Browse
Submit Data
Databases
API
Help

Dataset Information

30 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Functional TCR T cell screening using single-cell droplet microfluidics.

ABSTRACT: Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell activation upon recognition of target tumor cells. Notably, our platform includes a tracking system for each clone as well as a sorting procedure with 100% specificity validated by downstream single cell reverse-transcription PCR and sequencing of TCR chains. Our TCR screening prototype will facilitate immunotherapeutic screening and development of T cell therapies.

SUBMITTER: Segaliny AI

PROVIDER: S-EPMC6279597 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Functional TCR T cell screening using single-cell droplet microfluidics.

Segaliny Aude I AI Li Guideng G Kong Lingshun L Ren Ci C Chen Xiaoming X Wang Jessica K JK Baltimore David D Wu Guikai G Zhao Weian W

Lab on a chip 20181201 24

Adoptive T cell transfer, in particular TCR T cell therapy, holds great promise for cancer immunotherapy with encouraging clinical results. However, finding the right TCR T cell clone is a tedious, time-consuming, and costly process. Thus, there is a critical need for single cell technologies to conduct fast and multiplexed functional analyses followed by recovery of the clone of interest. Here, we use droplet microfluidics for functional screening and real-time monitoring of single TCR T cell a ...[more]

PMID: 30397689

Similar Datasets

Functional single-cell hybridoma screening using droplet-based microfluidics.

Project description:Monoclonal antibodies can specifically bind or even inhibit drug targets and have hence become the fastest growing class of human therapeutics. Although they can be screened for binding affinities at very high throughput using systems such as phage display, screening for functional properties (e.g., the inhibition of a drug target) is much more challenging. Typically these screens require the generation of immortalized hybridoma cells, as well as clonal expansion in microtiter plates over several weeks, and the number of clones that can be assayed is typically no more than a few thousand. We present here a microfluidic platform allowing the functional screening of up to 300,000 individual hybridoma cell clones within less than a day. This approach should also be applicable to nonimmortalized primary B-cells, as no cell proliferation is required: Individual cells are encapsulated into aqueous microdroplets and assayed directly for the release of antibodies inhibiting a drug target based on fluorescence. We used this system to perform a model screen for antibodies that inhibit angiotensin converting enzyme 1, a target for hypertension and congestive heart failure drugs. When cells expressing these antibodies were spiked into an unrelated hybridoma cell population in a ratio of 1:10,000 we observed a 9,400-fold enrichment after fluorescence activated droplet sorting. A wide variance in antibody expression levels at the single-cell level within a single hybridoma line was observed and high expressors could be successfully sorted and recultivated.

| S-EPMC3406880 | biostudies-other

Single-cell analysis and sorting using droplet-based microfluidics.

Project description:We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ?200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ?1 million cells, the microfluidic operations require 2-6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5-7 d.

| S-EPMC4128248 | biostudies-literature

Single cell kinase signaling assay using pinched flow coupled droplet microfluidics.

Project description:Droplet-based microfluidics has shown potential in high throughput single cell assays by encapsulating individual cells in water-in-oil emulsions. Ordering cells in a micro-channel is necessary to encapsulate individual cells into droplets further enhancing the assay efficiency. This is typically limited due to the difficulty of preparing high-density cell solutions and maintaining them without cell aggregation in long channels (>5 cm). In this study, we developed a short pinched flow channel (5 mm) to separate cell aggregates and to form a uniform cell distribution in a droplet-generating platform that encapsulated single cells with >55% encapsulation efficiency beating Poisson encapsulation statistics. Using this platform and commercially available Sox substrates (8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline), we have demonstrated a high throughput dynamic single cell signaling assay to measure the activity of receptor tyrosine kinases (RTKs) in lung cancer cells triggered by cell surface ligand binding. The phosphorylation of the substrates resulted in fluorescent emission, showing a sigmoidal increase over a 12 h period. The result exhibited a heterogeneous signaling rate in individual cells and showed various levels of drug resistance when treated with the tyrosine kinase inhibitor, gefitinib.

| S-EPMC4032411 | biostudies-other

High-resolution dose-response screening using droplet-based microfluidics.

