Dino Di Carlo

Continuous inertial focusing, ordering, and separation of particles in microchannels

Under laminar flow conditions, when no external forces are applied, particles are generally thought to follow fluid streamlines. Contrary to this perspective, we observe that flowing particles migrate across streamlines in a continuous, predictable, and accurate manner in microchannels experiencing laminar flows. The migration is attributed to lift forces on particles that are observed when inertial aspects of the flow become significant. We identified symmetric and asymmetric channel geometries that provide additional inertial forces that bias particular equilibrium positions to create continuous streams of ordered particles precisely positioned in three spatial dimensions. We were able to order particles laterally, within the transverse plane of the channel, with >80-nm accuracy, and longitudinally, in regular chains along the direction of flow. A fourth dimension of rotational alignment was observed for discoidal red blood cells. Unexpectedly, ordering appears to be independent of particle buoyant direction, suggesting only minor centrifugal contributions. Theoretical analysis indicates the physical principles are operational over a range of channel and particle length scales. The ability to differentially order particles of different sizes, continuously, at high rates, and without external forces in microchannels is expected to have a broad range of applications in continuous bioparticle separation, high-throughput cytometry, and large-scale filtration systems.

Label-free cell separation and sorting in microfluidic systems

AbstractCell separation and sorting are essential steps in cell biology research and in many diagnostic and therapeutic methods. Recently, there has been interest in methods which avoid the use of biochemical labels; numerous intrinsic biomarkers have been explored to identify cells including size, electrical polarizability, and hydrodynamic properties. This review highlights microfluidic techniques used for label-free discrimination and fractionation of cell populations. Microfluidic systems have been adopted to precisely handle single cells and interface with other tools for biochemical analysis. We analyzed many of these techniques, detailing their mode of separation, while concentrating on recent developments and evaluating their prospects for application. Furthermore, this was done from a perspective where inertial effects are considered important and general performance metrics were proposed which would ease comparison of reported technologies. Lastly, we assess the current state of these technologies and suggest directions which may make them more accessible. FigureA wide range of microfluidic technologies have been developed to separate and sort cells by taking advantage of differences in their intrinsic biophysical properties

Dynamic single cell culture array

It is important to quantify the distribution of behavior amongst a population of individual cells to reach a more complete quantitative understanding of cellular processes. Improved high-throughput analysis of single cell behavior requires uniform conditions for individual cells with controllable cell-cell interactions, including diffusible and contact elements. Uniform cell arrays for static culture of adherent cells have previously been constructed using protein micropatterning techniques but lack the ability to control diffusible secretions. Here we present a microfluidic-based dynamic single cell culture array that allows both arrayed culture of individual adherent cells and dynamic control of fluid perfusion with uniform environments for individual cells. In our device no surface modification is required and cell loading is done in less than 30 seconds. The device consists of arrays of physical U-shaped hydrodynamic trapping structures with geometries that are biased to trap only single cells. HeLa cells were shown to adhere at a similar rate in the trapping array as on a control glass substrate. Additionally, rates of cell death and division were comparable to the control experiment. Approximately 100 individual isolated cells were observed growing and adhering in a field of view spanning approximately 1 mm(2) with greater than 85% of cells maintained within the primary trapping site after 24 hours. Also, greater than 90% of cells were adherent and only 5% had undergone apoptosis after 24 hours of perfusion culture within the trapping array. We anticipate uses in single cell analysis of drug toxicity with physiologically relevant perfused dosages as well as investigation of cell signaling pathways and systems biology.

Hydrodynamic stretching of single cells for large population mechanical phenotyping

Cell state is often assayed through measurement of biochemical and biophysical markers. Although biochemical markers have been widely used, intrinsic biophysical markers, such as the ability to mechanically deform under a load, are advantageous in that they do not require costly labeling or sample preparation. However, current techniques that assay cell mechanical properties have had limited adoption in clinical and cell biology research applications. Here, we demonstrate an automated microfluidic technology capable of probing single-cell deformability at approximately 2,000 cells/s. The method uses inertial focusing to uniformly deliver cells to a stretching extensional flow where cells are deformed at high strain rates, imaged with a high-speed camera, and computationally analyzed to extract quantitative parameters. This approach allows us to analyze cells at throughputs orders of magnitude faster than previously reported biophysical flow cytometers and single-cell mechanics tools, while creating easily observable larger strains and limiting user time commitment and bias through automation. Using this approach we rapidly assay the deformability of native populations of leukocytes and malignant cells in pleural effusions and accurately predict disease state in patients with cancer and immune activation with a sensitivity of 91% and a specificity of 86%. As a tool for biological research, we show the deformability we measure is an early biomarker for pluripotent stem cell differentiation and is likely linked to nuclear structural changes. Microfluidic deformability cytometry brings the statistical accuracy of traditional flow cytometric techniques to label-free biophysical biomarkers, enabling applications in clinical diagnostics, stem cell characterization, and single-cell biophysics.

