Droplet-based Microfluidic System Research Articles

Fabrication of oxygen-releasing dextran microgels by droplet-based microfluidic method.

In the tissue engineering field, the supply of oxygen to three-dimensional (3D) tissues is an important aspect to avoid necrosis due to hypoxia. Although oxygen-releasing bulk materials containing calcium peroxide (CaO2, CP) have attracted much attention, micrometer-sized oxygen-releasing soft materials would be advantageous because of their highly controllable structures, which can be applied for cell scaffolds, injectable materials, and bioink components in 3D bioprinting. In this study, oxygen-releasing microgels were fabricated via a droplet-based microfluidic system. Homogeneous, monodisperse and stable oxygen-releasing microgels were obtained by photo-crosslinking of droplets composed of biocompatible dextran modified with methacrylate groups and CP nanoparticles as an oxygen source. We also used our microfluidic system for the in situ amorphous calcium carbonate (CaCO3, ACC) formation on the surface of CP nanoparticles to achieve the controlled release of oxygen from the microgel. Oxygen release from an ACC-CP microgel in a neutral cell culture medium was suppressed because incorporation of CP in the ACC suppressed the reaction with water. Strikingly, stimuli to dissolve ACC such as a weak acidic conditions triggered the oxygen release from microgels loaded with ACC-CP, as the dissolution of CaCO3 allows CP to react. Taken together, applications of this new class of biomaterials for tissue engineering are greatly anticipated. In addition, the developed microfluidic system can be used for a variety of oxygen-releasing microgels by changing the substrates of the hydrogel network.

Formation Pattern Analysis of Spheroids Formed by a Droplet-Based Microfluidic System for Predicting the Aggressiveness of Tumor Cells.

Examining tumor heterogeneity is essential for selecting an appropriate anticancer treatment for an individual. This study aimed to distinguish low- and high-aggressive tumor cells by analyzing the formation patterns of spheroids. The droplet-based microfluidic system was employed for the formation of each spheroid from four different subtypes of breast tumor cells. Additionally, heterotypic spheroids with T lymphocytes and cancer-associated fibroblasts (CAFs) were produced, and distinctions between low- and high-aggressive tumor cells were explored through the analysis of formation patterns using circularity, convexity, and cell distributions. In both homotypic spheroids and heterotypic spheroids with T lymphocytes, spheroids formed from low-aggressive tumor cells exhibited high circularity and convexity. On the other hand, spheroids formed from high-aggressive tumor cells had relatively low circularity and convexity. In the case of heterotypic spheroids with CAFs, circularity and convexity did not exhibit clear differences between low- and high-aggressive tumor cells, but distinct variations were observed in cell distributions. CAFs and low-aggressive tumor cells were evenly distributed, whereas the CAFs were predominantly located in the inner layer, and high-aggressive tumor cells were primarily located in the outer layer. This finding can offer valuable insights into predicting the aggressiveness of unknown tumor cells.

Miniaturized droplets flow reactor for one-step highly controlled synthesis of SnO2 quantum dots at room temperature

In recent years, the conventional methods of synthesizing nanomaterials have been surpassed by the emergence of the microfluidics route, which has brought forth numerous advantages and transformed the domain of nanomaterial synthesis. However, the synthesis of semiconducting oxide nanomaterials, specifically Tin oxide (SnO2), remains a crucial area of research due to its remarkable advantages as a viable alternative to toxic and costly materials. Additionally, SnO2 quantum dots (QDs) exhibit immense potential across a diverse range of applications due to their exceptional optical and electrical properties. The existing synthesis methods for SnO2 QDs are either time-consuming or involve high-temperature conditions. To address these challenges, the droplet-based microfluidic technique has emerged as a promising approach for the controlled synthesis of various semiconducting nanomaterials. This article highlights the synthesis of SnO2 QDs with tunable size through the utilization of a droplet-based microfluidic technique, providing precise control over droplet volumes. X-ray diffraction analysis verified the presence of rutile-type tetragonal structure in SnO2 QDs. From the transmission electron microscopy analysis, the average particle size was calculated to be 1.90 nm, 2.09 nm and 2.63 nm for the volume of droplet corresponding to 33.25 μl, 27.84 μl and 18.25 μl respectively. Furthermore, with a decrease in particle size a tunabilty in optical bandgap, from 4.60 eV to 4.00 eV was observed. This work provides insights into the influence of droplet volume on the particle size which in turn affects the associated properties of SnO2 QDs in a droplet-based microfluidic synthesis system.

