scholarly journals Design and Optimizations of a High-throughput Valve-based Microfluidic Device for Single Cell Compartmentalization and Analysis

2020 ◽  
Author(s):  
Jonathan Briones ◽  
Wilfred Espulgar ◽  
Shohei Koyama ◽  
Hyota Takamatsu ◽  
Eiichi Tamiya ◽  
...  
Keyword(s):  
Protein Expression ◽  
Single Cell ◽  
Microfluidic Device ◽  
High Throughput ◽  
Single Cells ◽  
Enzymatic Assay ◽  
Fluid Exchange ◽  
Genomics And Proteomics ◽  
Sealing Pressure ◽  
Cell Compartmentalization

Abstract The need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

scholarly journals A design and optimization of a high throughput valve based microfluidic device for single cell compartmentalization and analysis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonathan Briones ◽  
Wilfred Espulgar ◽  
Shohei Koyama ◽  
Hyota Takamatsu ◽  
Eiichi Tamiya ◽  
...  
Keyword(s):  
Protein Expression ◽  
Single Cell ◽  
Microfluidic Device ◽  
High Throughput ◽  
Single Cells ◽  
Enzymatic Assay ◽  
Fluid Exchange ◽  
Genomics And Proteomics ◽  
Sealing Pressure ◽  
Cell Compartmentalization

AbstractThe need for high throughput single cell screening platforms has been increasing with advancements in genomics and proteomics to identify heterogeneity, unique cell subsets or super mutants from thousands of cells within a population. For real-time monitoring of enzyme kinetics and protein expression profiling, valve-based microfluidics or pneumatic valving that can compartmentalize single cells is advantageous by providing on-demand fluid exchange capability for several steps in assay protocol and on-chip culturing. However, this technique is throughput limited by the number of compartments in the array. Thus, one big challenge lies in increasing the number of microvalves to several thousand that can be actuated in the microfluidic device to confine enzymes and substrates in picoliter volumes. This work explores the design and optimizations done on a microfluidic platform to achieve high-throughput single cell compartmentalization as applied to single-cell enzymatic assay for protein expression quantification. Design modeling through COMSOL Multiphysics was utilized to determine the circular microvalve’s optimized parameters, which can close thousands of microchambers in an array at lower sealing pressure. Multiphysical modeling results demonstrated the relationships of geometry, valve dimensions, and sealing pressure, which were applied in the fabrication of a microfluidic device comprising of up to 5000 hydrodynamic traps and corresponding microvalves. Comparing the effects of geometry, actuation media and fabrication technique, a sealing pressure as low as 0.04 MPa was achieved. Applying to single cell enzymatic assay, variations in granzyme B activity in Jurkat and human PBMC cells were observed. Improvement in the microfluidic chip’s throughput is significant in single cell analysis applications, especially in drug discovery and treatment personalization.

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.

A microfluidic device enabling deterministic single cell trapping and release

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Huichao Chai ◽  
Yongxiang Feng ◽  
Fei Liang ◽  
Wenhui Wang
Keyword(s):  
Chemical Analysis ◽  
Single Cell ◽  
Microfluidic Device ◽  
Single Cells ◽  
Cell Isolation ◽  
Cell Trapping ◽  
Analysis Techniques ◽  
Single Cell Trapping ◽  
Single Cell Isolation ◽  
Made In

Successful single-cell isolation is a pivotal technique for subsequent biological and chemical analysis of single cells. Although significant advances have been made in single-cell isolation and analysis techniques, most passive...

scholarly journals P02.10 FocuSCOPE: a single cell, multi-omics solution to simultaneously analyze tumor variants and microenvironment

2021 ◽  
Vol 9 (Suppl 1) ◽  
pp. A12.1-A12
Author(s):  
Y Arjmand Abbassi ◽  
N Fang ◽  
W Zhu ◽  
Y Zhou ◽  
Y Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Microenvironment ◽  
Single Cell ◽  
High Throughput ◽  
Immune Cells ◽  
Genetic Variants ◽  
Expression Profiles ◽  
Single Cells ◽  
Gene Expression Profiles ◽  
Single Cell Sequencing