Controlled encapsulation of single-cells into monodisperse picolitre drops

Encapsulation of cells within picolitre-size monodisperse drops provides new means to perform quantitative biological studies on a single-cell basis for large cell populations. Variability in the number of cells per drop due to stochastic cell loading is a major barrier to these techniques. We overcome this limitation by evenly spacing cells as they travel within a high aspect-ratio microchannel; cells enter the drop generator with the frequency of drop formation.

Equilibrium Separation and Filtration of Particles Using Differential Inertial Focusing

Rapid separation and filtration of particles in solution has a wide range of applications including blood cell separation, ultrasound contrast agent preparation, and purification of fermentation products. However, current techniques that provide quick processing rates are high in complexity. We present a rapid microfluidic filtration technology capable of separating particles based on size, with purities from 90 to 100% and high-volume throughputs of 1 mL/min. Data for separation of rigid particles, deformable emulsions, and platelets from whole blood are presented. The system is based upon differential inertial focusing of particles of varying sizes and allows continuous separation based only on intrinsic hydrodynamic forces developed in a flow through an asymmetrically curved channel. A theoretical description of the underlying forces is developed, and in combination with data determining a size cutoff for separation, a semiempirical relationship describing how channel geometry is related to this cutoff is shown. Cascading separations in series is shown to be useful for increasing purity and yield. This type of microfluidic system can filter deformable particles, is largely independent of particle density, and can provide throughputs typical of macroscale filtration in a compact format, enabling applications in blood filtration and particle concentration.

High-throughput size-based rare cell enrichment using microscale vortices

Cell isolation in designated regions or from heterogeneous samples is often required for many microfluidic cell-based assays. However, current techniques have either limited throughput or are incapable of viable off-chip collection. We present an innovative approach, allowing high-throughput and label-free cell isolation and enrichment from heterogeneous solution using cell size as a biomarker. The approach utilizes the irreversible migration of particles into microscale vortices, developed in parallel expansion-contraction trapping reservoirs, as the cell isolation mechanism. We empirically determined the critical particle∕cell diameter D(crt) and the operational flow rate above which trapping of cells∕particles in microvortices is initiated. Using this approach we successfully separated larger cancer cells spiked in blood from the smaller blood cells with processing rates as high as 7.5×10(6) cells∕s. Viable long-term culture was established using cells collected off-chip, suggesting that the proposed technique would be useful for clinical and research applications in which in vitro culture is often desired. The presented technology improves on current technology by enriching cells based on size without clogging mechanical filters, employing only a simple single-layered microfluidic device and processing cell solutions at the ml∕min scale.

Continuous scalable blood filtration device using inertial microfluidics

Cell separation is broadly useful for applications in clinical diagnostics, biological research, and potentially regenerative medicine. Recent attention has been paid to label‐free size‐based techniques that may avoid the costs or clogging issues associated with centrifugation and mechanical filtration. We present for the first time a massively parallel microfluidic device that passively separates pathogenic bacteria cells from diluted blood with macroscale performance. The device was designed to process large sample volumes in a high‐throughput, continuous manner using 40 single microchannels placed in a radial array with one inlet and two rings of outlets. Each single channel consists of a short focusing, gradual expansion and collection region and uses unique differential transit times due to size‐dependent inertial lift forces as a method of cell separation. The gradual channel expansion region is shown to manipulate cell equilibrium positions close to the microchannel walls, critical for higher efficiency collection. We demonstrate >80% removal of pathogenic bacteria from blood after two passes of the single channel system. The massively parallel device can process 240 mL/h with a throughput of 400 million cells/min. We expect that this parallelizable, robust, and label‐free approach would be useful for filtration of blood as well as for other cell separation and concentration applications from large volume samples. Biotechnol. Bioeng. 2010;107: 302–311.

High-throughput single-microparticle imaging flow analyzer

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Automated cellular sample preparation using a Centrifuge-on-a-Chip

The standard centrifuge is a laboratory instrument widely used by biologists and medical technicians for preparing cell samples. Efforts to automate the operations of concentration, cell separation, and solution exchange that a centrifuge performs in a simpler and smaller platform have had limited success. Here, we present a microfluidic chip that replicates the functions of a centrifuge without moving parts or external forces. The device operates using a purely fluid dynamic phenomenon in which cells selectively enter and are maintained in microscale vortices. Continuous and sequential operation allows enrichment of cancer cells from spiked blood samples at the mL min(-1) scale, followed by fluorescent labeling of intra- and extra-cellular antigens on the cells without the need for manual pipetting and washing steps. A versatile centrifuge-analogue may open opportunities in automated, low-cost and high-throughput sample preparation as an alternative to the standard benchtop centrifuge in standardized clinical diagnostics or resource poor settings.