RESUMO
The hydraulic permeability of the lipid bilayer membrane of a single cell, a very important parameter in biological and medical fields, has been attracting increasing attention. To date, methods developed to determine this permeability are either operation-complicated or time-consuming. Therefore, we developed a chip for automatically and rapidly determining the permeability of cells that integrates microfluidics and cell impedance analysis. The chip is designed to automatically identify a single cell, capture the cell, and record the volume change in that cell. We confirmed the abilities of single-cell identification and capture with the upper and lower voltage thresholds determined, validated the performance of the differential electrode design for accurate cell volume measurements, deduced the extracellular osmotic pressure change in the presence of a hypertonic solution according to fluorescence intensity, and demonstrated the single-cell volume change recorded by the chip. Then, the accuracy of the permeability determined with the chip was verified using HeLa cells. Finally, the permeability of human-induced pluripotent stem cells (hiPSCs) was determined to be 0.47 ± 0.03 µm/atm/min. Using the chip, the permeability can be determined within 5 min. This study provides insights for the new design of an automatic single-cell identification and capture chip for single cell-related studies. Graphical abstract.
Assuntos
Permeabilidade da Membrana Celular , Tamanho Celular , Dispositivos Lab-On-A-Chip , Análise de Célula Única/instrumentação , Linhagem Celular , Impedância Elétrica , Desenho de Equipamento , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Técnicas Analíticas Microfluídicas/instrumentaçãoRESUMO
The identification of individuals in a mixture of two semen samples usually involves an analysis of autosomal and Y chromosomal short tandem repeats (STR) which can exclude unrelated individuals but cannot achieve the purpose of individual identification. In sperm cells, there are multiple copies of mitochondrial DNAs (mtDNA) which exhibit genetic polymorphisms in different matrilineal-related individuals. Single-cell capture technology can be applied to obtain some single sperm cells in a mixed semen sample, then polymerase chain reaction can be employed to amplify the mtDNA hypervariable region I (HVR I) from each cell. By pooling the cells with the same HVR I sequence, we can obtain the sufficient nuclear DNA for STR typing.
Assuntos
Impressões Digitais de DNA/métodos , DNA Mitocondrial/análise , Sêmen/citologia , Espermatozoides/citologia , DNA Mitocondrial/genética , Humanos , Masculino , Pinças Ópticas , Reação em Cadeia da Polimerase , Polimorfismo Genético , Análise de Célula ÚnicaRESUMO
To obtain single-source short tandem repeat (STR) profiles in trace female/male blood mixture samples, we combined florescence in situ hybridization (FISH), laser microdissection, and low volume PCR (LV-PCR) to isolate male/female cells and improve sensitivity. The results showed that isolation of as few as 10 leukocytes was sufficient to yield full STR profiles in fresh female or male blood samples for 32 independent tests with a low additional alleles rate (3.91%) and drop-out alleles rate (5.01%). Moreover, this procedure was tested in two fresh blood mixture series at three ratios (1:5, 1:10, and 1:20), two mock female/male blood mixture casework samples, and one practical casework sample. Male and female STR profiles were successfully detected in all of these samples, showing that this procedure could be used in forensic casework in the future.
Assuntos
Impressões Digitais de DNA/métodos , Leucócitos/citologia , Repetições de Microssatélites , Contagem de Células Sanguíneas , Cromossomos Humanos X , Cromossomos Humanos Y , Feminino , Humanos , Hibridização in Situ Fluorescente , Microdissecção e Captura a Laser , Masculino , Reação em Cadeia da PolimeraseRESUMO
The capture of individual cells using microfluidic chips represents a widely adopted and efficient approach for investigating the biochemical microenvironment of singular cells. While conventional methods reliant on boundary effects pose challenges in precisely manipulating individual cells, single-cell capture grounded in the principle of stagnation point flow offers a solution to this limitation. Nevertheless, such capture mechanisms encounter inconsistency due to the instability of the flow field and stagnation point. In this study, a microfluidic device for the stable capture of single cells was designed, integrating the principle of fluid mechanics by amalgamating stagnation point flow and boundary effects. This innovative microfluidic chip transcended the limitations associated with single methodologies, leveraging the strengths of both stagnation point flow and boundary effects to achieve reliable single-cell capture. Notably, the incorporation of capture ports at the stagnation point not only harnessed boundary effects but also enhanced capture efficiency significantly, elevating it from 31.9% to 83.3%, thereby augmenting capture stability. Furthermore, computational simulations demonstrated the efficacy of the capture ports in entrapping particles of varying diameters, including 9 µm, 14 µm, and 18 µm. Experiment validation underscored the capability of this microfluidic system to capture single cells within the chip, maintaining stability even under flow rate perturbations spanning from 60 µL/min to 120 µL/min. Consequently, cells with dimensions between 8 µm and 12 µm can be reliably captured. The designed microfluidic system not only furnishes a straightforward and efficient experimental platform but also holds promise for facilitating deeper investigations into the intricate interplay between individual cells and their surrounding microenvironment.