Study on the velocity of droplet at steady state in contraction microchannels by numerical simulation

The droplet-based microfluidic system is increasingly advancing and widely applied in various fields of analytical techniques and experiments. To optimize and advance this system, droplet dynamics is of utmost concern. The velocity of droplets is highly significant as it aids in precise droplet control and manipulation, ultimately leading to the optimization of device design and performance. This paper utilizes numerical simulations to explore the influence of flow characteristics, fluid properties, and geometric parameters of the contraction microchannel on the velocity of droplets while they are in a stable state. The findings indicate that the droplet velocity is influenced by factors such as viscosity ratio (λ), capillary number (Ca), and contraction ratio (C).

Comparative study of microscale and macroscale technique for encapsulation of Calotropis gigantea extract in metal-conjugated nanomatrices for invasive ductal carcinoma

The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV–Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments.

Design and Validation of a Droplet-based Microfluidic System To Study Non-Photochemical Laser-Induced Nucleation of Potassium Chloride Solutions.

Non-photochemical laser-induced nucleation (NPLIN) has emerged as a promising primary nucleation control technique offering spatiotemporal control over crystallization with potential for polymorph control. So far, NPLIN was mostly investigated in milliliter vials, through laborious manual counting of the crystallized vials by visual inspection. Microfluidics represents an alternative to acquiring automated and statistically reliable data. Thus we designed a droplet-based microfluidic platform capable of identifying the droplets with crystals emerging upon Nd:YAG laser irradiation using the deep learning method. In our experiments, we used supersaturated solutions of KCl in water, and the effect of laser intensity, wavelength (1064, 532, and 355 nm), solution supersaturation (S), solution filtration, and intentional doping with nanoparticles on the nucleation probability is quantified and compared to control cooling crystallization experiments. Ability of dielectric polarization and the nanoparticle heating mechanisms proposed for NPLIN to explain the acquired results is tested. Solutions with lower supersaturation (S = 1.05) exhibit significantly higher NPLIN probabilities than those in the control experiments for all laser wavelengths above a threshold intensity (50 MW/cm2). At higher supersaturation studied (S = 1.10), irradiation was already effective at lower laser intensities (10 MW/cm2). No significant wavelength effect was observed besides irradiation with 355 nm light at higher laser intensities (≥50 MW/cm2). Solution filtration and intentional doping experiments showed that nanoimpurities might play a significant role in explaining NPLIN phenomena.

Synthesis and characterization of peanut hull modified chitosan beads.

The incorporation of plant materials is an effective method to improve the stability of chitosan beads, as it further increases the adsorption of toxic dyes and metals from aqueous systems. In the present study, chitosan gels were impregnated with a novel type of powder as the groundnut hull powder in order to form composite beads by using a simple droplet-based microfluidic system. The beads were then characterised through various techniques such as SEM, TGA, FTIR, and XRD. Microscopic imaging revealed a change in the surface morphology of the composite beads, which became rough and wrinkled with more valley-like features and irregular cracks. FTIR data suggest that the impregnation of groundnut hull powder led to an increase in functional groups. The thermal analysis allowed for the assessment of composite bead hydration contents and indicated the presence of groundnut hull entrapped in the loaded beads, which was corroborated by vibrational spectroscopy. XRD analysis allows us to conclude that there is an involvement of groundnut hull in the chitosan gels, and the consequence of that is the formation of amorphous beads, which would make them suitable for the adsorption of toxic dyes and metals from water systems.

Dispersive phase microscopy incorporated with droplet-based microfluidics for biofactory-on-a-chip.

Biomolecular imaging of intracellular structures of a single cell and subsequent screening of the cells are of high demand in metabolic engineering to develop strains with the desired phenotype. However, the capability of current methods is limited to population-scale identification of cell phenotyping. To address this challenge, we propose to utilize dispersive phase microscopy incorporated with a droplet-based microfluidic system that combines droplet volume-on-demand generation, biomolecular imaging, and droplet-on-demand sorting, to achieve high-throughput screening of cells with an identified phenotype. Particularly, cells are encapsulated in homogeneous environments with microfluidic droplet formation, and the biomolecule-induced dispersive phase can be investigated to indicate the biomass of a specific metabolite in a single cell. The retrieved biomass information consequently guides the on-chip droplet sorting unit to screen cells with the desired phenotype. To demonstrate the proof of concept, we showcase the method by promoting the evolution of the Haematococcus lacustris strain toward a high production of natural antioxidant astaxanthin. The validation of the proposed system with on-chip single-cell imaging and droplet manipulation functionalities reveals the high-throughput single-cell phenotyping and selection potential that applies to many other biofactory scenarios, such as biofuel production, critical quality attribute control in cell therapy, etc.

