microscale-vortex-assisted-electroporator-for-sequential-molecular

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Harvard University

Rapid and accurate differentiation of cell types within a heterogeneous solution is a challenging but important task for various applications in biological research and medicine. Flow cytometry is the gold standard in cell analysis and is regularly used for blood analysis (i.e., complete blood counts). Flow cytometry, however, lacks sufficient throughput to analyze rare cells in blood or other dilute solutions in a reasonable time period because it is an inherently serial process. In this study, we exploit inertial effects for label- and sheath-free parallel flow cytometry with extreme throughput. We demonstrate a microfluidic device that consists of 256 high-aspect (W = 16 microm, H = 37 microm) parallel channels yielding a sample rate up to 1 million cells s(-1), only limited by the field-of-view of our high-speed optical interrogation method. The particles or cells flowing through the channels are focused to one uniform z-position (SD = +/-1.81 microm) with uniform downstream velocity (U(ave) = 0.208 +/- 0.004 m s(-1)) to reduce the probability of overlap and out-of-focus blur and provide similar cell signature images for accurate detection and analysis. To demonstrate a proof-of-concept application of our system operating at these throughputs, we conducted automated RBC and leukocyte counts on diluted whole blood and achieved high counting sensitivity and specificity (86-97%) compared to visual inspection of raw images. As no additional external forces are required to create ordered streams of cells, this approach has the potential for future applications in cost-effective hematology or rare-cell analysis platforms with extreme throughput capabilities when integrated with suitable large field-of view imaging or interrogation methods.

Sheathless inertial cell ordering for extreme throughput flow cytometry.

Cell 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.

Label-free cell separation and sorting in microfluidic systems.

The ability to detect and isolate rare target cells from heterogeneous samples is in high demand in cell biology research, immunology, tissue engineering and medicine. Techniques allowing label-free cell enrichment or detection are especially important to reduce the complexity and costs towards clinical applications. Single-cell deformability has recently been recognized as a unique label-free biomarker for cell phenotype with implications for assessment of cancer invasiveness. Using a unique combination of fluid dynamic effects in a microfluidic system, we demonstrate high-throughput continuous label-free cell classification and enrichment based on cell size and deformability. The system takes advantage of a balance between deformability-induced and inertial lift forces as cells travel in a microchannel flow. Particles and droplets with varied elasticity and viscosity were found to have separate lateral dynamic equilibrium positions due to this balance of forces. We applied this system to successfully classify various cell types using cell size and deformability as distinguishing markers. Furthermore, using differences in dynamic equilibrium positions, we adapted the system to conduct passive, label-free and continuous cell enrichment based on these markers, enabling off-chip sample collection without significant gene expression changes. The presented method has practical potential for high-throughput deformability measurements and cost-effective cell separation to obtain viable target cells of interest in cancer research, immunology, and regenerative medicine.

Deformability-based cell classification and enrichment using inertial microfluidics.

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.

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

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 size-based rare cell enrichment using microscale vortices.

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.

High-throughput single-microparticle imaging flow analyzer.

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.

Label-free enrichment of adrenal cortical progenitor cells using inertial microfluidics.

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 system's 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.

Article	Year
Sheathless inertial cell ordering for extreme throughput flow cytometry. Lab on a chip\| PubMed ID: 20090998	2010
Label-free cell separation and sorting in microfluidic systems. Analytical and bioanalytical chemistry\| PubMed ID: 20419490	2010
Deformability-based cell classification and enrichment using inertial microfluidics. Lab on a chip\| PubMed ID: 21271000	2011
Automated cellular sample preparation using a Centrifuge-on-a-Chip. Lab on a chip\| PubMed ID: 21804970	2011
High-throughput size-based rare cell enrichment using microscale vortices. Biomicrofluidics\| PubMed ID: 21918676	2011
High-throughput single-microparticle imaging flow analyzer. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 22753513	2012
Label-free enrichment of adrenal cortical progenitor cells using inertial microfluidics. PloS one\| PubMed ID: 23056341	2012
Sequential multi-molecule delivery using vortex-assisted electroporation. Lab on a chip\| PubMed ID: 23727978	2013

Soojung Claire Hur

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