Separation of Lung Cancer Cells From Mixed Cell Samples Using Aptamer-Modified Magnetic Beads and Permalloy Micromagnets.

This study involved the design and fabrication of a microfluidic chip integrated with permalloy micromagnets. The device was used with aptamer-modified magnetic beads (MBs) of various sizes to successfully separate lung cancer cells from a mixture of other cells. The overall separation efficiency was evaluated based on the ratios of cells in the different outlets and inlets of the chip. The results showed efficiencies ranging from 43.4% to 50.2% for MB sizes between 1.36 and 4.50 µm. Interestingly, efficiency slightly decreased as the size of the MBs increased, contrary to predictions. Further examination revealed that larger MBs exerted gravitational force on the cell-bound MBs at low flow rates, causing the targets to settle before reaching the main microchannel region. This was attributed to fluidic resistance caused by a size mismatch between the inlet tube and the microfluidic conduit. An increase in cell accumulation at the inlet was observed with larger MB sizes due to gravity. Therefore, the definition of effective separation efficiency was revised to exclude the effect of cell accumulation at the inlet. Effective separation efficiencies were found to be 71.6%, 76.4%, and 79.4% for MB sizes of 1.36, 3.00, and 4.50 µm, respectively. The study concluded that larger MBs interacted more with the magnetic force, resulting in better separation. However, cells with smaller MBs were more likely to evade the magnetic force. The investigation provides valuable insights into isolating lung cancer cells using this method, with the potential for clinical application in cancer diagnosis and treatment.

Protocol for muscle fiber type and cross-sectional area analysis in cryosections of whole lower mouse hindlimbs.

We outline a protocol to visualize all mouse lower hindlimb skeletal muscles simultaneously. We describe procedures for orientating the whole lower hindlimb in gum tragacanth prior to freezing, simplifying the proceeding experimental steps, and enhancing the comprehensiveness of characterizations. We then detail steps for quantifying muscle fiber size and fiber type characteristics in a single cryosection using histochemistry and immunofluorescence. This protocol can be applied to histological and (immuno)histochemical evaluations such as muscle regeneration, fibrosis, enzymatic activity, and glycogen content.

Ultra-Stretchable Microfluidic Devices for Optimizing Particle Manipulation in Viscoelastic Fluids.

Viscoelastic microfluidics leverages the unique properties of non-Newtonian fluids to manipulate and separate micro- or submicron particles. Channel geometry and dimension are crucial for device performance. Traditional rigid microfluidic devices require numerous iterations of fabrication and testing to optimize these parameters, which is time-consuming and costly. In this work, we developed a flexible microfluidic device using ultra-stretchable and biocompatible Flexdym material to overcome this issue. Our device allows for simultaneous modification of channel dimensions by external stretching. We fabricated a stretchable device with an initial square microchannel (30 µm × 30 µm), and the channel aspect ratio can be adjusted from 1 to 5 by external stretching. Next, the effects of aspect ratio, particle size, flow rate, and poly(ethylene oxide) (PEO) concentration that make the fluid viscoelastic on particle migration were investigated. Finally, we demonstrated the feasibility of our approach by testing channels with an aspect ratio of 3 for the separation of both particles and cells.

RBC subpopulations in RCCs affected by donor factors.

Understanding red blood cell (RBC) subpopulations is crucial for comprehending donor variability and enhancing transfusion outcomes. This review highlights the significance of RBC subpopulations, focusing on the properties of biologically young and old RBCs and underscores how donor variability impacts transfusion outcomes. The role of senescent RBCs in adverse transfusion reactions and the emerging significance of circulating erythroid cells (CECs) is discussed. RBC aging and the role of oxidative stress and aging mechanisms is highlighted. Changes in RBC flexibility, calcium homeostasis, band 3 protein modifications, membrane microvesiculation, 2,3-diphosphoglycerate (2,3-DPG) levels, and immunological markers like CD47 and CD55 contribute to RBC clearance and erythrophagocytosis. Also, methods of characterizing / separating of biologically young and old RBC subpopulations is introduced. This review emphasizes the importance of RBC subpopulations in understanding donor variability and improving transfusion outcomes.

µREACT: A microfluidic system for rapid evaluation of trans-kingdom interactions.