Microfluidic synthesis of monodisperse and size-tunable CsPbBr3 supraparticles.

The highly controlled, microfluidic template-assisted self-assembly of CsPbBr3 nanocrystals into spherical supraparticles is presented, achieving precise control over average supraparticle size through the variation of nanocrystal concentration and droplet size; thus facilitating the synthesis of highly monodisperse, sub-micron supraparticles (with diameters between 280 and 700 nm).

Directed Evolution of Laccase for Improved Thermal Stability Facilitated by Droplet-Based Microfluidic Screening System.

Laccases are attractive biocatalysts for industry due to their broad substrate spectrum, the use of oxygen as final electron acceptor, and water as the sole byproduct. Increasing efforts have been devoted to the engineering of laccases to improve their properties. The droplet-based microfluidic screening (DMFS) technology can accelerate the screening procedure and probe the large sequence space. In this study, a DMFS system including a heating step and picoinjection was used to sort large laccase libraries, yielding 12 variants with enhanced thermotolerance. All the obtained amino acid substitutions are distributed on the surface of the laccase. Interestingly, recombination of three identified substitutions of Asp to Asn on the surface resulted in the best variant M20, exhibiting 24.0-fold higher remaining activity at 58.8 °C and 1.9-3.4-fold higher remaining activity after incubation in organic solvents solution (20% (v/v) methanol and ethanol) and ionic liquid solution (20% (v/v) 1-ethyl-3-methylimidazolium ethyl sulfate) for 12 h. Furthermore, molecular dynamic simulations revealed that the recombination of the three beneficial substitutions, Asp98Asn, Asp474Asn, and Asp340Asn on the surface introduced more hydrogen bonds compared to the wild type, which made M20 more thermostable. This study highlighted the importance of the DMFS system for an efficient identification of beneficial long-distance amino acid substitutions.

Droplet Microfluidics-Based Fabrication of Monodisperse Poly(ethylene glycol)-Fibrinogen Breast Cancer Microspheres for Automated Drug Screening Applications.

Spheroidal cancer microtissues are highly advantageous for a wide range of biomedical applications, including high-throughput drug screening, multiplexed target validation, mechanistic investigation of tumor-extracellular matrix (ECM) interactions, among others. Current techniques for spheroidal tissue formation rely heavily on self-aggregation of single cancer cells and have substantial limitations in terms of cell-type-specific heterogeneities, uniformity, ease of production and handling, and most importantly, mimicking the complex native tumor microenvironmental conditions in simplistic models. These constraints can be overcome by using engineered tunable hydrogels that closely mimic the tumor ECM and elucidate pathologically relevant cell behavior, coupled with microfluidics-based high-throughput fabrication technologies to encapsulate cells and create cancer microtissues. In this study, we employ biosynthetic hybrid hydrogels composed of poly(ethylene glycol diacrylate) (PEGDA) covalently conjugated to natural protein (fibrinogen) (PEG-fibrinogen, PF) to create monodisperse microspheres encapsulating breast cancer cells for 3D culture and tumorigenic characterization. A previously developed droplet-based microfluidic system is used for rapid, facile, and reproducible fabrication of uniform cancer microspheres with either MCF7 or MDA-MB-231 (metastatic) breast cancer cells. Cancer cell-type-dependent variations in cell viability, metabolic activity, and 3D morphology, as well as microsphere stiffness, are quantified over time. Particularly, MCF7 cells grew as tight cellular clusters in the PF microspheres, characteristic of their epithelial morphology, while MDA-MB-231 cells displayed elongated and invasive morphology, characteristic of their mesenchymal and metastatic nature. Finally, the translational potential of the cancer microsphere platform toward high-throughput drug screening is also demonstrated. With high uniformity, scalability, and control over engineered microenvironments, the established cancer microsphere model can be potentially used for mechanistic studies, fabrication of modular cancer microtissues, and future drug-testing applications.

060 Heterogeneity and lineage development of memory CD8+ T cells after viral infection of skin

To better understand CD8 memory T cell development, we analyzed the profile of CD8+ T cells infiltrating skin as well as their circulating counterparts in the dLN after the same VACV skin infection at days 2-60, using the droplet-based microfluidic system Chromium (10X Genomics) single cell RNA sequencing platform. Using the Leiden algorithm, Ag-specific T cells were clustered and mapped through uniform manifold approximation and projection. Early activated T cells clustered together, showing a unique transcriptional profile, and skin and LN effectors began to diverge at day 5.

