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

2020 ◽  
Vol 92 (19) ◽  
pp. 13262-13270
Author(s):  
Kara K. Brower ◽  
Margarita Khariton ◽  
Peter H. Suzuki ◽  
Chris Still ◽  
Gaeun Kim ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Double Emulsion ◽  
Cell Encapsulation ◽  
Single Cell Encapsulation

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

Micropillar array embedded system for single cell encapsulation in hydrogel

2015 ◽  
Vol 1724 ◽  
Author(s):  
Kyun Joo Park ◽  
Kyoung G. Lee ◽  
Seunghwan Seok ◽  
Bong Gill Choi ◽  
Seok Jae Lee ◽  
...  
Keyword(s):  
Embedded System ◽  
Single Cell ◽  
Single Cells ◽  
Cell Encapsulation ◽  
Polyethylene Glycol Diacrylate ◽  
Glycol Diacrylate ◽  
Practical Applications ◽  
Cell Clusters ◽  
Single Cell Encapsulation ◽  
Micropillar Array

ABSTRACTA cylindrical-shaped micropillar array embedded microfluidic device was proposed to enhance the dispersion of cell clusters and the efficiency of single cell encapsulation in hydrogel. Different sizes of micropillar arrays act as a sieve to break Escherichia coli (E. coli) aggregates into single cells in polyethylene glycol diacrylate (PEGDA) solution. We applied the external force for the continuous breakup of cell clusters, resulting in the production of more than 70% of single cells into individual hydrogel particles. This proposed strategy and device will be a useful platform to utilize genetically modified microorganisms in practical applications.

scholarly journals Deterministic trapping, encapsulation and retrieval of single-cells

Lab on a Chip ◽  
2017 ◽  
Vol 17 (13) ◽  
pp. 2186-2192 ◽  
Author(s):  
M. Sauzade ◽  
E. Brouzes
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Cell Encapsulation ◽  
Single Cell Encapsulation ◽  
Passive Strategy

Passive strategy for efficient true single-cell encapsulation.

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>

High-Throughput Generation of Durable Droplet Arrays for Single-Cell Encapsulation, Culture, and Monitoring

2018 ◽  
Vol 90 (7) ◽  
pp. 4303-4309 ◽  
Author(s):  
Han Wu ◽  
Xinlian Chen ◽  
Xinghua Gao ◽  
Mengying Zhang ◽  
Jinbo Wu ◽  
...  
Keyword(s):  
Single Cell ◽  
High Throughput ◽  
Cell Encapsulation ◽  
Single Cell Encapsulation

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 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.

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