Trans-kingdom interactions between cells play pivotal roles in shaping intricate ecological and biological networks. However, our grasp of these interactions remains incomplete. Specifically, the vast phylogenetic spectrum of microorganisms capable of interacting with a given host cell type remains obscure, primarily due to the absence of efficient, high-throughput, single-cell resolution systems that can rapidly decipher these interactions. Here, we introduce µREACT (Microfluidic system for Rapid Evaluation of bacterial Adherence and Communication in Trans-kingdom interactions), a microfluidic system designed to analyze interkingdom interactions. µREACT not only unveiled both recognized and previously unknown interactions but also enabled their detailed characterization. The system features the use of microfluidic dielectrophoretic separation of bacteria that adhere to host cells at single-cell (digital) resolution, and enabled the sorting of 107 adherent microorganisms per hour, representing a comparable throughput to conventional flow cytometry systems, but without requiring any labeling. The analysis of soil microbial samples using µREACT revealed several bacterial species previously known to have high adherence to mammalian host cells, as well as new interactions involving strains that displayed hallmarks of emerging endosymbiosis. Taken together, µREACT serves as a formidable tool for identifying and characterizing webs of interkingdom interactions. Its implications extend beyond discovery of such interactions, where it has the potential to provide new insights into fundamental mechanisms driving ecosystem dynamics and biological processes.

Effective Boundary Correction for Deterministic Lateral Displacement Microchannels to Improve Cell Separation: A Numerical and Experimental Study.

Particle separation and sorting techniques based on microfluidics have found extensive applications and are increasingly gaining prominence. This research presents the design and fabrication of a microfluidic device for separating cells using deterministic lateral displacement (DLD), enabling accuracy and continuity while being size-based. Nevertheless, it remains demanding, to completely reverse the detrimental effects of the boundaries that disturb the fluidic flow in the channel and reduce particle separation efficiency. This study introduces a novel approach to enhance the boundary structure of channels. By using this design, separation efficiency is boosted, and the fluid behavior around the walls is improved. The boundary correction (BC) enhances the operation of the microchannel and is very effective in microchannels. With boundary correction, the device exhibited improved separation efficiencies, but in its absence, separation efficiencies dropped. The collected microscopic images of the isolation of prostate cancer cell lines and red blood cells revealed promising outcomes. The efficiency of circulating tumor cell (CTC) throughput in the microfluidic channel, quantified as the ratio or proportion of tumor cells exiting the channel to cells entering it, exceeds 93%. Moreover, the efficiency of CTC isolation, expressed as the proportion of tumor cells from the upper outlet of the microfluidic channel to all cells, is over 89%. Additionally, the efficiency of red blood cell isolation, evaluated as the ratio of red blood cells from the lower outlet of the microfluidic channel to all cells, surpasses 77%. While using the same DLD separator without boundary correction reduced the separation efficiency by around 5%.

Recent Advances in Dielectrophoretic Manipulation and Separation of Microparticles and Biological Cells.

Dielectrophoresis (DEP) is an advanced microfluidic manipulation technique that is based on the interaction of polarized particles with the spatial gradient of a non-uniform electric field to achieve non-contact and highly selective manipulation of particles. In recent years, DEP has made remarkable progress in the field of microfluidics, and it has gradually transitioned from laboratory-scale research to high-throughput manipulation in practical applications. This paper reviews the recent advances in dielectric manipulation and separation of microparticles and biological cells and discusses in detail the design of chip structures for the two main methods, direct current dielectrophoresis (DC-DEP) and alternating current dielectrophoresis (AC-DEP). The working principles, technical implementation details, and other improved designs of electrode-based and insulator-based chips are summarized. Functional customization of DEP systems with specific capabilities, including separation, capture, purification, aggregation, and assembly of particles and cells, is then performed. The aim of this paper is to provide new ideas for the design of novel DEP micro/nano platforms with the desired high throughput for further development in practical applications.

A Novel Size-Based Centrifugal Microfluidic Design to Enrich and Magnetically Isolate Circulating Tumor Cells from Blood Cells through Biocompatible Magnetite-Arginine Nanoparticles.

