scholarly journals Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  
Keyword(s):  
Head Start ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Droplet Microfluidics ◽  
Metagenomic Data ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Technical Details ◽  
Droplet Sorting

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

scholarly journals Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics: An Engineering Head Start

2021 ◽  
Author(s):  
David Conchouso ◽  
Amani Al-Ma’abadi ◽  
Hayedeh Behzad ◽  
Mohammed Alarawi ◽  
Masahito Hosokawa ◽  
...  
Keyword(s):  
Head Start ◽  
Single Cell ◽  
High Throughput ◽  
Single Cells ◽  
Droplet Microfluidics ◽  
Metagenomic Data ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Technical Details ◽  
Droplet Sorting

<p>Droplet microfluidics techniques have shown promising results to study single-cells at high throughput. However, their adoption in laboratories studying “-omics” sciences is still irrelevant because of the field’s complex and multidisciplinary nature. To facilitate their use, here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput. First, a device encapsulating single-cells in droplets at a rate of ~ 250 Hz is described considering droplet size and cell growth. Then, we expand on previously reported fluorescent activated droplet sorting (FADS) systems to integrate the use of 4 independent fluorescence-exciting lasers (e.g., 405, 488, 561, 637 nm) in a single platform to make it compatible with different fluorescence-emitting biosensors. For this sorter, both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz. Then, a passive droplet merger was also integrated into our method to enable adding new reagents to already made droplets at a rate of 200 Hz. Finally, we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools. Because of the overall integration and the technical details presented here, our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput (> 50,000 cells/day) capabilities to mining and bioprospecting metagenomic data.</p>

scholarly journals Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based Microfluidics

2021 ◽  
Vol 9 ◽  
Author(s):  
B. M. Tiemeijer ◽  
M. W. D. Sweep ◽  
J. J. F. Sleeboom ◽  
K. J. Steps ◽  
J. F. van Sprang ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Biological Function ◽  
Single Cells ◽  
Macrophage Polarization ◽  
Cell Polarization ◽  
Droplet Microfluidics ◽  
Cellular Heterogeneity ◽  
Adherent Cell ◽  
M2 Polarization

Human immune cells intrinsically exist as heterogenous populations. To understand cellular heterogeneity, both cell culture and analysis should be executed with single-cell resolution to eliminate juxtacrine and paracrine interactions, as these can lead to a homogenized cell response, obscuring unique cellular behavior. Droplet microfluidics has emerged as a potent tool to culture and stimulate single cells at high throughput. However, when studying adherent cells at single-cell level, it is imperative to provide a substrate for the cells to adhere to, as suspension culture conditions can negatively affect biological function and behavior. Therefore, we combined a droplet-based microfluidic platform with a thermo-reversible polyisocyanide (PIC) hydrogel, which allowed for robust droplet formation at low temperatures, whilst ensuring catalyzer-free droplet gelation and easy cell recovery after culture for downstream analysis. With this approach, we probed the heterogeneity of highly adherent human macrophages under both pro-inflammatory M1 and anti-inflammatory M2 polarization conditions. We showed that co-encapsulation of multiple cells enhanced cell polarization compared to single cells, indicating that cellular communication is a potent driver of macrophage polarization. Additionally, we highlight that culturing single macrophages in PIC hydrogel droplets displayed higher cell viability and enhanced M2 polarization compared to single macrophages cultured in suspension. Remarkably, combining phenotypical and functional analysis on single cultured macrophages revealed a subset of cells in a persistent M1 state, which were undetectable in conventional bulk cultures. Taken together, combining droplet-based microfluidics with hydrogels is a versatile and powerful tool to study the biological function of adherent cell types at single-cell resolution with high throughput.

scholarly journals Optimized double emulsion flow cytometry with high-throughput single droplet isolation

2019 ◽  
Author(s):  
Kara K. Brower ◽  
Catherine Carswell-Crumpton ◽  
Sandy Klemm ◽  
Bianca Cruz ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Single Cell ◽  
High Throughput ◽  
Single Cell Analysis ◽  
Double Emulsion ◽  
Droplet Microfluidics ◽  
Cell Analysis ◽  
Single Droplet ◽  
Emulsion Droplets ◽  
Droplet Sorting

