Cell Sorting Applications Research Articles

Field‐free, sheathless cell focusing in exponentially expanding hydrophoretic channels for microflow cytometry

Cell focusing into a narrow stream is an essential step prior to counting and sorting cells in microfluidic devices for flow cytometry and cell sorting applications. Hydrodynamic focusing techniques, however, rely on the need for large volumes of sheath liquid and complex mechanical setup for flow control, preventing miniaturization of the systems. Although microfluidic methods based on active or passive particle control offer sheathless and efficient focusing, they often accompany fabrication complexities or bulky external setups, and operate in a certain range of flow rates. We present here a microfluidic device to focus cells into a narrow stream. The device employs hydrophoresis to guide cells by locally patterned slanted grooves, and channel expansion to improve focusing efficiency and produce a narrow stream of cells. This device principle allows easy improvement of focusing efficiency by adding more expansion steps. Adjusting channel expansion also ensures successful cell focusing without defocusing by inertial effects even at high Reynolds numbers. Using this device, we successfully produced a narrow stream of cells having size variation of >11% in a coefficient of variation (CV), achieving a narrow cell stream with a focusing variation below CV of 3.0%.

Quantitative analysis of spherical microbubble cavity array formation in thermally cured polydimethylsiloxane for use in cell sorting applications

Microbubbles are spherical cavities formed in thermally cured polydimethylsiloxane (PDMS) using the gas expansion molding technique. Microbubble cavity arrays are generated by casting PDMS over a silicon wafer mold containing arrays of deep etched pits. To be useful in various high throughput cell culture and sorting applications it is imperative that uniform micron-sized cavities can be formed over large areas (in(2)). This paper provides an in-depth quantitative analysis of the fabrication parameters that effect the microbubble cavity formation efficiency and size. These include (1) the hydrophobic coating of the mold, (2) the mold pit dimensions, (3) the spatial arrangement of the pit openings, (4) the curing temperature of PDMS pre-polymer, (5) PDMS thickness, and (6) the presence and composition of residual gas in the PDMS pre-polymer mixture. Results suggest that the principles of heterogeneous nucleation and gas diffusion govern microbubble cavity formation, and that surface tension prevents detachment of the vapor bubble that forms in the PDMS over the pit. Paramerters are defined that enable the fabrication of large format arrays with uniform cavity size over 6 in(2) with a coefficient-of-variation <10 %. The architecture of the microbubble cavity is uniquely advantageous for cell culture. Large format arrays provide a highly versatile system that can be adapted for use in various high-throughput cell sorting applications. Herein, we demonstrate the use of microbubble cavity arrays to dissect the cellular heterogeneity that exists in a tumorigenic cutaneous squamous cell carcinoma cell line at the single cell level.

Size-regulated group separation of CoFe2O4 nanoparticles using centrifuge and their magnetic resonance contrast properties

Magnetic nanoparticle (MNP)-based magnetic resonance imaging (MRI) contrast agents (CAs) have been the subject of extensive research over recent decades. The particle size of MNPs varies widely and is known to influence their physicochemical and pharmacokinetic properties. There are two commonly used methods for synthesizing MNPs, organometallic and aqueous solution coprecipitation. The former has the advantage of being able to control the particle size more effectively; however, the resulting particles require a hydrophilic coating in order to be rendered water soluble. The MNPs produced using the latter method are intrinsically water soluble, but they have a relatively wide particle size distribution. Size-controlled water-soluble MNPs have great potential as MRI CAs and in cell sorting and labeling applications. In the present study, we synthesized CoFe2O4 MNPs using an aqueous solution coprecipitation method. The MNPs were subsequently separated into four groups depending on size, by the use of centrifugation at different speeds. The crystal shapes and size distributions of the particles in the four groups were measured and confirmed by transmission electron microscopy and dynamic light scattering. Using X-ray diffraction analysis, the MNPs were found to have an inverse spinel structure. Four MNP groups with well-selected semi-Gaussian-like diameter distributions were obtained, with measured T2 relaxivities (r2) at 4.7 T and room temperature in the range of 60 to 300 mM−1s−1, depending on the particle size. This size regulation method has great promise for applications that require homogeneous-sized MNPs made by an aqueous solution coprecipitation method. Any group of the CoFe2O4 MNPs could be used as initial base cores of MRI T2 CAs, with almost unique T2 relaxivity owing to size regulation. The methodology reported here opens up many possibilities for biosensing applications and disease diagnosis.PACS75.75.Fk, 78.67.Bf, 61.46.Df

Glucosamine‐Conjugated Nanoparticles for the Separation of Insulin‐Secreting Beta Cells

A small molecule, glucosamine, is used as targeting moiety for insulin-secreting beta cell separation in artificial cell mixtures and tissue samples. The specificity of glucosamine allows it to be used in cell sorting applications. In addition, a thrombin-specific cleavable peptide was used as an intermediary to release nanoparticles from cell surfaces to facilitate cell attachment and proliferation.

