Soojung Claire Hur

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

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.

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.

Inertial focusing of non-spherical microparticles

We have investigated the focusing and dynamics of non-spherical polymeric particles in microfluidic flows at finite Reynolds number. The rotational diameter, Dmax, of a particle, regardless of its cross-sectional shape, was found to determine the final focused position, except for the case of asymmetric disks. Additionally, elongated particles with larger Dmax exhibited longer residence times in a horizontal orientation than those with smaller Dmax. These findings inform approaches to hydrodynamically control shaped and barcoded particles for multiplexed biochemical assays.

Label-Free Enrichment of Adrenal Cortical Progenitor Cells Using Inertial Microfluidics

Passive and label-free isolation of viable target cells based on intrinsic biophysical cellular properties would allow for cost savings in applications where molecular biomarkers are known as well as potentially enable the separation of cells with little-to-no known molecular biomarkers. We have demonstrated the purification of adrenal cortical progenitor cells from digestions of murine adrenal glands utilizing hydrodynamic inertial lift forces that single cells and multicellular clusters differentially experience as they flow through a microchannel. Fluorescence staining, along with gene expression measurements, confirmed that populations of cells collected in different outlets were distinct from one another. Furthermore, primary murine cells processed through the device remained highly viable and could be cultured for 10 days in vitro. The proposed target cell isolation technique can provide a practical means to collect significant quantities of viable intact cells required to translate stem cell biology to regenerative medicine in a simple label-free manner.

Sequential multi-molecule delivery using vortex-assisted electroporation.

We developed an on-chip microscale electroporation system that enables sequential delivery of multiple molecules with precise and independent dosage controllability into pre-selected identical populations of target cells. The ability to trap cells with uniform size distribution contributed to enhanced molecular delivery efficiency and cell viability. Additionally, the system provides real-time monitoring ability of the entire delivery process, allowing timely and independent modification of cell- and molecule-specific electroporation parameters. The precisely controlled amount of inherently membrane-impermeant molecules was transferred into human cancer cells by varying electric field strengths and molecule injection durations. The proposed microfluidic electroporation systems improved viability and comparable gene transfection efficiency to that of commercial systems suggest that the current system has great potential to expand the research fields that on-chip electroporation techniques can be used in.

Towards an Integrated Chip-Scale Plasmonic Biosensor

Biosensing allows researchers to detect tiny amounts of harmful chemicals before they become major threats. These researchers are using advanced optical technologies to develop the biosensor of the future-a plasmonic-based chip-scale device that will allow for compact, inexpensive, ubiquitous and sensitive detection.

Intra-aneurysmal flow reductions in a thin film nitinol flow diverter

A novel hyper-elastic thin film nitinol (HE-TFN) covered stent has been developed to promote aneurysm occlusion by diminishing flow in the aneurysm. Laboratory aneurysm models were used to assess the flow changes produced by stents covered with different patterns of HE-TFN placed across the aneurysm neck in the parent vessel. The flow diverters were constructed by covering Wingspan stents (Boston Scientific) with different HE-TFNs (i.e., of 82% and 77% porosity) and deployed in both in vitro wide-neck and fusiform glass aneurysm models. In wide-neck aneurysms, the 82% porous HE-TFN stent reduced mean flow velocity in the middle of the sac by 86 ± 1%, while the 77% porous stent reduced the velocity by 93 ± 5% (n = 3). Local wall shear rates were also significantly reduced by about 98% in this model after device placement. Tests conducted on the fusiform aneurysm revealed smaller intra-aneurysmal flow velocity reduction by 48 ± 3% for the 82% porous stent and by 59 ± 7% for the 77% porous stent. The wall shear was reduced by approximately 50% by HE-TFN stents in fusiform models. These results suggest that HE-TFN covered stents have the potential to promote thrombosis in both wide-neck and fusiform aneurysm sacs.

Pulsed laser triggered high speed fluorescence activated microfluidic switch

We report a high speed fluorescence activated microfluidic switch capable of achieving a switching time of 50 µsec with a detection efficiency of 86.6% and a switching efficiency of 86.5% at a particle flow speed of ∼0.7 m/s. The switching mechanism is realized by exciting dynamic vapor bubbles with focused laser pulses in a microfluidic PDMS channel. The explosive bubble expansion generates fast fluid flows which are directed into a neighboring particle channel to switch the particle flow. Fluorescence activated switching of 10 micron polystyrene microspheres in a Y channel has been demonstrated. This ultrafast laser triggered switching mechanism has the potential to advance the sorting speed of the state-of-the-art microscale fluorescent activated cell sorting devices.