Droplet microfluidics has made large impacts in diverse areas such as enzyme evolution, chemical product screening, polymer engineering, and single-cell analysis. However, while droplet reactions have become increasingly sophisticated, phenotyping droplets by a fluorescent signal and sorting them to isolate variants-of-interest remains a field-wide bottleneck. Here, we present an optimized double emulsion workflow, sdDE-FACS, that enables high-throughput phenotyping, selection, and sorting of droplets using standard flow cytometers. Using a 130 μm nozzle, we demonstrate robust post-sort recovery of intact droplets, with little to no shear-induced droplet breakage, at high sort frequency (12-14 kHz) across two industry-standard FACS instruments. We report the first quantitative plate statistics for double emulsion droplet isolation and demonstrate single droplet recovery with >70% efficiency. In addition, we establish complete downstream recovery of nucleic acids from single, sorted double emulsion droplets, an advance in droplet sorting comparable with the capabilities of single-cell FACS. This work resolves several hurdles in the field of high-throughput droplet analysis and paves the way for a variety of new droplet assays, including rare variant isolation and multiparameter single-cell analysis, marrying the full power of flow cytometry with droplet microfluidics.

scholarly journals Double Emulsion Picoreactors for High-Throughput Single-Cell Encapsulation and Phenotyping via FACS

2020 ◽  
Author(s):  
Kara K. Brower ◽  
Margarita Khariton ◽  
Peter H. Suzuki ◽  
Chris Still ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Lines ◽  
High Throughput ◽  
Single Cells ◽  
Double Emulsion ◽  
Cell Encapsulation ◽  
Functional Screening ◽  
Cellular Phenotype ◽  
Cell Loading ◽  
Single Cell Encapsulation

ABSTRACTIn the past five years, droplet microfluidic techniques have unlocked new opportunities for the high-throughput genome-wide analysis of single cells, transforming our understanding of cellular diversity and function. However, the field lacks an accessible method to screen and sort droplets based on cellular phenotype upstream of genetic analysis, particularly for large and complex cells. To meet this need, we developed Dropception, a robust, easy-to-use workflow for precise single-cell encapsulation into picoliter-scale double emulsion droplets compatible with high-throughput phenotyping via fluorescence-activated cell sorting (FACS). We demonstrate the capabilities of this method by encapsulating five standardized mammalian cell lines of varying size and morphology as well as a heterogeneous cell mixture of a whole dissociated flatworm (5 - 25 μm in diameter) within highly monodisperse double emulsions (35 μm in diameter). We optimize for preferential encapsulation of single cells with extremely low multiple-cell loading events (<2% of cell-containing droplets), thereby allowing direct linkage of cellular phenotype to genotype. Across all cell lines, cell loading efficiency approaches the theoretical limit with no observable bias by cell size. FACS measurements reveal the ability to discriminate empty droplets from those containing cells with good agreement to single-cell occupancies quantified via microscopy, establishing robust droplet screening at single-cell resolution. High-throughput FACS phenotyping of cellular picoreactors has the potential to shift the landscape of single-cell droplet microfluidics by expanding the repertoire of current nucleic acid droplet assays to include functional screening.ABSTRACT FIGURE

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.

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.

Inertial-ordering-assisted droplet microfluidics for high-throughput single-cell RNA-sequencing

Lab on a Chip ◽  
2018 ◽  
Vol 18 (5) ◽  
pp. 775-784 ◽  
Author(s):  
Hui-Sung Moon ◽  
Kwanghwi Je ◽  
Jae-Woong Min ◽  
Donghyun Park ◽  
Kyung-Yeon Han ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
High Throughput ◽  
Droplet Microfluidics ◽  
Single Cell Rna Sequencing ◽  
Spiral Microchannel

We developed a modified high-throughput droplet barcoding technique for single-cell Drop-Seq via introduction of hydrodynamic ordering in a spiral microchannel.

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.

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