Hydrodynamic shuttling for deterministic high-efficiency multiple single-cell capture in a microfluidic chip

Lab on a Chip ◽  
2019 ◽  
Vol 19 (8) ◽  
pp. 1370-1377 ◽  
Author(s):  
Cheng-Kun He ◽  
Ya-Wen Chen ◽  
Ssu-Han Wang ◽  
Chia-Hsien Hsu
Keyword(s):  
Single Cell ◽  
Biomedical Research ◽  
Microfluidic Chip ◽  
High Efficiency ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Cell Capture

A new microfluidics technique for high-efficiency paring and analyzing multiple single cells can facilitate cellular heterogeneity studies important for biological and biomedical research.

scholarly journals Microfluidic Cell Trapping for Single-Cell Analysis

Micromachines ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 409 ◽  
Author(s):  
Bing Deng ◽  
Heyi Wang ◽  
Zhaoyi Tan ◽  
Yi Quan
Keyword(s):  
Fluid Dynamics ◽  
Cell Growth ◽  
Single Cell ◽  
Microfluidic Chip ◽  
Cultured Cells ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cell Analysis ◽  
Cell Capture ◽  
Culture Process

The single-cell capture microfluidic chip has many advantages, including low cost, high throughput, easy manufacturing, integration, non-toxicity and good stability. Because of these characteristics, the cell capture microfluidic chip is increasingly becoming an important carrier on the study of life science and pharmaceutical analysis. Important promises of single-cell analysis are the paring, fusion, disruption and analysis of intracellular components for capturing a single cell. The capture, which is based on the fluid dynamics method in the field of micro fluidic chips is an important way to achieve and realize the operations mentioned above. The aim of this study was to compare the ability of three fluid dynamics-based microfluidic chip structures to capture cells. The effects of cell growth and distribution after being captured by different structural chips and the subsequent observation and analysis of single cells on the chip were compared. It can be seen from the experimental results that the microfluidic chip structure most suitable for single-cell capture is a U-shaped structure. It enables single-cell capture as well as long-term continuous culture and the single-cell observation of captured cells. Compared to the U-shaped structure, the cells captured by the microcavity structure easily overlapped during the culture process and affected the subsequent analysis of single cells. The flow shortcut structure can also be used to capture and observe single cells, however, the shearing force of the fluid caused by the chip structure is likely to cause deformation of the cultured cells. By comparing the cell capture efficiency of the three chips, the reagent loss during the culture process and the cell growth state of the captured cells, we are provided with a theoretical support for the design of a single-cell capture microfluidic chip and a reference for the study of single-cell capture in the future.

scholarly journals Time Sequential Single-Cell Patterning with High Efficiency and High Density

Sensors ◽  
2018 ◽  
Vol 18 (11) ◽  
pp. 3672 ◽  
Author(s):  
Yang Liu ◽  
Dahai Ren ◽  
Xixin Ling ◽  
Weibin Liang ◽  
Jing Li ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Capture Efficiency ◽  
Jurkat Cells ◽  
Objective Lens ◽  
Cell Capture ◽  
Cell Trapping ◽  
Loop Channel ◽  
Single Cell Trapping

Single-cell capture plays an important role in single-cell manipulation and analysis. This paper presents a microfluidic device for deterministic single-cell trapping based on the hydrodynamic trapping mechanism. The device is composed of an S-shaped loop channel and thousands of aligned trap units. This arrayed structure enables each row of the device to be treated equally and independently, as it has row periodicity. A theoretical model was established and a simulation was conducted to optimize the key geometric parameters, and the performance was evaluated by conducting experiments on MCF-7 and Jurkat cells. The results showed improvements in single-cell trapping ability, including loading efficiency, capture speed, and the density of the patterned cells. The optimized device can achieve a capture efficiency of up to 100% and single-cell capture efficiency of up to 95%. This device offers 200 trap units in an area of 1 mm2, which enables 100 single cells to be observed simultaneously using a microscope with a 20× objective lens. One thousand cells can be trapped sequentially within 2 min; this is faster than the values obtained with previously reported devices. Furthermore, the cells can also be recovered by reversely infusing solutions. The structure can be easily extended to a large scale, and a patterned array with 32,000 trap sites was accomplished on a single chip. This device can be a powerful tool for high-throughput single-cell analysis, cell heterogeneity investigation, and drug screening.

scholarly journals A Microfluidic Array Device for Single Cell Capture and Intracellular Ca2+ Response Analysis Induced by Dynamic Biochemical Stimulus

2021 ◽  
Author(s):  
Wenbo Wei ◽  
Miao Zhang ◽  
Zhongyuan Xu ◽  
Weifeng Li ◽  
Lixin Cheng ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
K562 Cells ◽  
Cellular Heterogeneity ◽  
Response Analysis ◽  
Cell Capture ◽  
Cell Trapping ◽  
Dynamic Stimulus ◽  
Single Cell Trapping ◽  
Varying Environments