Recent advances of high-throughput single cell sequencing technologies have greatly improved our understanding of the complex biological systems. Heterogeneous samples such as tumor tissues commonly harbor cancer cell-specific genetic variants and gene expression profiles, both of which have been shown to be related to the mechanisms of disease development, progression, and responses to treatment. Furthermore, stromal and immune cells within tumor microenvironment interact with cancer cells to play important roles in tumor responses to systematic therapy such as immunotherapy or cell therapy. However, most current high-throughput single cell sequencing methods detect only gene expression levels or epigenetics events such as chromatin conformation. The information on important genetic variants including mutation or fusion is not captured. To better understand the mechanisms of tumor responses to systematic therapy, it is essential to decipher the connection between genotype and gene expression patterns of both tumor cells and cells in the tumor microenvironment. We developed FocuSCOPE, a high-throughput multi-omics sequencing solution that can detect both genetic variants and transcriptome from same single cells. FocuSCOPE has been used to successfully perform single cell analysis of both gene expression profiles and point mutations, fusion genes, or intracellular viral sequences from thousands of cells simultaneously, delivering comprehensive insights of tumor and immune cells in tumor microenvironment at single cell resolution.Disclosure InformationY. Arjmand Abbassi: None. N. Fang: None. W. Zhu: None. Y. Zhou: None. Y. Chen: None. U. Deutsch: None.

scholarly journals Microfluidic guillotine for single-cell wound repair studies

2017 ◽  
Vol 114 (28) ◽  
pp. 7283-7288 ◽  
Author(s):  
Lucas R. Blauch ◽  
Ya Gai ◽  
Jian Wei Khor ◽  
Pranidhi Sood ◽  
Wallace F. Marshall ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Wound Repair ◽  
Time Course ◽  
Single Cells ◽  
Repair Process ◽  
Gene Knockdown ◽  
Viscous Stress ◽  
Repair Capacity ◽  
Stentor Coeruleus

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

High-throughput deterministic single-cell encapsulation and droplet pairing, fusion, and shrinkage in a single microfluidic device

2013 ◽  
Vol 35 (2-3) ◽  
pp. 385-392 ◽  
Author(s):  
Rogier M. Schoeman ◽  
Evelien W.M. Kemna ◽  
Floor Wolbers ◽  
Albert van den Berg
Keyword(s):  
Single Cell ◽  
Microfluidic Device ◽  
High Throughput ◽  
Cell Encapsulation ◽  
Single Cell Encapsulation

scholarly journals Microfluidic Techniques for Single-Cell Protein Expression Analysis

2006 ◽  
Vol 52 (6) ◽  
pp. 1080-1088 ◽  
Author(s):  
Ethan Fitzpatrick ◽  
Sterling McBride ◽  
Jonathan Yavelow ◽  
Saltanat Najmi ◽  
Peter Zanzucchi ◽  
...  
Keyword(s):  
Cell Surface ◽  
Protein Expression ◽  
Cancer Cells ◽  
Microfluidic Device ◽  
Single Cells ◽  
Surface Protein ◽  
Surface Proteins ◽  
Cell Protein ◽  
Surface Protein Expression ◽  
Mcf 7

Abstract Background: The analysis of single cells obtained from needle aspirates of tumors is constrained by the need for processing. To this end, we investigated two microfluidic approaches to measure the expression of surface proteins in single cancer cells or in small populations (<50 cells). Methods: One approach involved indirect fluorescence labeling of cell-surface proteins and channeling of cells in a microfluidic device past a fluorescence detector for signal quantification and analysis. A second approach channeled cells in a microfluidic device over detection zones coated with ligands to surface proteins and measured rates of passage and of retardation based on transient interactions between surface proteins and ligands. Results: The fluorescence device detected expression of integrin α5 induced by basic fibroblast growth factor (FGF-2) treatment in MCF-7 cells and that of Her-2/neu in SK-BR-3 cells compared with controls. Experiments measuring passage retardation showed significant differences in passage rates between FGF-2–treated and untreated MCF-7 cells over reaction regions coated with fibronectin and antibody to integrin α5β1 compared with control regions. Blocking peptides reversed the retardation, demonstrating specificity. Conclusions: Immunofluorescence detection in a microfluidic channel demonstrates the potential for assaying surface protein expression in a few individual cells and will permit the development of future iterations not requiring cell handling. The flow retardation device represents the first application of this technology for assessing cell-surface protein expression in cancer cells and may provide a way for analyzing expression profiles of single cells without preanalytical manipulation.