Microfluidic screening and genomic mutation identification for enhancing cellulase production in Pichia pastoris

BackgroundPichia pastoris is a widely used host organism for heterologous production of industrial proteins, such as cellulases. Although great progress has been achieved in improving protein expression in P. pastoris, the potential of the P. pastoris expression system has not been fully explored due to unknown genomic impact factors. Recently, whole-cell directed evolution, employing iterative rounds of genome-wide diversity generation and high-throughput screening (HTS), has been considered to be a promising strategy in strain improvement at the genome level.ResultsIn this study, whole-cell directed evolution of P. pastoris, employing atmospheric and room temperature plasma (ARTP) mutagenesis and droplet-based microfluidic HTS, was developed to improve heterogenous cellulase production. The droplet-based microfluidic platform based on a cellulase-catalyzed reaction of releasing fluorescence was established to be suitable for methanol-grown P. pastoris. The validation experiment showed a positive sorting efficiency of 94.4% at a sorting rate of 300 droplets per second. After five rounds of iterative ARTP mutagenesis and microfluidic screening, the best mutant strain was obtained and exhibited the cellulase activity of 11,110 ± 523 U/mL, an approximately twofold increase compared to the starting strain. Whole-genome resequencing analysis further uncovered three accumulated genomic alterations in coding region. The effects of point mutations and mutant genes on cellulase production were verified using reconstruction of point mutations and gene deletions. Intriguingly, the point mutation Rsc1G22V was observed in all the top-performing producers selected from each round, and gene deletion analysis confirmed that Rsc1, a component of the RSC chromatin remodeling complex, might play an important role in cellulase production.ConclusionsWe established a droplet-based microfluidic HTS system, thereby facilitating whole-cell directed evolution of P. pastoris for enhancing cellulase production, and meanwhile identified genomic alterations by whole-genome resequencing and genetic validation. Our approaches and findings would provide guides to accelerate whole-cell directed evolution of host strains and enzymes of high industrial interest.

Scalable and automated CRISPR-based strain engineering using droplet microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on a chip. The microfluidic device contains a 10 × 10 element array, and each element contains sets of electrodes for two electric field-actuated operations: electrowetting for merging droplets to mix reagents and electroporation for transformation. This device can perform up to 100 genetic modification reactions in parallel, providing a scalable platform for generating the large number of engineered strains required for the combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the system’s capabilities through the CRISPR-based engineering of two test cases: (1) disruption of the function of the enzyme galactokinase (galK) in E. coli and (2) targeted engineering of the glutamine synthetase gene (glnA) and the blue-pigment synthetase gene (bpsA) to improve indigoidine production in E. coli.

Ultrahigh-throughput screening of industrial enzyme-producing strains by droplet-based microfluidic system.

Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultrahigh throughput, but its widespread application remains limited by the accessibility of a droplet microfluidic high-throughput screening (HTS) platform, especially to common laboratories having no background in microfluidics. Here, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; and (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed, and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing Bacillus licheniformis strains from a mutant library generated by atmospheric and room temperature plasma mutagenesis, and clones displaying over 50% improvement in α-amylase productivity were isolated. This droplet screening system could be applied to the engineering of other industrially valuable strains.

Comparison of Acetaminophen Degradation by Laccases Immobilized by Two Different Methods via a Continuous Flow Microreactor Process Scheme.

The presence of micropollutants in wastewater is one of the most significant environmental challenges. Particularly, pollutants such as pharmaceutical residues present high stability and resistance to conventional physicochemical and biological degradation processes. Thus, we aimed at immobilizing a laccase enzyme by two different methods: the first one was based on producing alginate-laccase microcapsules through a droplet-based microfluidic system; the second one was based on covalent binding of the laccase molecules on aluminum oxide (Al2O3) pellets. Immobilization efficiencies approached 92.18% and 98.22%, respectively. Laccase immobilized by the two different methods were packed into continuous flow microreactors to evaluate the degradation efficiency of acetaminophen present in artificial wastewater. After cyclic operation, enzyme losses were found to be up to 75 µg/mL and 66 µg/mL per operation cycle, with a maximum acetaminophen removal of 72% and 15% and a retention time of 30 min, for the laccase-alginate microcapsules and laccase-Al2O3 pellets, respectively. The superior catalytic performance of laccase-alginate microcapsules was attributed to their higher porosity, which enhances retention and, consequently, increased the chances for more substrate–enzyme interactions. Finally, phytotoxicity of the treated water was lower than that of the untreated wastewater, especially when using laccase immobilized in alginate microcapsules. Future work will be dedicated to elucidating the routes for scaling-up and optimizing the process to assure profitability.