A microfluidic array was constructed for trapping single cell and loading identical dynamic biochemical stimulation for gain a better understanding of Ca2+ signalling in single cells by applying extracellular dynamic biochemical stimulus. 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 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 array chip, the captured single cells in each trapping channels were able to receive identical extracellular dynamic biochemical stimuli which being transmitted from the entrance at 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 in single cells to temporally varying environments by modulating the stimulation signal in terms of concentration, pattern, and duration of exposure.

scholarly journals Cryopreservation of human cancers conserves tumour heterogeneity for single-cell multi-omics analysis

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sunny Z. Wu ◽  
Daniel L. Roden ◽  
Ghamdan Al-Eryani ◽  
Nenad Bartonicek ◽  
Kate Harvey ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Tumour Heterogeneity ◽  
Fresh Tissue ◽  
Human Cancers ◽  
Cryopreserved Cell ◽  
Single Cell Rna Sequencing

Abstract Background High throughput single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for exploring cellular heterogeneity among complex human cancers. scRNA-Seq studies using fresh human surgical tissue are logistically difficult, preclude histopathological triage of samples, and limit the ability to perform batch processing. This hindrance can often introduce technical biases when integrating patient datasets and increase experimental costs. Although tissue preservation methods have been previously explored to address such issues, it is yet to be examined on complex human tissues, such as solid cancers and on high throughput scRNA-Seq platforms. Methods Using the Chromium 10X platform, we sequenced a total of ~ 120,000 cells from fresh and cryopreserved replicates across three primary breast cancers, two primary prostate cancers and a cutaneous melanoma. We performed detailed analyses between cells from each condition to assess the effects of cryopreservation on cellular heterogeneity, cell quality, clustering and the identification of gene ontologies. In addition, we performed single-cell immunophenotyping using CITE-Seq on a single breast cancer sample cryopreserved as solid tissue fragments. Results Tumour heterogeneity identified from fresh tissues was largely conserved in cryopreserved replicates. We show that sequencing of single cells prepared from cryopreserved tissue fragments or from cryopreserved cell suspensions is comparable to sequenced cells prepared from fresh tissue, with cryopreserved cell suspensions displaying higher correlations with fresh tissue in gene expression. We showed that cryopreservation had minimal impacts on the results of downstream analyses such as biological pathway enrichment. For some tumours, cryopreservation modestly increased cell stress signatures compared to freshly analysed tissue. Further, we demonstrate the advantage of cryopreserving whole-cells for detecting cell-surface proteins using CITE-Seq, which is impossible using other preservation methods such as single nuclei-sequencing. Conclusions We show that the viable cryopreservation of human cancers provides high-quality single-cells for multi-omics analysis. Our study guides new experimental designs for tissue biobanking for future clinical single-cell RNA sequencing studies.

Testing of solid oxide cells at high current densities

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
André Weber
Keyword(s):  
Single Cell ◽  
High Performance ◽  
High Efficiency ◽  
Electrochemical Impedance ◽  
Single Cells ◽  
Operating Conditions ◽  
Solid Oxide ◽  
Current Densities ◽  
Cell Testing ◽  
The Impact

Abstract Solid Oxide Cells (SOCs) have gained an increasing interest as electrochemical energy converters due to their high efficiency, fuel flexibility and ability of reversible fuel cell/electrolysis operation. During the development process as well as in quality assurance tests, the performance of single cells and cell stacks is commonly evaluated by means of current/voltage- (CV-) characteristics. Despite of the fact that the measurement of a CV-characteristic seems to be simple compared to more complex, dynamic methods as electrochemical impedance spectroscopy or current interrupt techniques, the resulting performance strongly depends on the test setup and the chosen operating conditions. In this paper, the impact of different single cell testing environments and operating conditions on the CV-characteristic of high performance cells is discussed. The influence of cell size, contacting and current collection, contact pressure, fuel flow rate and composition on the achievable cell performance is presented and limitations arising from the test bed and testing conditions will be pointed out. As today’s high performance cells are capable of delivering current densities of several ampere per cm2 a special emphasis will be laid on single cell testing in this current range.

scholarly journals Integration of marker-free selection of single cells at a wireless electrode array with parallel fluidic isolation and electrical lysis

2019 ◽  
Vol 10 (5) ◽  
pp. 1506-1513 ◽  
Author(s):  
Min Li ◽  
Robbyn K. Anand
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Electrode Array ◽  
Cell Capture ◽  
Bipolar Electrodes ◽  
Manual Intervention ◽  
Electrical Lysis ◽  
Marker Free ◽  
Selection Of

We present integration of selective single-cell capture at an array of wireless electrodes (bipolar electrodes, BPEs) with transfer into chambers, reagent exchange, fluidic isolation and rapid electrical lysis in a single platform, thus minimizing sample loss and manual intervention steps.

