Microfluidic Cell Sorting Research Articles

Cost-effective microfluidic flow cytometry for precise and gentle cell sorting.

Microfluidic flow cytometry (MFCM) is considered to be an effective substitute for traditional flow cytometry, because of its advantages in terms of higher integration, smaller device size, lower cost, and higher cell sorting activity. However, MFCM still faces challenges in balancing parameters such as sorting throughput, viability, sorting efficiency, and cost. Here, we demonstrate a cost-effective and high-performance microfluidic cytometry cell sorting system, along with a customized microfluidic chip that integrates hydrodynamic focusing, droplet encapsulation, and sorting for precise cell manipulation. An innovative photon incremental counting-based fluorescence detection method is proposed, which requires only one-fiftieth of the data compared to traditional methods. This significantly simplifies the structure of the system and substantially reduces costs. The system exhibits detection recoveries exceeding 95% across sample solution flow rates ranging from 10 to 80 μL min-1. Moreover, it accurately achieves individual droplet deflections at a droplet generation frequency of 1600 Hz. Ultimately, our cell sorting system offers an impressive sorting efficiency of 90.7% and a high cell viability of 94.3% when operating at a droplet generation frequency of 1316 Hz, highlighting its accuracy and gentleness throughout the entire process. Our work will enhance advances in the life sciences, thereby creating a boom in great applications in single-cell cloning, single-cell analysis, drug screening, etc.

High-Density Microporous Drainage-Integrating Sheath Flow Generator for Streamlining Microfluidic Cell Sorting Systems.

Tremendous efforts have been made to develop practical and efficient microfluidic cell and particle sorting systems; however, there are technological limitations in terms of system complexity and low operability. Here, we propose a sheath flow generator that can dramatically simplify operational procedures and enhance the usability of microfluidic cell sorters. The device utilizes an embedded polydimethylsiloxane (PDMS) sponge with interconnected micropores, which is in direct contact with microchannels and seamlessly integrated into the microfluidic platform. The high-density micropores on the sponge surface facilitated fluid drainage, and the drained fluid was used as the sheath flow for downstream cell sorting processes. To fabricate the integrated device, a new process for sponge-embedded substrates was developed through the accumulation, incorporation, and dissolution of PMMA microparticles as sacrificial porogens. The effects of the microchannel geometry and flow velocity on the sheath flow generation were investigated. Furthermore, an asymmetric lattice-shaped microchannel network for cell/particle sorting was connected to the sheath flow generator in series, and the sorting performances of model particles, blood cells, and spiked tumor cells were investigated. The sheath flow generation technique developed in this study is expected to streamline conventional microfluidic cell-sorting systems as it dramatically improves versatility and operability.

Numerical and experimental characterization of a piezoelectric actuator for microfluidic cell sorting

Piezoelectric actuators offer great opportunities for precise and low-cost control of fluids at the microscale. Microfluidic systems with integrated piezoelectric actuators find application as droplet generators, micropumps, and microsorters. To accelerate device design and optimization, modeling and simulation approaches represent an attractive tool, but there are challenges arising from the multiphysics nature of the problem. Simple, potentially real-time approaches to experimentally characterize the fluid response to piezoelectric actuation are also highly desirable. In this work, we propose a strategy for the numerical and experimental characterization of a piezoelectric microfluidic cell sorter. Specifically, we present a 3D coupled multiphysics finite-element model of the system and an easy image-based approach for flow monitoring. Sinusoidal and pulse actuation are considered as case studies to test the proposed methodology. The results demonstrate the validity of the approach as well as the suitability of the system for cell sorting applications.

Microfluidic pumps for cell sorting.