Project description:A critical early step in drug discovery is the screening of a chemical library. Typically, promising compounds are identified in a primary screen and then more fully characterized in a dose-response analysis with 7-10 data points per compound. Here, we describe a robust microfluidic approach that increases the number of data points to approximately 10,000 per compound. The system exploits Taylor-Aris dispersion to create concentration gradients, which are then segmented into picoliter microreactors by droplet-based microfluidics. The large number of data points results in IC(50) values that are highly precise (± 2.40% at 95% confidence) and highly reproducible (CV = 2.45%, n = 16). In addition, the high resolution of the data reveals complex dose-response relationships unambiguously. We used this system to screen a chemical library of 704 compounds against protein tyrosine phosphatase 1B, a diabetes, obesity, and cancer target. We identified a number of novel inhibitors, the most potent being sodium cefsulodine, which has an IC(50) of 27 ± 0.83 ?M.

| S-EPMC3258639 | biostudies-literature

Automated Chemotactic Sorting and Single-cell Cultivation of Microbes using Droplet Microfluidics.

Project description:We report a microfluidic device for automated sorting and cultivation of chemotactic microbes from pure cultures or mixtures. The device consists of two parts: in the first part, a concentration gradient of the chemoeffector was built across the channel for inducing chemotaxis of motile cells; in the second part, chemotactic cells from the sample were separated, and mixed with culture media to form nanoliter droplets for encapsulation, cultivation, enumeration, and recovery of single cells. Chemotactic responses were assessed by imaging and statistical analysis of droplets based on Poisson distribution. An automated procedure was developed for rapid enumeration of droplets with cell growth, following with scale-up cultivation on agar plates. The performance of the device was evaluated by the chemotaxis assays of Escherichia coli (E. coli) RP437 and E. coli RP1616. Moreover, enrichment and isolation of non-labelled Comamonas testosteroni CNB-1 from its 1:10 mixture with E. coli RP437 was demonstrated. The enrichment factor reached 36.7 for CNB-1, based on its distinctive chemotaxis toward 4-hydroxybenzoic acid. We believe that this device can be widely used in chemotaxis studies without necessarily relying on fluorescent labelling, and isolation of functional microbial species from various environments.

| S-EPMC4831006 | biostudies-literature

Massively parallel whole genome amplification for single-cell sequencing using droplet microfluidics.

Project description:Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

| S-EPMC5507899 | biostudies-literature

Drug screening of cancer cell lines and human primary tumors using droplet microfluidics.

Project description:Precision Medicine in Oncology requires tailoring of therapeutic strategies to individual cancer patients. Due to the limited quantity of tumor samples, this proves to be difficult, especially for early stage cancer patients whose tumors are small. In this study, we exploited a 2.4 × 2.4 centimeters polydimethylsiloxane (PDMS) based microfluidic chip which employed droplet microfluidics to conduct drug screens against suspended and adherent cancer cell lines, as well as cells dissociated from primary tumor of human patients. Single cells were dispersed in aqueous droplets and imaged within 24 hours of drug treatment to assess cell viability by ethidium homodimer 1 staining. Our results showed that 5 conditions could be screened for every 80,000 cells in one channel on our chip under current circumstances. Additionally, screening conditions have been adapted to both suspended and adherent cancer cells, giving versatility to potentially all types of cancers. Hence, this study provides a powerful tool for rapid, low-input drug screening of primary cancers within 24 hours after tumor resection from cancer patients. This paves the way for further technological advancement to cutting down sample size and increasing drug screening throughput in advent to personalized cancer therapy.

| S-EPMC5567315 | biostudies-literature

Single nuclei RNA-seq using droplet microfluidics (DroNc-seq)

Project description:We report the application of DroNc-seq on archived tissue from mouse adult hippocampus and cortex barin regions

2017-10-06 | GSE104525 | GEO

High throughput single cell counting in droplet-based microfluidics.

Project description:Droplet-based microfluidics is extensively and increasingly used for high-throughput single-cell studies. However, the accuracy of the cell counting method directly impacts the robustness of such studies. We describe here a simple and precise method to accurately count a large number of adherent and non-adherent human cells as well as bacteria. Our microfluidic hemocytometer provides statistically relevant data on large populations of cells at a high-throughput, used to characterize cell encapsulation and cell viability during incubation in droplets.

| S-EPMC5431057 | biostudies-literature

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

Project description: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.

| S-EPMC8552120 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data