Centrifugal Microfluidics Traps for Parallel Isolation and Imaging of Single Cells

Analysis at the single cell level has becoming an increasingly important procedure to diagnose cancer tissue biopsies. These tissue samples are often heterogeneous and consist of 1000–15,000 cells. We study the use of centrifugal microfluidics to isolate single cells into micro chambers. We describe the optimization of our microfluidics flow device, characterize its performance using both polystyrene beads as a cell analogue and MCF-7 breast cancer cells, and discuss potential applications for the device. Our results show rapid isolation of ~2000 single cell aliquots in ~20 min. We were able to occupy 65% of available chambers with singly occupied cancer cells, and observed capture efficiencies as high as 80% using input samples ranging from 2000 to 15,000 cells in 20 min. We believe our device is a valuable research tool that addresses the unmet need for massively parallel single cell level analysis of cell populations.

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A method of quantifying centrosomes at the single-cell level in human normal and cancer tissue

Molecular Biology of the Cell ◽

10.1091/mbc.e18-10-0651 ◽

2019 ◽

Vol 30 (7) ◽

pp. 811-819 ◽

Cited By ~ 3

Author(s):

Mengdie Wang ◽

Beatrice S. Knudsen ◽

Raymond B. Nagle ◽

Gregory C. Rogers ◽

Anne E. Cress

Keyword(s):

Single Cell ◽

Human Tissue ◽

Single Cells ◽

Cancer Tissue ◽

Single Cell Level ◽

Cell Level ◽

Tissue Samples ◽

Tumor Subtypes ◽

Different Types ◽

Pericentriolar Material

Centrosome abnormalities are emerging hallmarks of cancer. The overproduction of centrosomes (known as centrosome amplification) has been reported in a variety of cancers and is currently being explored as a promising target for therapy. However, to understand different types of centrosome abnormalities and their impact on centrosome function during tumor progression, as well as to identify tumor subtypes that would respond to the targeting of a centrosome abnormality, a reliable method for accurately quantifying centrosomes in human tissue samples is needed. Here, we established a method of quantifying centrosomes at a single-cell level in different types of human tissue samples. We tested multiple anti-centriole and pericentriolar-material antibodies to identify bona fide centrosomes and multiplexed these with cell border markers to identify individual cells within the tissue. High-resolution microscopy was used to generate multiple Z-section images, allowing us to acquire whole cell volumes in which to scan for centrosomes. The normal cells within the tissue serve as internal positive controls. Our method provides a simple, accurate way to distinguish alterations in centrosome numbers at the level of single cells.

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Abstract P1-07-03: Quantification of HER2 expression at the single cell level and HER2 intratumoral heterogeneity of breast cancer tissue samples using automated image analysis

10.1158/0008-5472.sabcs12-p1-07-03 ◽

2012 ◽

Cited By ~ 1

Author(s):

E Geretti ◽

V Paragas ◽

M Onsum ◽

A Kudla ◽

S Moulis ◽

...

Keyword(s):

Breast Cancer ◽

Image Analysis ◽

Single Cell ◽

Breast Cancer Tissue ◽

Automated Image Analysis ◽

Cancer Tissue ◽

Single Cell Level ◽

Her2 Expression ◽

Cell Level ◽

Tissue Samples

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Efficient analysis of a small number of cancer cells at the single-cell level using an electroactive double-well array

Lab on a Chip ◽

10.1039/c6lc00241b ◽

2016 ◽

Vol 16 (13) ◽

pp. 2440-2449 ◽

Cited By ~ 26

Author(s):

Soo Hyeon Kim ◽

Teruo Fujii

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Single Cells ◽

Capture Efficiency ◽

Single Cell Level ◽

Cell Capture ◽

Cell Level ◽

Cell Trapping ◽

Highly Efficient ◽

Single Cell Trapping

The electroactive double well-array consists of trap-wells for highly efficient single-cell trapping using dielectrophoresis (cell capture efficiency of 96 ± 3%) and reaction-wells that confine cell lysates for analysis of intracellular materials from single cells.

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LINE-1 Retrotransposon expression in cancerous, epithelial and neuronal cells revealed by 5’-single cell RNA-Seq

10.1101/2021.01.19.427347 ◽

2021 ◽

Author(s):

Wilson McKerrow ◽

Shane A. Evans ◽

Azucena Rocha ◽

John Sedivy ◽

Nicola Neretti ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Early Development ◽

Immune Cells ◽

Copy Number ◽

Single Cells ◽

Single Cell Level ◽

Rna Seq ◽

Cell Level ◽

Mouse Hippocampus

AbstractLINE-1 retrotransposons are known to be expressed in early development, in tumors and in the germline. Less is known about LINE-1 expression at the single cell level, especially outside the context of cancer. Because LINE-1 elements are present at a high copy number, many transcripts that are not driven by the LINE-1 promoter nevertheless terminate at the LINE-1 3’ UTR. Thus, 3’ targeted single cell RNA-seq datasets are not appropriate for studying LINE-1. However, 5’ targeted single cell datasets provide an opportunity to analyze LINE-1 expression at the single cell level. Most LINE-1 copies are 5’ truncated, and a transcript that contains the LINE-1 5’ UTR as its 5’ end is likely to have been transcribed from its promoter. We developed a method, L1-sc (LINE-1 expression for single cells), to quantify LINE-1 expression in 5’ targeted 10x genomics single cell RNA-seq datasets. Our method confirms that LINE-1 expression is high in cancer cells, but low or absent from immune cells. We also find that LINE-1 expression is elevated in epithelial compared to immune cells outside of the context of cancer and that it is also elevated in neurons compared to glia in the mouse hippocampus.