Microfluidic cell sorting has shown promising advantages over traditional bulky cell sorting equipment and has demonstrated wide-reaching applications in biological research and medical diagnostics. The most important characteristics of a microfluidic cell sorter are its throughput, ease of use, and integration of peripheral equipment onto the chip itself. In this review, we discuss the six most common methods for pumping fluid samples in microfluidic cell sorting devices, present their advantages and drawbacks, and discuss notable examples of their use. Syringe pumps are the most commonly used method for fluid actuation in microfluidic devices because they are easily accessible but they are typically too bulky for portable applications, and they may produce unfavorable flow characteristics. Peristaltic pumps, both on- and off-chip, can produce reversible flow but they suffer from pulsatile flow characteristics, which may not be preferable in many scenarios. Gravity-driven pumping, and similarly hydrostatic pumping, require no energy input but generally produce low throughputs. Centrifugal flow is used to sort cells on the basis of size or density but requires a large external rotor to produce centrifugal force. Electroosmotic pumping is appealing because of its compact size but the high voltages required for fluid flow may be incompatible with live cells. Emerging methods with potential for applications in cell sorting are also discussed. In the future, microfluidic cell sorting methods will trend toward highly integrated systems with high throughputs and low sample volume requirements.

Multistage microfluidic cell sorting method and chip based on size and stiffness

High performance sorting of circulating tumor cells (CTCs) from peripheral blood is key to liquid biopsies. Size-based deterministic lateral displacement (DLD) technique is widely used in cell sorting. But conventional microcolumns have poor fluid regulation ability, which limits the sorting performance of DLD. When the size difference between CTCs and leukocytes is small (e.g., less than 3 μm), not only DLD, many size-based separation techniques fail due to low specificity. CTCs have been confirmed to be softer than leukocytes, which could serve as a basis for sorting. In this study, we presented a multistage microfluidic CTCs sorting method, first sorting CTCs using a size-based two-array DLD chip, then purifying CTCs mixed by leukocytes using a stiffness-based cone channel chip, and finally identifying cell types using Raman techniques. The entire CTCs sorting and analysis process was label free, highly pure, high-throughput and efficient. The two-array DLD chip employed a droplet-shaped microcolumn (DMC) developed by optimization design rather than empirical design. Attributed to the excellent fluid regulation capability of DMC, the CTCs sorter system developed by parallelizing four DMC two-array DLD chips was able to process a sample of 2.5 mL per minute with a recovery efficiency of 96.30 ± 2.10% and a purity of 98.25 ± 2.48%. To isolate CTCs mixed dimensionally by leukocytes, a cone channel sorting method and chip were developed based on solid and hydrodynamic coupled analysis. The cone channel chip allowed CTCs to pass through the channel and entrap leukocytes, improving the purity of CTCs mixed by leukocytes by 1.8-fold.

Abstract B014: Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS

Abstract Kirsten rat sarcoma viral oncogene homolog (KRAS) is the most frequently mutated oncogene detected in 30% of all human cancers. Drugs that can impede the function of oncogenic KRAS offer exceptional therapeutic value. However, direct targeting KRAS is challenging due to its protein structure that lacks deep binding pockets for small-molecule inhibitors. Despite the recent success in targeting the G12C allele, targeted therapy for another hotspot mutant – G12V mutant KRAS has not been described. Here, we utilize CRISPR-Cas9 based genome-wide knockout screens and leverage a high-throughput microfluidic cell sorting platform to identify genes that regulate mutant KRAS harboring G12V mutation. The screens were conducted in two colorectal cancer cell lines HT29 and SW480, expressing wild-type KRAS and homozygous G12V KRAS, respectively. We have identified a list of genes whose loss-of-function reduces wild-type or mutant G12V KRAS protein expression. Our top gene identified from the mutant KRAS screen - denoted as selective-mutant KRAS modulator (SMKM) has shown promising results in selectively decreasing G12V KRAS protein expression. Through multiple assays including live cell imaging, western blot, flow cytometry, and proteomic analysis in SMKM knockout cells and SMKM inhibitor-treated cells. We found highly selective reduction in G12V KRAS protein expression. More importantly, genetically or pharmacologically inhibiting SMKM has demonstrated anti-tumor activity. We conducted cell viability assays including CellTiter-glo, MTT, and clonogenic assay. We treated a panel of G12V KRAS mutant and wild-type KRAS cell lines with SMKM inhibitor and found significantly reduced cell viability in G12V KRAS mutant cell lines across colorectal and lung cancer. To further demonstrate the downstream impact on mutant KRAS signaling, secondary assays including western blot and Lumit immunoassay were conducted. We observe a reduction in the aberrant phospho-ERK and phospho-AKT signaling in SMKM inhibitor treated cells. Our in vivo efficacy trial using heterozygous G12V KRAS lung cancer NCI-H441 xenograft model has shown SMKM inhibition significantly reduces tumor growth and increases the survival rate. Together, these data indicate the feasibility of selectively targeting G12V KRAS with SMKM inhibition. Lastly, our results provide a first-in-class small molecule oral inhibitor based selective protein clearance as an effective anti-G12V mutant KRAS therapeutic strategy. Citation Format: Xiyue Hu, Randy Singh, Shana Kelley. Phenotypic CRISPR genome-wide screening for the discovery of genetic regulators of allele-specific mutant KRAS [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr B014.