Capillary Force Driven Single-Cell Spiking Apparatus for Studying Circulating Tumor Cells

2018 ◽  
Author(s):  
Jacob Amontree ◽  
Kangfu Chen ◽  
Jose Varillas ◽  
Z. Hugh Fan
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Surface Membrane ◽  
Critical Element ◽  
Cell Capture ◽  
Biomedical Sciences ◽  
Low Concentrations

The characterization of single cells within heterogeneous populations has great impact on both biomedical sciences and cancer research. By investigating cellular compositions on a broad scale, pertinent outliers may be lost in the sample set. Alternatively, an investigation focused on the behavior of specific cells, such as circulating tumor cells (CTCs), will reveal genetic biomarkers or phenotypic characteristics associated with cancer and metastasis. On average, CTC concentration in peripheral blood is extremely low, as few as one to two per billion of healthy blood cells. Consequently, the critical element lacking in many methods of CTC detection is accurate cell capture efficiency at low concentrations. To simulate CTC isolation, researchers usually spike small amounts of tumor cells to healthy blood for separation. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. We report our study on an innovative apparatus and method designed for low-cost, precise, and replicable single-cell spiking (SCS). Our SCS method operates solely from capillary aspiration without the reliance on external laboratory equipment. To ensure that our method does not affect the viability of each cell, we investigated the effects of surface membrane tensions induced by aspiration. Finally, we performed affinity-based CTC isolation using human acute lymphoblastic leukemia cells (CCRF-CEM) spiked into healthy whole blood with the SCS technique. The results of the isolation experiments demonstrate the reliability of our method in generating low-concentration cell samples.

Integration of a microfluidic chip with a size-based cell bandpass filter for reliable isolation of single cells

Lab on a Chip ◽  
2015 ◽  
Vol 15 (21) ◽  
pp. 4128-4132 ◽  
Author(s):  
Hojin Kim ◽  
Sanghyun Lee ◽  
Jae-hyung Lee ◽  
Joonwon Kim
Keyword(s):  
Single Cell ◽  
Microfluidic Chip ◽  
Bandpass Filter ◽  
Single Cells ◽  
Cell Array ◽  
Novel Approach ◽  
Reliable Isolation

A novel approach for reliable arraying of single cells is presented using a size-based cell bandpass filter integrated with a microfluidic single-cell array chip.

scholarly journals Computational approaches towards reducing contamination in single-cell RNA-seq data

2020 ◽  
Author(s):  
Siamak Yousefi ◽  
Hao Chen ◽  
Jesse F. Ingels ◽  
Melinda S. McCarty ◽  
Arthur G. Centeno ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Real Life ◽  
Cell Types ◽  
Cell Capture ◽  
Rna Seq ◽  
Sequence Analyses ◽  
Cell Functions ◽  
Biological Interpretation ◽  
Different Cell Types

SUMMARYSingle cell RNA sequencing has enabled quantification of single cells and identification of different cell types and subtypes as well as cell functions in different tissues. Single cell RNA sequence analyses assume acquired RNAs correspond to cells, however, RNAs from contamination within the input data are also captured by these assays. The sequencing of background contamination as well as unwanted cells making their way to the final assay Potentially confound the correct biological interpretation of single cell transcriptomic data. Here we demonstrate two approaches to deal with background contamination as well as profiling of unwanted cells in the assays. We use three real-life datasets of whole-cell capture and nucleotide single-cell captures generated by Fluidigm and 10x technologies and show that these methods reduce the effect of contamination, strengthen clustering of cells and improves biological interpretation.

scholarly journals High-throughput and high-efficiency sample preparation for single-cell proteomics using a nested nanowell chip

2021 ◽  
Author(s):  
Jongmin Woo ◽  
Sarah M. Williams ◽  
Victor Aguilera-Vazquez ◽  
Ryan L. Sontag ◽  
Ronald J. Moore ◽  
...  
Keyword(s):  
Single Cell ◽  
High Efficiency ◽  
Single Cells ◽  
Protein Recovery ◽  
Processing Capacity ◽  
Protein Abundance ◽  
Proteome Coverage ◽  
Low Protein ◽  
Functional Differences ◽  
Cell Proteome

AbstractGlobal quantification of protein abundances in single cells would provide more direct information on cellular function phenotypes and complement transcriptomics measurements. However, single-cell proteomics (scProteomics) is still immature and confronts technical challenges, including limited proteome coverage, poor reproducibility, as well as low throughput. Here we describe a nested nanoPOTS (N2) chip to dramatically improve protein recovery, operation robustness, and processing throughput for isobaric-labeling-based scProteomics workflow. The N2 chip allows reducing cell digestion volume to <30 nL and increasing processing capacity to > 240 single cells in one microchip. In the analysis of ∼100 individual cells from three different cell lines, we demonstrate the N2 chip-based scProteomics platform can robustly quantify ∼1500 proteins and reveal functional differences. Our analysis also reveals low protein abundance variations (median CVs < 16.3%), highlighting the utility of such measurements, and also suggesting the single-cell proteome is highly stable for the cells cultured under identical conditions.

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