Modulation of aspect ratio for complete separation in an inertial microfluidic channel

Inertial microfluidics has been attracting considerable interest in recent years due to immensely promising applications in cell separations and sorting. Despite the intense attention, the moderate efficiencies and low purity of the reported devices have hindered their widespread acceptance. In this work, we report on a simple inertial microfluidic system with high efficiency (>99%) and purity (>90%). Our system builds on the concept of two-stage inertial migration which permits precise prediction of particle or cell position within the microchannel. Our design manipulates the inertial equilibrium positions by modulating channel aspect ratio to achieve a complete separation. Here, we successfully demonstrate a complete separation of particles and isolation of rare cells in blood spiked with human prostate epithelial tumor (HPET) cells. Based on the planar structure, large separation spacing and predictable focusing, we envision promising applications and easy integration of our system with existing lab-on-a-chip systems for cell separations.

Preparation and characterization of CS–Fe3O4@ZnS:Mn magnetic-fluorescent nanoparticles in aqueous media

Chitosan (CS) coated magnetic-fluorescent nanoparticles (CS–Fe3O4@ZnS:Mn) were synthesized in aqueous media under ambient pressure and characterized by X-ray diffraction, UV–vis absorption, fluorescence emission, transmission electron microscope and energy dispersion spectrum. The effects of experimental conditions on the fluorescence properties of CS–Fe3O4@ZnS:Mn were studied. It was found that the chitosan coating on the surface of Fe3O4 could effectively suppress the interaction between Fe3O4 and ZnS:Mn, as well as providing appropriate functional groups for the synthesis and modification of ZnS:Mn semiconductor nanocrystals. The obtained multifunctional nanoparticles are promising candidates for applications in simultaneous biolabeling, imaging, cell sorting and separation.

Precise and programmable manipulation of microbubbles by two-dimensional standing surface acoustic waves

A microfluidic device is developed to transport microbubbles (MBs) along a desired trajectory in fluid by introducing the phase-shift to a planar standing surface acoustic wave (SSAW). The radiation force of SSAW due to the acoustic pressure gradient modulated by a phase-shift can move MBs to anticipated potential wells in a programmable manner. The resolution of the transportation is approximately 2.2 µm and the estimated radiation force on the MBs is on the order of 10−9 N. This device can be used for manipulation of bioparticles, cell sorting, tissue engineering, and other biomedical applications.

LewisX: A neural stem cell specific glycan?

LewisX (LeX) detecting antibodies are routinely used for cell sorting of neural stem- and progenitor cells (NSPCs). Applications include the enrichment of NSPCs after neural differentiation of human induced pluripotent- or embryonic stem cells, as well as their direct isolation from mouse neural tissue. Nevertheless, only little is known about the role of LeX in the central nervous system. Here we review the current knowledge on LeX-containing glycans expressed by neural stem cells and their progeny. New LeX-carrier proteins and ligands have recently been identified which reveal further insights into the potential function(s) of LeX-glycans. Moreover, evidence accumulates that individual LeX detecting antibody clones vary in their suitability as neural stem cell specific biomarker. Each antibody clone detects a unique LeX-containing glycan epitope. This allows a versatile utilization of anti-LeX antibodies that goes beyond neural stem cell sorting applications.

Systematic investigation of the aqueous processing of CdSe quantum dots and CuS nanoparticles for potential bio-medical applications.

ABSTRACTSemiconductor quantum dots are considered very promising candidates for bio-imaging and diagnosis applications because of their tunable optical properties and good optical stability in aqueous phase. Any practical application of these materials will rely on the viability of their simple and direct synthesis in aqueous phase with no need for toxic and unstable organic media. The optical properties of CdSe quantum dots and CuS nanoparticles are desirable in bio-imaging and cell sorting applications because of their tunable photoluminescence at the visible range. The present work addresses the synthesis of CdSe quantum dots and CuS nanoparticles via an optimized, simple and scalable aqueous processing route at low temperatures. The tunability of the optical properties was achieved by a suitable control of the citrate/Cd mole ratio, temperature of synthesis (20-90°C) and reaction time (0-1 hour). In the case of CuS, the strong plasmonic absorption offers the opportunity to investigate this material as a photothermal coupling agent for photothermal therapy. The intensity of the plasmonic absorption was enhanced by selecting an appropriate sulfide precursor (Na2S, Thioglycolic acid), temperature of synthesis (90-120°C) and reaction time. Nanocrystals were characterized by x-ray diffraction, UV-VIS, photoluminescence (PL) spectroscopy techniques and electron microscopy. The effects of the synthesis conditions on the crystal size and the corresponding functional properties of synthesized quantum dots are presented and discussed.

Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ∼ 5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ∼ 10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

A self-immobilizing and fluorogenic unnatural amino acid that mimics phosphotyrosine

Synthesis of the first self-immobilizing, fluorogenic unnatural amino acid that mimics phosphotyrosine (pTyr) is reported. By using solid-phase peptide synthesis, it was subsequently incorporated into peptide-based probes which found applications in bioimaging and fluorescence-activated cell sorting (FACS).

Modeling cell entry into a micro-channel

Cell entry into a micro-channel has potential applications in cell sorting and cancer diagnostics. In this paper, we numerically model breast cancer cell entry into a constricted micro-channel. Our results indicate that the cell velocity decreases during entry and increases after entry, an observation in agreement with experiments. We found that the cell entry time depend strongly on the cortical stiffness and is minimum at some critical cortical elasticity. In addition, we found that for the same entry time, a stiff nucleus is displaced toward the cell front, whereas a viscous nucleus is displaced toward the rear. In comparison, the nucleus is less sensitive to the viscosity of the cytoplasm. These observations suggest that specific intra-cellular properties can be deduced non-invasively during cell entry, through the inspection of the nucleus using suitable illumination techniques, such as fluorescent labeling.

A multicomponent recognition and separation system established via fluorescent, magnetic, dualencoded multifunctional bioprobes

Accurate and rapid recognition and separation of multiple types of biological targets such as molecules, cells, bacteria or viruses from complex sample mixtures is of great importance for a wide range of diagnostic and therapeutic strategies. To achieve this goal, a set of fluorescent, magnetic, dual-encoded multifunctional bioprobes has been constructed by co-embedding different-sized quantum dots and varying amounts of γ-Fe 2O 3 magnetic nanoparticles into swollen poly(styrene/acrylamide) copolymer nanospheres. The dual-encoded bioprobes, which possessed different photoluminescent property and magnetic susceptibility, were proven to be capable of simultaneously recognizing and separating multiple components from a complex sample when three kinds of lectins were used as the targets. The lectins were separated with high efficiency and kept their bioactivity during the process. Compared to the conventional batchwise separation, this method does not require a large number of sequential reaction steps, which is economical of time and can be very reagent-saving. By combining the multiplexing capability of quantum dots with the superparamagnetic properties of iron oxide nanoparticles, this dual-encoded technique is expected to open new opportunities in high-throughput and multiplex bioassays, such as cell sorting, proteomical and genomical applications, drug screening etc.

Small but mighty: How the MACS®‐technology based on nanosized superparamagnetic particles has helped to analyze the immune system within the last 20 years

Today, magnetic cell sorting and flow cytometric cell sorting both are state-of-the-art technologies, with a plethora of applications in biology and biomedicine. Both technologies have their stand-alone applications, but they also perfectly complement each other, in particular for the analysis and isolation of fragile and rare cells. The technological evolution from simple magnets and steel wool separation columns to sophisticated instrumentation and automated procedures has paved the way for magnetic cell separation in the scientific community. This review will focus on applications of magnetic cell sorting with commercially available paramagnetic MACS(R)-nanoparticles (Miltenyi Biotech, Bergisch-Gladbach, Germany) that have allowed unprecedented approaches to the exploration of the immune system in basal and clinical research of humans and rodents. Thus, during the last 20 years this technology has been continuously developed so that the widest array of leukocyte subsets, stem cells, and connective tissue cells can be addressed by MACS(R)-conjugated antibodies directed against cell-specific surface antigens or secreted cytokines for research and clinical applications.

Complex display of putative tumor stem cell markers in the NCI60 tumor cell line panel.

Tumor stem cells or cancer initiating cells (CICs) are single tumor cells that can regenerate a tumor or a metastasis. The identification and isolation of CICs remain challenging, and a variety of putative CIC markers have been described. We hypothesized that cell lines of the NCI60 panel contain CICs and express putative CIC markers. We investigated expression of putative CIC surface markers (CD15, CD24, CD44, CD133, CD166, CD326, PgP) and the activity of aldehyde dehydrogenase in the NCI60 panel singly and in combination by six-color fluorescence-activated cell sorting analysis. All investigated markers were expressed in cell lines of the NCI60 panel. Expression levels of individual markers varied widely across the 60 cell lines, and neither single marker expression nor simple combinations nor co-expression patterns correlated with the colony-formation capacity of cell lines. Rather, marker expression patterns correlated with tumor types in multidimensional analysis. Whereas some expression patterns correlated with tumor entities such as basal breast cancer, other expression patterns occurred across different tumor types and largely related to expression of a more mesenchymal phenotype in individual breast, lung, renal, and melanoma cell lines. Our data for the first time demonstrate that tumor cell lines display CIC markers in a complex pattern that relates to the tumor type. The complexity and tumor type specificity of marker display creates challenges for the application of cell sorting and other approaches to isolation of putative tumor stem cell populations and suggests that therapeutic targeting strategies will need to take this into account.