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A step towards enzyme-free tissue dissociation

Current Directions in Biomedical Engineering ◽

10.1515/cdbme-2019-0137 ◽

2019 ◽

Vol 5 (1) ◽

pp. 545-548

Author(s):

Stefan Scheuermann ◽

Armin Schäfer ◽

Jens Langejürgen ◽

Christian Reis

Keyword(s):

Single Cell ◽

Stromal Cells ◽

Processing Time ◽

Single Cells ◽

Single Cell Level ◽

Cell Level ◽

Tissue Samples ◽

Tissue Dissociation ◽

Tissue Matrix ◽

Generation Sequencing

AbstractThe future of personalized diagnostics commences on the single cell level. Even high-end technologies like Next Generation Sequencing can be improved if applied on pure single cell populations (e.g., tumor cells without contaminating stromal cells) or on a single cell level (DNA/RNA sequencing). The vast majority of these technologies need individual and preferably undistorted cells for the analytical process. Thus, decisive prerequisite for high-end analytics is to remove cells from their tissue matrix as gently as possible. This can be accomplished by an enzyme-free, fast and reproducible approach of generating pure and individual single cells from tissue samples. In this study we demonstrate the utility of a semi-automated Tissue Grinder that is compatible with standard 50 ml centrifuge tubes and standard cell strainer for mechanically, nonenzymatic and parallel processing of tissue samples. We show that without enzymatic treatment viable single-cell yields match or exceed reference enzymatic methods, while reducing processing time by at least 80%.

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Microfluidic filter device with nylon mesh membranes efficiently dissociates cell aggregates and digested tissue into single cells

Lab on a Chip ◽

10.1039/c8lc00507a ◽

2018 ◽

Vol 18 (18) ◽

pp. 2776-2786 ◽

Cited By ~ 4

Author(s):

Xiaolong Qiu ◽

Jeremy A. Lombardo ◽

Trisha M. Westerhof ◽

Marissa Pennell ◽

Anita Ng ◽

...

Keyword(s):

Single Cell ◽

Single Cells ◽

Cell Suspensions ◽

Cell Aggregates ◽

Single Cell Level ◽

Cell Level ◽

Tissue Samples ◽

Nylon Mesh

Tissues are increasingly being analyzed at this single cell level. We present a simple and inexpensive microfluidic filter device that can rapidly and effectively improve the quality of single cell suspensions obtained from digested tissue samples.

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Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽

10.3390/cells11020285 ◽

2022 ◽

Vol 11 (2) ◽

pp. 285

Author(s):

Eszter Széles ◽

Krisztina Nagy ◽

Ágnes Ábrahám ◽

Sándor Kovács ◽

Anna Podmaniczki ◽

...

Keyword(s):

Chlamydomonas Reinhardtii ◽

Single Cell ◽

Single Cells ◽

Fluorescence Induction ◽

Photochemical Quenching ◽

Support Surface ◽

Single Cell Level ◽

Microfluidic Platforms ◽

Cell Level ◽

Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

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High-resolution label-free 3D mapping of extracellular pH of single living cells

Nature Communications ◽

10.1038/s41467-019-13535-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 7

Author(s):

Yanjun Zhang ◽

Yasufumi Takahashi ◽

Sung Pil Hong ◽

Fengjie Liu ◽

Joanna Bednarska ◽

...

Keyword(s):

Single Cell ◽

Cancer Cells ◽

Extracellular Ph ◽

Single Cell Level ◽

Ph Sensing ◽

Label Free ◽

Cell Level ◽

Spatiotemporal Resolution ◽

Ion Conductance ◽

Scanning Ion Conductance Microscopy

AbstractDynamic mapping of extracellular pH (pHe) at the single-cell level is critical for understanding the role of H+ in cellular and subcellular processes, with particular importance in cancer. While several pHe sensing techniques have been developed, accessing this information at the single-cell level requires improvement in sensitivity, spatial and temporal resolution. We report on a zwitterionic label-free pH nanoprobe that addresses these long-standing challenges. The probe has a sensitivity > 0.01 units, 2 ms response time, and 50 nm spatial resolution. The platform was integrated into a double-barrel nanoprobe combining pH sensing with feedback-controlled distance dependance via Scanning Ion Conductance Microscopy. This allows for the simultaneous 3D topographical imaging and pHe monitoring of living cancer cells. These classes of nanoprobes were used for real-time high spatiotemporal resolution pHe mapping at the subcellular level and revealed tumour heterogeneity of the peri-cellular environments of melanoma and breast cancer cells.

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Dissecting CLL through high-dimensional single-cell technologies

Blood ◽

10.1182/blood-2018-09-835389 ◽

2019 ◽

Vol 133 (13) ◽

pp. 1446-1456

Author(s):

Satyen H. Gohil ◽

Catherine J. Wu

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Single Cell ◽

Cancer Cells ◽

Lymphocytic Leukemia ◽

Response To Treatment ◽

High Dimensional ◽

Single Cell Level ◽

Disease Course ◽

Cell Level ◽

New Techniques

Abstract We now have the potential to undertake detailed analysis of the inner workings of thousands of cancer cells, one cell at a time, through the emergence of a range of techniques that probe the genome, transcriptome, and proteome combined with the development of bioinformatics pipelines that enable their interpretation. This provides an unprecedented opportunity to better understand the heterogeneity of chronic lymphocytic leukemia and how mutations, activation states, and protein expression at the single-cell level have an impact on disease course, response to treatment, and outcomes. Herein, we review the emerging application of these new techniques to chronic lymphocytic leukemia and examine the insights already attained through this transformative technology.

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