RESUMO
Recent advancements in micro/nanofabrication techniques have led to the development of portable devices for high-throughput single-cell analysis through the isolation of individual target cells, which are then paired with functionalized microbeads. Compared with commercially available benchtop instruments, portable microfluidic devices can be more widely and cost-effectively adopted in single-cell transcriptome and proteome analysis. The sample utilization and cell pairing rate (â¼33%) of current stochastic-based cell-bead pairing approaches are fundamentally limited by Poisson statistics. Despite versatile technologies having been proposed to reduce randomness during the cell-bead pairing process in order to statistically beat the Poisson limit, improvement of the overall pairing rate of a single cell to a single bead is typically based on increased operational complexity and extra instability. In this article, we present a dielectrophoresis (DEP)-assisted dual-nanowell array (ddNA) device, which employs an innovative microstructure design and operating process that decouples the bead- and cell-loading processes. Our ddNA design contains thousands of subnanoliter microwell pairs specifically tailored to fit both beads and cells. Interdigitated electrodes (IDEs) are placed below the microwell structure to introduce a DEP force on cells, yielding high single-cell capture and pairing rates. Experimental results with human embryonic kidney cells confirmed the suitability and reproducibility of our design. We achieved a single-bead capture rate of >97% and a cell-bead pairing rate of >75%. We anticipate that our device will enhance the application of single-cell analysis in practical clinical use and academic research.
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Single-cell technologies have become critical tools to understand and characterize the complex dynamics that govern biological systems, from embryonic development to cancer heterogeneity. In this context, identification and capture of live individual cells in heterogenous ensembles typically rely on genetic manipulations that encode fluorescent probes. However, a precise understanding of how several molecular components interact to yield the phenotype of interest is a prerequisite to distinguishing and isolating such target cells based on fluorescence alone. Indeed, cellular phenotypes associated with migration, shape, location, or intracellular protein distribution play critical and well-understood roles in cancer biology, but the technologies to tag and isolate cells based on information obtained from imaging are not readily available.Cell labeling via photobleaching (CLaP) and single-cell magneto-optical capture (scMOCa) represent convenient and cost-effective systems for labeling, capturing, and expanding single cells from a heterogenous population, without altering cellular physiology and therefore enabling not only transcriptomic profiling but also biological characterization of target cells. Both techniques allow capturing cells after observation and permit researchers to choose target cells based on information obtained from images. The implementation of these technologies only needs the lasers of a confocal microscope and low-cost, commercially available chemical reagents. Here, we describe a detailed protocol to set up and perform CLaP and scMOCa and highlight critical points for optimal performance.
Assuntos
Corantes Fluorescentes , Luz , Corantes Fluorescentes/química , Fotodegradação , LasersRESUMO
Single-cell RNA sequencing (sc-RNAseq) has become a critical approach for the analysis of immune cell function and heterogeneity. So far, the immune cell isolation, based on surface marker expression predicted by the RNA expression profiles, is often limited by the poor correlation between transcript and protein expression patterns. To overcome these difficulties, novel single-cell multi-omic approaches based on the combined analysis of transcript and surface protein expression have been developed. One of the major benefits of these technologies is the possibility to use a high number of antibodies conjugated with oligonucleotide (AbOs) for the surface marker detection, thus overcoming the limit of using few surface markers as occurs in flow cytometry. Here we describe the BD Rhapsody single-cell analysis system protocol for 3' mRNA whole transcriptome analysis (WTA), combined with AbO- and Sample Tag library preparation.