DeNAno: Selectable deoxyribonucleic acid nanoparticle libraries

DNA nanoparticles of approximately 250 nm were produced by rolling circle replication of circular oligonucleotide templates which results in highly condensed DNA particulates presenting concatemeric sequence repeats. Using templates containing randomized sequences, high diversity libraries of particles were produced. A biopanning method that iteratively screens for binding and uses PCR to recover selected particles was developed. The initial application of this technique was the selection of particles that bound to human dendritic cells (DCs). Following nine rounds of selection the population of particles was enriched for particles that bound DCs, and individual binding clones were isolated and confirmed by flow cytometry and microscopy. This process, which we have termed DeNAno, represents a novel library technology akin to aptamer and phage display, but unique in that the selected moiety is a multivalent nanoparticle whose activity is intrinsic to its sequence. Cell targeted DNA nanoparticles may have applications in cell imaging, cell sorting, and cancer therapy.

Magnetic–Fluorescent Colloidal Nanobeads: Preparation and Exploitation in Cell Separation Experiments

Nanostructures displaying fluorescence and magnetic properties at the same time are potentially useful for achieving simultaneous bio-separation and bio-sensing (e.g., magnetic separation coupled with multiplexing optical detection of different tumour cell populations). Spherical nanobeads that display both fluorescent and magnetic features are reported; they are fabricated by grafting fluorescent oligothiophene molecules to an amphiphilic polymer that is then used to enwrap iron oxide nanoparticles, which acts as the magnetic domain. By tuning experimental conditions, control over the number of magnetic nanoparticles per bead and over the bead diameter (30-400 nm) was achieved. A cell separation efficiency of the level required for cell sorting applications is also reported.

Microfabrication of Bubbular Cavities in PDMS for Cell Sorting and Microcell Culture Applications

We describe a novel technique, low surface energy Gas Expansion Molding (GEM), to fabricate microbubble arrays in polydimethylsiloxane (PDMS) which are incorporated into parallel plate flow chambers and tested in cell sorting and microcell culture applications. This architecture confers several operational advantages that distinguish this technology approach from currently used methods. Herein we describe the GEM process and the parameters that are used to control microbubble formation and a Vacuum-Assisted Coating (VAC) process developed to selectively and spatially alter the PDMS surface chemistry in the wells and on the microchannel surface. We describe results from microflow image visualization studies conducted to investigate fluid streams above and within microbubble wells and conclude with a discussion of cell culture studies in PDMS.

Designing Electroactive Biointerface for Spatiotemporal Control of Cell Attachment and Release

In this paper, we demonstrate the use of individually addressable microelectrodes for cell sorting and cell micropatterning applications. Microelectrodes were modified with cell adhesive or non-adhesive molecules and then electrically stimulated to selectively adsorb or desorb proteins and/or mammalian cells. The switching of the surface properties was achieved by the electrochemical desorption of protein-functionalized thiols and poly(ethylene glycol) PEG silane from gold and indium tin oxide (ITO) electrodes respectively. The thiol surfaces were modified with anti-CD4 antibodies and used to capture T-cells. Upon electrical activation of the microelectrodes, both the antibodies and the T-cells were removed from the specific locations on the substrate. In addition, ITO electrodes were modified with cell- resistive PEG silane which was later electrochemically desorbed to make the surface adhesive to proteins or cells. This technique was employed to pattern two different cell types on the same substrate.

Double-layer silica core-shell nanospheres with superparamagnetic and fluorescent functionalities

Superparamagnetic nanocrystals and QDs have attracted much attention due to their great potential in biomedical applications. In this Letter, the interest arises from the possibility of positioning magnetic and optical particles at different layers in a silica core-shell structure. In this double layered core-shell structure, QDs are sandwiched between the core and shell silica layers, whereas Fe 3O 4 nanoparticles are entrapped in the core. This structure was designed with the idea of preventing direct contact between QDs and Fe 3O 4 nanoparticles, thus minimizing the ‘quenching effect’ and achieving high quality magnetic-fluorescent dual functionality. The obtained double-layer silica nanospheres are promising candidates for applications in simultaneous biolabeling, imaging, cell sorting and separation.