This paper presents a novel centrifugal microfluidic approach (so-called lab-on-a-CD) for magnetic circulating tumor cell (CTC) separation from the other healthy cells according to their physical and acquired chemical properties. This study enhances the efficiency of CTC isolation, crucial for cancer diagnosis, prognosis, and therapy. CTCs are cells that break away from primary tumors and travel through the bloodstream; however, isolating CTCs from blood cells is difficult due to their low numbers and diverse characteristics. The proposed microfluidic device consists of two sections: a passive section that uses inertial force and bifurcation law to sort CTCs into different streamlines based on size and shape and an active section that uses magnetic forces along with Dean drag, inertial, and centrifugal forces to capture magnetized CTCs at the downstream of the microchannel. The authors designed, simulated, fabricated, and tested the device with cultured cancer cells and human cells. We also proposed a cost-effective method to mitigate the surface roughness and smooth surfaces created by micromachines and a unique pulsatile technique for flow control to improve separation efficiency. The possibility of a device with fewer layers to improve the leaks and alignment concerns was also demonstrated. The fabricated device could quickly handle a large volume of samples and achieve a high separation efficiency (93%) of CTCs at an optimal angular velocity. The paper shows the feasibility and potential of the proposed centrifugal microfluidic approach to satisfy the pumping, cell sorting, and separating functions for CTC separation.

Spiral microchannels with concave cross-section for enhanced cancer cell inertial separation.

Inertial microfluidic technologies have proven effective for particle focusing and separation in many microchannels, typically the channels with the rectangular and trapezoidal shapes. To advance particle focusing in complex channels, we propose a spiral channel combining rectangular and concave cross-sections for high-resolution particle and cell focusing and separation. Numerical simulations were conducted to illustrate the effects of channel geometry on secondary flow distribution and particle focusing positions. The simulation shows the concave cross-section generates two asymmetrical Dean vortices skewing towards the inner and outer channel walls, resulting to stronger flow velocity magnitudes near the walls than the channel center. Consequently, larger particles focus near the inner wall, while smaller particles are trapped closer to the outer wall under the influence of the stronger velocity magnitude near the walls. A microfluidic chip with the proposed channel geometry, along with a traditional rectangular channel, was fabricated by 3D printing and PDMS casting. Fluorescent microbeads were used to investigate inertial focusing and separation behaviors in the microfluidic chips. Experimental results show that the concave channel facilitates particle focusing or trapping much closer to the walls than the traditional rectangular channel, achieving better separation resolution. Finally, the proposed channel was applied to separate lung cancer A549 cells from human blood, achieving a cancer cell recovery rate of ~ 84.78% (enrichment ratio over 820-fold) and a blood cell rejection rate of ~ 99.88%. This innovative channel design in inertial microfluidics offers new insights for enhanced particle focusing and holds significant promise for cell manipulation with improved separation resolution.

Protocol for optimized nasal mucosa sample processing to obtain high-quality scRNA-seq and scATAC-seq data.

Examining nasal mucosa samples is crucial for nasal cavity disease research and diagnosis. Simultaneously obtaining high-quality data for single-cell transcriptomics (single-cell RNA sequencing [scRNA-seq]) and epigenomics (single-cell assay for transposase-accessible chromatin using sequencing [scATAC-seq]) of nasal mucosa tissues is challenging. Here, we present a protocol for processing human nasal mucosa samples to obtain data for both scRNA-seq and scATAC-seq. We describe steps for extracting human nasal mucosa tissue, mechanical and enzymatic dissociation, lysis of red blood cells, and a viability assay. We then detail procedures for library preparation and quality control.

Protocol for isolating leukemia-derived extracellular vesicles from the spleen of preclinical models of leukemia using ultracentrifugation.

Here, we present a protocol for the direct isolation of small extracellular vesicles (sEVs) from the spleen of preclinical murine models of leukemia using ultracentrifugation. We describe steps for tissue collection, sample preparation, ultracentrifugation-based isolation, and sEV characterization. This protocol allows for efficient enrichment of both leukemia and its microenvironment-derived sEV (LME-sEV), providing a valuable tool for studying their composition and functional roles. Potential applications include investigating the role of sEV in leukemia progression and identifying biomarkers. For complete details on the use and execution of this protocol, please refer to Gargiulo et al.1.