Droplet-based valveless microfluidic system for phage-display screening against spheroids.

In this study, we proposed a droplet-based valveless microfluidic system that has the necessary functions to perform the binding, washing, eluting, and collecting processes of phage-display screening against spheroids, which can be expected to present a similar repertoire and number of membrane proteins as in vivo. Although spheroids have much larger sizes than single cells, spheroids are difficult to manipulate through manual operation. The proposed microfluidic system actively controls the position and velocity of droplets using a camera, three air pumps, and three liquid pumps to perform the processes for phage-display screening. The cross section of the microchannel is large in width and height for the passage of spheroids. Valves that can close such a large cross-sectional microchannel are not readily available. Thus, we proposed valveless flow control using liquid pumps. In addition, the proposed microfluidic system involves complex flow channels with airflow subchannels to perform phage-display screening. For washing, nonspecific-binding phages remaining in the flow channels must be minimized. The proposed microfluidic system can perform selective blocking and flush washing. Selective blocking can prevent the airflow channels from becoming hydrophilic with blocking liquid, and flush washing can flush phages remaining in the flow channel. We experimentally verified the functions of the developed microfluidic device based on the proposed system.

Single cell transfection of human-induced pluripotent stem cells using a droplet-based microfluidic system.

Microfluidic tools have recently made possible many advances in biological and biomedical research. Research in fields such as physics, engineering, chemistry and biology have combined to produce innovation in microfluidics which has positively impacted diverse areas such as nucleotide sequencing, functional genomics, single-cell studies, single molecules assays and biomedical diagnostics. Among these areas, regenerative medicine and stem cells have benefited from microfluidics since these tools have had a profound impact on their applications. In this study, we present a high-performance droplet-based system for transfecting individual human-induced pluripotent stem cells. We will demonstrate that this system has great efficiency in single cells and captured droplets, like other microfluidic methods but with lower cost. Moreover, this microfluidic approach can be associated with the PiggyBac transposase-based system to increase its transfection efficiency. Our results provide a starting point for subsequent applications in more complex transfection systems, single-cell differentiation interactions, cell subpopulations and cell therapy, among other potential applications.

Generation of droplets with adjustable chemical concentrations based on fixed potential induced-charge electro-osmosis

The effective control of the sample concentration within droplets is essential in a broad range of assays in chemistry and biochemistry. Here we provide an electrical method for producing batches of aqueous droplets with various chemical concentrations by exploiting fixed-potential induced-charge electroosmosis (ICEO) flow around a bipolar electrode. By applying an AC electric signal to the bipolar electrode and changing the zeta potential on it, the bipolar electrode acts as a gate electrode for generating asymmetric ICEO flow. The ICEO flow induced transverse vortexes interact with two parallel laminar streams with different chemical compositions. Controlled mixing of the aqueous solutions can be achieved by adjusting the shape and size of the asymmetric vortexes via altering the electric signal applied to the gate electrode. The mixed streams are split at a bifurcation, and one of the streams with a desired controlled concentration is pumped into a flow-focusing geometry to generate droplets with adjustable chemical concentrations. The in-droplet concentration increases in the range of 0.412-1.404 mM, as the applied voltage increases in the range of 0-70 mV at 15 kHz. This approach offers a promising method for on-chip control of chemical concentrations within droplets without labor-intensive dilutions while minimizing the sample consumption, showing great potential for next generation droplet-based applications.

Microfluidic encapsulation of supramolecular optical chemosensors for high-throughput analysis and screening

The binding fluorescence dye by supramolecular interactions to form optical chemosensors is a powerful sensing method allowing ultra-high selectivity for the target analyte and fast response of the detection. While notable success has been made to develop various supramolecular optical chemosensors, there is little effects contributed to the high throughput assays. Here we introduce a droplet-based microfluidic detection and screening system using supramolecular optical chemosensors to measure the enzyme activity and elucidate their prospects for high throughput screening. The supramolecular optical chemosensors derives from cucurbit[8]uril (CB[8])-mediated host-guest chemistry for macrocycle-dye complex formation. The encapsulation of CB[8]-dye in millions of picolitre microdroplets provides an efficient platform for handing a large number of parallel experiments, allowing for shorter processing time and reducing the needs for analyte volume. Further, the supramolecular optical chemosensors in microdroplets are used in measuring of the biocatalysis efficiency of enzyme immobilized hydrogel microparticles (HMPs), which also has been applied for high throughput screening. This label-free and robust approach of supramolecular optical chemosensors in microdroplets platform could be transferable to single-cell analysis and rapid selection of cells.