Assuntos
Análise de Célula Única , Perfilação da Expressão Gênica , RNA , Análise de Sequência de RNA , TranscriptomaRESUMO
A microfluidic array was constructed for trapping single cell and loading identical dynamic biochemical stimulation for gain a better understanding of Ca2+ signaling at single cell resolution in the present study. This microfluidic array consists of multiple radially aligned flow channels with equal intersection angles, which was designed by a combination of stagnation point flow and physical barrier. Numerical simulation results and trajectory analysis have shown the effectiveness of this single cell trapping device. Fluorescent experiment results demonstrated the effects of flow rate and frequency of dynamic stimulus on the profiles of biochemical concentration which exposed on captured cells. In this microarray, the captured single cells in each trapping channels were able to receive identical extracellular dynamic biochemical stimuli which being transmitted from the entrance in the middle of the microfluidic array. Besides, after loading dynamic Adenosine Triphosphate (ATP) stimulation on captured cells by this device, consistent average intracellular Ca2+ dynamics phase and cellular heterogeneity were observed in captured single K562 cells. Furthermore, this device is able to be used for investigating cellular respond on single cell resolution to temporally varying environments by modulating the stimulation signal in terms of concentration, pattern, and duration of exposure.
Assuntos
Trifosfato de Adenosina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Célula Única , Simulação por Computador , Humanos , Células K562 , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Análise Numérica Assistida por ComputadorRESUMO
The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.
Assuntos
Separação Celular/métodos , Campos Magnéticos , Coloração e Rotulagem/métodos , Estreptavidina/metabolismo , Animais , Linhagem Celular , HumanosRESUMO
Circulating tumor cells (CTCs) are regarded as a strong biomarker which includes clinically valuable information. However, CTCs are very rare and require precise separation and detection for effective clinical applications. Furthermore, downstream analysis has become necessary to identify the distinct sub-population of CTCs that causes metastasis. Here, we report a flow-restricted microfluidic trap array capable of deterministic single-cell capture of CTCs. The extent of flow restriction, correlating with the device geometry, was then optimized using a highly invasive breast cancer cell line (LM2 MDA-MB-231) to achieve 97% capture efficiency with a single-cell capture rate of 99%. Single-cell capture of CTCs from mice with full-blown metastasis was also demonstrated. The single-CTC capturing ability of the flow-restricted trap array not only showed cell enumerating ability but also high prospects for application in future automated downstream analysis.
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We developed a three-dimensional biointerface of graphene-based electrical impedance sensor for metastatic cancer diagnosis at single-cell resolution. Compared with traditional impedance sensor with two-dimensional interface, the graphene biointerface mimiced the topography and somatotype features of cancer cells, achieving more comprehensive and thorough single cell signals in the three-dimensional space. At the nodes of physiological behavior change of single cell, namely cell capture, adhesion, migration and proliferation, the collected electrical signals from graphene biointerface were about two times stronger than those from the two-dimensional gold interface due to the substantial increase in contact area and significant improvement of topographical interaction between cells and graphene electrode. Simultaneous CCD recording and electrical signal extraction from the entrapped single cell on the graphene biointerface enabled to investigate multidimensional cell-electrode interactions and predict cancerous stage and pathology.
Assuntos
Técnicas Biossensoriais/instrumentação , Grafite/química , Neoplasias/diagnóstico , Análise de Célula Única/instrumentação , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Eletrodos , Ouro/química , HumanosRESUMO
Our understanding of the diversity of cells that escape the primary tumor and seed micrometastases remains rudimentary, and approaches for studying circulating and disseminated tumor cells have been limited by low throughput and sensitivity, reliance on single parameter sorting, and a focus on enumeration rather than phenotypic and genetic characterization. Here, we utilize a highly sensitive microfluidic and dielectrophoretic approach for the isolation and genetic analysis of individual tumor cells. We employed fluorescence labeling to isolate 208 single cells from spiking experiments conducted with 11 cell lines, including 8 neuroblastoma cell lines, and achieved a capture sensitivity of 1 tumor cell per 10(6) white blood cells (WBCs). Sample fixation or freezing had no detectable effect on cell capture. Point mutations were accurately detected in the whole genome amplification product of captured single tumor cells but not in negative control WBCs. We applied this approach to capture 144 single tumor cells from 10 bone marrow samples of patients suffering from neuroblastoma. In this pediatric malignancy, high-risk patients often exhibit wide-spread hematogenous metastasis, but access to primary tumor can be difficult or impossible. Here, we used flow-based sorting to pre-enrich samples with tumor involvement below 0.02%. For all patients for whom a mutation in the Anaplastic Lymphoma Kinase gene had already been detected in their primary tumor, the same mutation was detected in single cells from their marrow. These findings demonstrate a novel, non-invasive, and adaptable method for the capture and genetic analysis of single tumor cells from cancer patients.