Isolation of tumour-reactive lymphocytes from peripheral blood via microfluidic immunomagnetic cell sorting.

The clinical use of tumour-infiltrating lymphocytes for the treatment of solid tumours is hindered by the need to obtain large and fresh tumour fractions, which is often not feasible in patients with unresectable tumours or recurrent metastases. Here we show that circulating tumour-reactive lymphocytes (cTRLs) can be isolated from peripheral blood at high yield and purity via microfluidic immunomagnetic cell sorting, allowing for comprehensive downstream analyses of these rare cells. We observed that CD103 is strongly expressed by the isolated cTRLs, and that in mice with subcutaneous tumours, tumour-infiltrating lymphocytes isolated from the tumours and rapidly expanded CD8+CD103+ cTRLs isolated from blood are comparably potent and respond similarly to immune checkpoint blockade. We also show that CD8+CD103+ cTRLs isolated from the peripheral blood of patients and co-cultured with tumour cells dissociated from their resected tumours resulted in the enrichment of interferon-γ-secreting cell populations with T-cell-receptor clonotypes substantially overlapping those of the patients' tumour-infiltrating lymphocytes. Therapeutically potent cTRLs isolated from peripheral blood may advance the clinical development of adoptive cell therapies.

Recent advances in deformation-assisted microfluidic cell sorting technologies.

Cell sorting is an essential prerequisite for cell research and has great value in life science and clinical studies. Among the many microfluidic cell sorting technologies, label-free methods based on the size of different cell types have been widely studied. However, the heterogeneity in size for cells of the same type and the inevitable size overlap between different types of cells would result in performance degradation in size-based sorting. To tackle such challenges, deformation-assisted technologies are receiving more attention recently. Cell deformability is an inherent biophysical marker of cells that reflects the changes in their internal structures and physiological states. It provides additional dimensional information for cell sorting besides size. Therefore, in this review, we summarize the recent advances in deformation-assisted microfluidic cell sorting technologies. According to how the deformability is characterized and the form in which the force acts, the technologies can be divided into two categories: (1) the indirect category including transit-time-based and image-based methods, and (2) the direct category including microstructure-based and hydrodynamics-based methods. Finally, the separation performance and the application scenarios of each method, the existing challenges and future outlook are discussed. Deformation-assisted microfluidic cell sorting technologies are expected to realize greater potential in the label-free analysis of cells.

Label-free microfluidic cell sorting and detection for rapid blood analysis.