Protocol for the isolation and purification of endoplasmic reticulum-plasma membrane junctions from the mouse brain.

Dynamic communication between intracellular organelles often takes place at specialized membrane contact sites that form between their membranes. Here we detail a procedure for the purification of endoplasmic reticulum-plasma membrane (ER-PM) junctions from the mouse brain. We describe steps for homogenizing isolated brain hemispheres and sequential centrifugation to remove the nuclear fraction from the other membrane fractions. We then detail procedures for separating the resulting crude membrane fractions by sucrose density gradients and purifying into their respective pellets. For complete details on the use and execution of this protocol, please refer to Weesner et al.1.

Protocol for simultaneous isolation of high-quality and high-quantity cardiomyocytes and non-myocyte cells from adult rat hearts.

Isolating high-quality different cell types is a powerful approach for understanding cellular compositions and features in the heart, but it is challenging. The available protocols typically focus on isolating one or two cell types. Here, we present a protocol to simultaneously isolate high-quality and high-quantity cardiomyocytes and non-myocyte cells, including immune cells, from adult rat hearts. We describe steps for purifying cells using bovine serum albumin. We also detail procedures for viability analysis and cell type identification using fluorescence-activated cell sorting. For complete details on the use and execution of this protocol, please refer to Zhang et al.,1 Valkov et al.,2 Vang et al.,3 and Li et al.4.

Protocol for the derivation and alveolar type 2 differentiation of late-stage lung tip progenitors from the developing human lungs.

Molecular and cellular mechanisms of human lung alveolar development are poorly understood due to a lack of in vitro model systems. This protocol details the isolation, derivation, and genetic modification of lung tip epithelial progenitors from human fetal lungs. It includes steps for isolating distal lung epithelial cells, expanding tip progenitor organoids, culturing tip organoids in vitro, and differentiating them into alveolar type 2 cells. This will aid in understanding alveolar differentiation mechanisms and neonatal diseases. For complete details on the use and execution of this protocol, please refer to Lim et al.1.

Interplay of size, deformability, and device layout on cell transport in microfluidics.

Microfluidics have been widely used for cell sorting and capture. In this work, numerical simulations of cell transport in microfluidic devices were studied considering cell sizes, deformability, and five different device designs. Among these five designs, deterministic lateral displacement device (DLD) and hyperuniform device (HU) performed better in promoting cell-micropost collision due to the continuously shifted micropost positions as compared with regular grid, staggered, and hexagonal layout designs. However, the grid and the hexagonal layouts showed best in differentiating cells by their size dependent velocity due to the size exclusion effect for cell transport in clear and straight paths in the flow direction. A systematic study of the velocity differentiation under different dimensionless groups was performed showing that the velocity difference is dominated by the micropost separation distance perpendicular to the direction of flow. Microfluidic experiments also confirmed the velocity differentiation results. The study can provide guiding principles for microfluidic design.

The single RRM domain-containing protein SARP1 is required for establishment of the separation zone in Arabidopsis.

Abscission is the shedding of plant organs in response to developmental and environmental cues. Abscission involves cell separation between two neighboring cell types, residuum cells (RECs) and secession cells (SECs) in the floral abscission zone (AZ) in Arabidopsis thaliana. However, the regulatory mechanisms behind the spatial determination that governs cell separation are largely unknown. The class I KNOTTED-like homeobox (KNOX) transcription factor BREVIPEDICELLUS (BP) negatively regulates AZ cell size and number in Arabidopsis. To identify new players participating in abscission, we performed a genetic screen by activation tagging a weak complementation line of bp-3. We identified the mutant ebp1 (enhancer of BP1) displaying delayed floral organ abscission. The ebp1 mutant showed a concaved surface in SECs and abnormally stacked cells on the top of RECs, in contrast to the precisely separated surface in the wild-type. Molecular and histological analyses revealed that the transcriptional programming during cell differentiation in the AZ is compromised in ebp1. The SECs of ebp1 have acquired REC-like properties, including cuticle formation and superoxide production. We show that SEPARATION AFFECTING RNA-BINDING PROTEIN1 (SARP1) is upregulated in ebp1 and plays a role in the establishment of the cell separation layer during floral organ abscission in Arabidopsis.