scholarly journals High-Throughput Single-Cell Cultivation on Microfluidic Streak Plates

2016 ◽  
Vol 82 (7) ◽  
pp. 2210-2218 ◽  
Author(s):  
Cheng-Ying Jiang ◽  
Libing Dong ◽  
Jian-Kang Zhao ◽  
Xiaofang Hu ◽  
Chaohua Shen ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Response Analysis ◽  
Bacterial Strains ◽  
Content Type ◽  
Chemical Gradients ◽  
Droplet Array ◽  
Polycyclic Aromatic ◽  
Single Cell Isolation

ABSTRACTThis paper describes the microfluidic streak plate (MSP), a facile method for high-throughput microbial cell separation and cultivation in nanoliter sessile droplets. The MSP method builds upon the conventional streak plate technique by using microfluidic devices to generate nanoliter droplets that can be streaked manually or robotically onto petri dishes prefilled with carrier oil for cultivation of single cells. In addition, chemical gradients could be encoded in the droplet array for comprehensive dose-response analysis. The MSP method was validated by using single-cell isolation ofEscherichia coliand antimicrobial susceptibility testing ofPseudomonas aeruginosaPAO1. The robustness of the MSP work flow was demonstrated by cultivating a soil community that degrades polycyclic aromatic hydrocarbons. Cultivation in droplets enabled detection of the richest species diversity with better coverage of rare species. Moreover, isolation and cultivation of bacterial strains by MSP led to the discovery of several species with high degradation efficiency, including fourMycobacteriumisolates and a previously unknown fluoranthene-degradingBlastococcusspecies.

scholarly journals RNA splicing programs define tissue compartments and cell types at single cell resolution

2021 ◽  
Author(s):  
Julia Eve Olivieri ◽  
Roozbeh Dehghannasiri ◽  
Peter Wang ◽  
SoRi Jang ◽  
Antoine de Morree ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
High Throughput ◽  
Rna Splicing ◽  
Single Cells ◽  
Cell Types ◽  
Mouse Lemur ◽  
Cell Type ◽  
Multiple Organs ◽  
Single Cell Pcr

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

scholarly journals Direct correlation between motile behavior and protein abundance in single cells

2016 ◽  
Author(s):  
Yann S Dufour ◽  
Sébastien Gillet ◽  
Nicholas W Frankel ◽  
Douglas B Weibel ◽  
Thierry Emonet
Keyword(s):  
Protein Expression ◽  
Single Cell ◽  
Phenotypic Diversity ◽  
Single Cells ◽  
Selective Advantage ◽  
Single Cell Protein ◽  
Cell Protein ◽  
Chemotaxis Proteins

AbstractUnderstanding how stochastic molecular fluctuations affect cell behavior requires the quantification of both behavior and protein numbers in the same cells. Here, we combine automated microscopy with in situ hydrogel polymerization to measure single-cell protein expression after tracking swimming behavior. We characterized the distribution of non-genetic phenotypic diversity in Escherichia coli motility, which affects single-cell exploration. By expressing fluorescently tagged chemotaxis proteins (CheR and CheB) at different levels, we quantitatively mapped motile phenotype (tumble bias) to protein numbers using thousands of single-cell measurements. Our results disagreed with established models until we incorporated the role of CheB in receptor deamidation and the slow fluctuations in receptor methylation. Beyond refining models, our central finding is that changes in numbers of CheR and CheB affect the population mean tumble bias and its variance independently. Therefore, it is possible to adjust the degree of phenotypic diversity of a population by adjusting the global level of expression of CheR and CheB while keeping their ratio constant, which, as shown in previous studies, confers functional robustness to the system. Since genetic control of protein expression is heritable, our results suggest that non-genetic diversity in motile behavior is selectable, supporting earlier hypotheses that such diversity confers a selective advantage.

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