Blood tests are considered as standard clinical procedures to screen for markers of diseases and health conditions. However, the complex cellular background (>99.9% RBCs) and biomolecular composition often pose significant technical challenges for accurate blood analysis. An emerging approach for point-of-care blood diagnostics is utilizing "label-free" microfluidic technologies that rely on intrinsic cell properties for blood fractionation and disease detection without any antibody binding. A growing body of clinical evidence has also reported that cellular dysfunction and their biophysical phenotypes are complementary to standard hematoanalyzer analysis (complete blood count) and can provide a more comprehensive health profiling. In this review, we will summarize recent advances in microfluidic label-free separation of different blood cell components including circulating tumor cells, leukocytes, platelets and nanoscale extracellular vesicles. Label-free single cell analysis of intrinsic cell morphology, spectrochemical properties, dielectric parameters and biophysical characteristics as novel blood-based biomarkers will also be presented. Next, we will highlight research efforts that combine label-free microfluidics with machine learning approaches to enhance detection sensitivity and specificity in clinical studies, as well as innovative microfluidic solutions which are capable of fully integrated and label-free blood cell sorting and analysis. Lastly, we will envisage the current challenges and future outlook of label-free microfluidics platforms for high throughput multi-dimensional blood cell analysis to identify non-traditional circulating biomarkers for clinical diagnostics.

Upgraded User-Friendly Image-Activated Microfluidic Cell Sorter Using an Optimized and Fast Deep Learning Algorithm.

Image-based cell sorting is essential in biological and biomedical research. The sorted cells can be used for downstream analysis to expand our knowledge of cell-to-cell differences. We previously demonstrated a user-friendly image-activated microfluidic cell sorting technique using an optimized and fast deep learning algorithm. Real-time isolation of cells was carried out using this technique with an inverted microscope. In this study, we devised a recently upgraded sorting system. The cell sorting techniques shown on the microscope were implemented as a real system. Several new features were added to make it easier for the users to conduct the real-time sorting of cells or particles. The newly added features are as follows: (1) a high-resolution linear piezo-stage is used to obtain in-focus images of the fast-flowing cells; (2) an LED strobe light was incorporated to minimize the motion blur of fast-flowing cells; and (3) a vertical syringe pump setup was used to prevent the cell sedimentation. The sorting performance of the upgraded system was demonstrated through the real-time sorting of fluorescent polystyrene beads. The sorter achieved a 99.4% sorting purity for 15 μm and 10 μm beads with an average throughput of 22.1 events per second (eps).

High efficiency sorting and outgrowth for single-cell cloning of mammalian cell lines

Single-cell selection and cloning is required for multiple bioprocessing and cell engineering workflows. Dispensing efficiency and outgrowth were optimized for multiple common suspension (CHO ES, Expi293F, and Jurkat) and adherent (MCF-7, A549, CHO-K1, and HEK293) cell lines. Single-cell sorting using a low pressure microfluidic cell sorter, the WOLF Cell Sorter, was compared with limiting dilution at 0.5 cells/well to demonstrate the increased efficiency of using flow cytometry selection of cells. In this work, there was an average single cell deposition on Day 0 of 89.1% across all the cell lines tested compared to 41.2% when using limiting dilution. After growth for 14 days, 66.7% of single-cell clones sorted with the WOLF Cell Sorter survived and only 23.8% when using limiting dilution. Using the WOLF Cell Sorter for cell line development results in higher viable single-cell colonies and the ability to select subpopulations of single-cells using multiple parameters.

Rapid switching and durable on-chip spark-cavitation-bubble cell sorter

Precise and high-speed sorting of individual target cells from heterogeneous populations plays an imperative role in cell research. Although the conventional fluorescence-activated cell sorter (FACS) is capable of rapid and accurate cell sorting, it occupies a large volume of the instrument and inherently brings in aerosol generation as well as cross-contamination among samples. The sorting completed in a fully enclosed and disposable microfluidic chip has the potential to eliminate the above concerns. However, current microfluidic cell sorters are hindered by the high complexities of the fabrication procedure and the off-chip setup. In this paper, a spark-cavitation-bubble-based fluorescence-activated cell sorter is developed to perform fast and accurate sorting in a microfluidic chip. It features a simple structure and an easy operation. This microfluidic sorter comprises a positive electrode of platinum and a negative electrode of tungsten, which are placed on the side of the main channel. By applying a high-voltage discharge on the pair of electrodes, a single spark cavitation bubble is created to deflect the target particle into the downstream collection channel. The sorter has a short switching time of 150 μs and a long lifespan of more than 100 million workable actions. In addition, a novel control strategy is proposed to dynamically adjust the discharge time to stabilize the size of the cavitation bubble for continuous sorting. The dynamic control of continuously triggering the sorter, the optimal delay time between fluorescence detection and cell sorting, and a theoretical model to predict the ideal sorting recovery and purity are studied to improve and evaluate the sorter performance. The experiments demonstrate that the sorting rate of target particles achieves 1200 eps, the total analysis throughput is up to 10,000 eps, the particles sorted at 4000 eps exhibit a purity greater than 80% and a recovery rate greater than 90%, and the sorting effect on the viability of HeLa cells is negligible.