The characteristics of autolysins associated with cell separation in Bacillus subtilis.

The peptidoglycan hydrolases responsible for the cell separation of Bacillus subtilis cells are collectively referred to as autolysins. However, the role of each autolysin in the cell separation of B. subtilis is not fully understood. In this study, we constructed a series of cell separation-associated autolysin deficient strains and strains overexpressing the transcription factors SlrR and SinR, and the morphological changes of these strains in liquid culture were observed. The results showed that the absence of D,L-endopeptidases CwlS and LytF only increased the cell chain length in the early exponential phase. The absence of D,L-endopeptidase LytE or N-acetylmuramyl-L-alanine amidase LytC can cause cells to form chains throughout the growth of B. subtilis, although the cell chain length was significantly shortened during the stationary phase. However, the absence of peptidoglycan N-acetylglucosaminidase LytD only caused minor defect in cell separation. Therefore, we concluded that LytE and LytC were the major autolysins that ensure the timely separation of B. subtilis daughter cells, whereas CwlS, LytF, and LytD were the minor autolysins. In addition, overexpression of the transcription factors SinR and SlrR in the cwlS lytF lytC lytE mutant enabled B. subtilis cells to form ultra-long chains in the vegetative phase, and its biomass level was basically the same as that of the wild type. This led to the conclusion that besides inhibiting the expression of lytC and lytF, the SinR-SlrR complex also has other potential mechanisms to inhibit cell separation.IMPORTANCEIn this study, the effects of CwlS, LytC, LytD, LytF, LytE, and SinR-SlrR complex on the cell separation of Bacillus subtilis at different growth phases were studied, and an ultra-long-chained B. subtilis strain was constructed. In microbial fermentation, due to its large cell size, this ultra-long-chained B. subtilis strain may be more likely to be precipitated or intercepted during the removal of bacterial process with centrifugation and membrane filtration as the main methods, which is crucial to improve the purity of the product.

A protocol for measuring the activity of protein kinase C-delta in murine bone-marrow-derived dendritic cells.

Protein kinase C-δ (PKC-δ) is a key enzyme controlling growth, differentiation, and apoptosis in various cells, including immune cells. Here, we present a protocol to determine PKC-δ activation in response to increased membrane-bound diacylglycerol or phorbol-12-myristate-13-acetate treatment in murine bone-marrow-derived dendritic cells. We describe steps for dendritic cell differentiation, the isolation of plasma membrane lipids, and the quantification of diacylglycerol. We then detail procedures for measuring PKC-δ kinase activity by in vitro assay, indirect immunofluorescence, and western blotting experiments. For complete details on the use and execution of this protocol, please refer to Parsons et al.1.

Proposal and performance evaluation of a new parallel plate continuous cell separation device using dielectrophoresis.

Along with the rapid development of cellular biological research in recent years, there has been an urgent need for a high-speed, high-precision method of separating target cells from a highly heterogeneous cell population. Among the various cell separation technologies proposed so far, dielectrophoresis (DEP)-based approaches have shown particular promise because they are noninvasive to cells. We have developed a new DEP-based device to separate large numbers of live and dead cells of the human mammary cell line MCF10A. In this study, we validated the separation performance of this device. The results showed the successful separation of a higher percentage of cells than in previous studies, with a separation efficiency higher than 90%. In the past, there have been no confirmed cases in which a separation rate of over 90% and high-speed processing of a large number of cells were simultaneously achieved. It was shown that the proposed device can process large numbers of cells at high speed and with high accuracy.

Protocol for qualitative analysis of lysosome immunoprecipitation from patient-derived glioblastoma stem-like cells.

Lysosomes are critical for the sustenance of glioblastoma stem-like cells (GSCs) properties. We present a protocol to enrich and purify lysosomes from patient-derived GSCs in culture. We describe the steps required to stably express a tagged lysosomal protein in GSCs, mechanically lyse cells, magnetically immunopurify lysosomes, and qualitatively assess these organelles. We then detail the procedure for retrieving intact and purified lysosomes from GSCs. We also specify cell culture conditions, storage procedures, and sample preparation for immunoblotting. For complete details on the use and execution of this protocol, please refer to Maghe et al.1.