust characterization of neutrophil migratory phenotypes in a manner independent of the donor species and performed directly in a droplet of whole blood. The assay relies on the particular ability of neutrophils to deform actively during chemotaxis through microscale channels that block the advance of other blood cells. Neutrophil migration is measured directly in blood, in the presence of other blood cells and serum factors. Our measurements reveal important differences among migration counts, velocity, and directionality among neutrophils from 2 common mouse strains, rats, and humans.

Flexible Octopus‐Shaped Hydrogel Particles for Specific Cell Capture

Multiarm hydrogel microparticles with varying geometry are fabricated to specifically capture cells expressing epithelial cell adhesion molecule. Results show that particle shape influences cell-capture efficiency due to differences in surface area, hydrodynamic effects, and steric constraints. These findings can lead to improved particle design for cell separation and diagnostic applications.

Microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.