Microfluidic cell sorting for gentle isolation of immune cells.

Abstract Isolation of specific immune cells is critical to biomedical research, however, techniques such as droplet-based fluorescence-activated cell sorting can mechanically irritate cells resulting in cell death or unwanted activation of immune cells that affect experimental analysis. Here we show that T cell subsets and neutrophils can be isolated using a low pressure (<2 psi) microfluidic cell sorter (WOLF G2®, NanoCellect®). These experiments show that this 11-parameter cell sorter can isolate complex cell types at high purity and high viability. In addition, due to the low-pressure microfluidic sorting mechanism, laminar flow reduces shear stress relative to traditional droplet sorters and cells remain functional and unaltered for subsequent experiments. Furthermore, we demonstrate high quality single-cell genomics data when combined downstream with single-cell partitioning and plate-based workflows. These findings demonstrate a novel workflow for isolating immune cells using the next generation of cell sorters.

Basic Principles and Recent Advances in Magnetic Cell Separation

Magnetic cell separation has become a key methodology for the isolation of target cell populations from biological suspensions, covering a wide spectrum of applications from diagnosis and therapy in biomedicine to environmental applications or fundamental research in biology. There now exists a great variety of commercially available separation instruments and reagents, which has permitted rapid dissemination of the technology. However, there is still an increasing demand for new tools and protocols which provide improved selectivity, yield and sensitivity of the separation process while reducing cost and providing a faster response. This review aims to introduce basic principles of magnetic cell separation for the neophyte, while giving an overview of recent research in the field, from the development of new cell labeling strategies to the design of integrated microfluidic cell sorters and of point-of-care platforms combining cell selection, capture, and downstream detection. Finally, we focus on clinical, industrial and environmental applications where magnetic cell separation strategies are amongst the most promising techniques to address the challenges of isolating rare cells.

Ultrathroughput immunomagnetic cell sorting platform.

High-throughput phenotypic cell sorting is critical to the development of cell-based therapies and cell screening discovery platforms. However, current cytometry platforms are limited by throughput, number of fractionated populations that can be isolated, cell viability, and cost. We present an ultrathroughput microfluidic cell sorter capable of processing hundreds of millions of live cells per hour per device based on protein expression. This device, a next-generation microfluidic cell sorter (NG-MICS), combines multiple technologies, including 3D printing, reversible clamp sealing, and superhydrophobic treatments to create a reusable and user-friendly platform ready for deployment. The utility of such a platform is demonstrated through the rapid isolation of mature natural killer cells from peripheral blood mononuclear cells, for use in CAR-NK therapies at clinically-relevant scale.

Microfluidic System for Cell Sorting

The need for efficient cell sorting, which is a useful technique in medical and biological research, has boosted the development of microfluidic cell sorter. This review paper concludes the basic principle of microfluidic sorter which covers active and passive sorting techniques. The work process, sorting efficiency, advantages, disadvantages, and avenue for improvement are explored for each type by introducing several typical sorters. The industrial implementation and potential development are also conveyed. In conclusion, developing efficient microfluidic cell sorter offers a greater control over cell distribution and is fundamental in realizing efficacious cell sorting systems.

Electricity-free hand-held inertial microfluidic sorter for size-based cell sorting

Conventional batch-top cell sorters are often bulky and expensive, and miniaturized microfluidic sorters available mostly require field generators and electricity-powered pumping systems. Therefore, the development of a low-cost, portable cell sorter that can be used in low resource settings is essential. In this study, we propose such an electricity-free hand-held inertial microfluidic sorter that can be used for the high-efficiency sorting of differently sized cells in a continuous and passive manner. The proposed hand-held sorter is composed of a wheel-shaped all-in-one syringe inertial microfluidic sorter (i-sorter) with flow stabilizer units and two spring-driven mechanical syringe drivers. The release of the compression spring in the mechanical syringe driver through a one-click operation provides the flow driving force. Passive flow stabilizer units in the i-sorter enable flow-rate-sensitive inertial cell separation for the unstable driving flow rate generated by the low-cost mechanical syringe driver. We successfully achieved sorting of differently sized particles and high-efficiency separation of rare tumor cells from the blood using the fabricated prototype. Our hand-held inertial microfluidic cell sorter has many advantages, including low device cost, simple electricity-free operation, compactness, and portability; additionally, samples do not need to be pre-labelled. Therefore, it has potential for use in low-resource settings.

Coprocytobiology: A Technical Review of Cytological Colorectal Cancer Screening in Fecal Samples.

This review discusses the field of coprocytobiology, defined as a combined method of cell preservation, isolation, and cytology, which has applications to the investigation of noninvasive fecal screening for colorectal cancer. In the decade since the field was last reviewed, cell isolation has progressed rapidly via the development of technologies such as microfluidic and magnetic cell sorting. The landscape of cytology has also advanced in this time with the emergence of novel cytological methods and cell preservation strategies. Previous reviews present an outdated and incomplete view of coprocytobiology, summarizing a limited number of early publications, ignoring the principle of cell preservation and focusing on a single method of isolation rather than the field as a whole. In contrast to these publications, this review presents an updated, comprehensive, and unbiased representation of the technical aspects of coprocytobiology and provides unique insight into the common methodological pitfalls, best practice, and future directions of cytological screening for colorectal cancer. Graphical Abstract

Label-free image-encoded microfluidic cell sorter with a scanning Bessel beam.

The microfluidic-based, label-free image-guided cell sorter offers a low-cost, high information content, and disposable solution that overcomes many limitations in conventional cell sorters. However, flow confinement for most microfluidic devices is generally only one-dimensional using sheath flow. As a result, the equilibrium distribution of cells spreads beyond the focal plane of commonly used Gaussian laser excitation beams, resulting in a large number of blurred images that hinder subsequent cell sorting based on cell image features. To address this issue, we present a Bessel–Gaussian beam image-guided cell sorter with an ultra-long depth of focus, enabling focused images of >85% of passing cells. This system features label-free sorting capabilities based on features extracted from the output temporal waveform of a photomultiplier tube (PMT) detector. For the sorting of polystyrene beads, SKNO1 leukemia cells, and Scenedesmus green algae, our results indicate a sorting purity of 97%, 97%, and 98%, respectively, showing that the temporal waveforms from the PMT outputs have strong correlations with cell image features. These correlations are also confirmed by off-line reconstructed cell images from a temporal–spatial transformation algorithm tailored to the scanning Bessel–Gaussian beam.

Extremely High-Throughput Parallel Microfluidic Vortex-Actuated Cell Sorting.

We demonstrate extremely high-throughput microfluidic cell sorting by making a parallel version of the vortex-actuated cell sorter (VACS). The set-up includes a parallel microfluidic sorter chip and parallel cytometry instrumentation: optics, electronics and control software. The result is capable of sorting lymphocyte-sized particles at 16 times the rate of our single-stream VACS devices, and approximately 10 times the rate of commercial cell sorters for an equivalent procedure. We believe this opens the potential to scale cell sorting for applications requiring the processing of much greater cell numbers than currently possible with conventional cell sorting.