Program at a Glance 20.04.2021
Fully-automated isolation of circulating tumor cells to enable microfluidic single cell analysis from patient blood
Fraunhofer IMM --- GERMANY
Single cell isolation of circulating tumor cells (CTCs) has become leading-edge in Liquid biopsy to propose personalized therapy approaches for cancer patients, as the number and the genetic fingerprint of tumorassociated cells in patient blood correlates with the disease progression. Here, we would like to showcase a fully-automated CTC isolation platform to dispense CTCs in single droplets through a fluorescence-based microfluidic chip after immunomagnetic positive cell selection from 7.5 mL whole blood. Model systems in breast cancer and squamous cell carcinoma confirmed the device functionality and “CTCelect” enabled single cell isolation from head and neck cancer patients in first studies.
Microfluidic device for high-frequency dielectrophoresis characterization and single-cell discrimination
Institut de recherche (Xlim) --- FRANCE
Glioblastoma is one of the most aggressive solid tumors, particularly due to the presence of cancer stem cells. Today the characterization of those type of cells with an efficient, fast and low-cost method stays an issue. Hence, we have developed an innovative microfluidic lab-on-chip based on the use of Ultra High Frequency electromagnetic waves able to interrogate their intracellular content and so to recognize such cells. Based on dielectrophoresis electromanipulation principle, this label-free system allows us to handle and analyze single cell. Thanks to it, we succeeded at discriminating the undifferentiated from the differentiated phenotypes of U87-MG cells.
Characterizing Cellular Hierarchies Using Quantitative Single-Cell Proteomics
Technical University of Denmark --- DENMARK
We have taken a primary AML patient sample, "8227", and subjected it to a newly developed single-cell proteomics strategy to identify the protein landscapes of individual cells within this patient. By using the latest state-of-the-art LC-MS instrumentation with intelligent data acquisition, this has resulted in an unprecedented map of protein expression in individual AML cells. Furthermore, we developed a computational pipeline (SCeptre) that effectively normalizes the data, clusters the cells, integrates available FACS data and permits the extraction of cell-specific proteins. The approach presented here lays a solid foundation for implementing global single-cell proteomics studies in labs across the world.
Oscillating microchannels for label-free multiparametric cell characterization
Institute of Micro and Nanotechnology (IMN-CNM-CSIC) --- SPAIN
Measuring physical parameters have been demonstrated as a promising technique for label-free cell discerning. In this work we introduce the novel approach of transparent microcapillary resonators (TMR). This approach consists on a doubly-clamped transparent commercial silica tube which oscillates in a guitar-string mode. By means of an interferometric readout system, this technique allows combining mechanical, optical and hydrodynamic analysis obtaining three independent parameters of each individual cell. We demonstrate TMR approach is able to discern between healthy (MCF-10A) and tumoral (MCF-7) cells of human breast tissue. Moreover, this technique allows measuring interesting biophysical parameters as cell mass density.
FocuSeq: A multi-omics single cell sequencing solution to detect mutation and gene expression in individual cells
Singleron BioTechnologies GmbH --- GERMANY
Heterogeneity in mutations and cellular gene expression profiles plays an important role in cancer development and treatment responses . We will present FocuSeq, a high-throughput, multi-omics single cell sequencing library prep solution that can detect both genetic variants and gene expression patterns in single cells, effectively linking genotype to phenotype. The FocuSeq single cell library prep can be performed both manually with a portable microfluidic chip, or automated on SingleronMatrix instrument.
Noncontact Multiphysics Probe for Spatiotemporal Resolved Single-Cell Manipulation and Analyses
NYU Abu Dhabi --- UAE
In this talk, we discuss the noncontact multiphysics probe (NMP) as a novel microfluidic tool for precisely performing multiple probing procedures on living single-cells in tissue-like culture. The tool performs electropermealization-based molecular transfer into single-cells in adherent culture, with fully integrated scanning capability. The technology also opens the door for controllable cytoplasm extraction from living single-cells, where multiple time point biopsies on viable adherent cells can be executed. Further, the NMP is capable of manipulating whole single-cells for total expression assays. The NMP is a multifunctional tool with unprecedented minimally-invasive probing features for spatiotemporal single-cell analysis within tissue samples.
Label-Free Detection of Circulating Tumor Cells with Machine Learning by Brightfield Image and Deformability Analysis
Lehigh University --- USA
This work aims to develop a machine learning-based platform for label-free detection and sorting of Circulating tumor cells (CTCs). This is an improvement of our previous label-free detection model which implements the deformability of the CTC using narrow microfluidic channels. The device deforms the CTCs either by physical contact or by the shear stress inside the channels without damaging the cells. By tracking the velocity of the cell during the flow, deformability and recovery of the cells can be depicted. The velocity data along with the brightfield images can be used to classify the cells more accurately.
Nanopipette Technology: A new tool for Single-Cell Analysis
Yokogawa Deutschland GmbH --- GERMANY
Approaching sub-cellular biological problems requires the incorporation of new technologies and readouts. Due to their low-invasiveness, nanotechnology-based tools hold great promise for single-cell manipulation. In this talk, we discuss the incorporation of electrical measurements into nanopipette technology. Here we will introduce a newly developed single-cell manipulation platform (SU-10) using a nanopipette for single-cell injection into living cells. This newly developed technology positions its nanopipette with nanoscale precision allowing injection of minute amounts of material (drugs, proteins, CRISPR Cas9) into individual cells without comprising cell viability. This strategy will help in basic researchers in drug discovery.
Analyzing killing efficiency in CAR-T cell assays by combining Artificial Intelligence and Micropatterning
ibidi GmbH --- GERMANY
To address single cell assays, ibidi merged two state of the art technologies, Micropatterning and CAR-T cells. These later ones, represent a promising new cancer therapy tool. Merging these tools with live cell imaging and an Artificial Intelligence Algorithm allowed to identify and track cellular behavior. That way we evaluate CAR-T cell potency on a single cell level. Applying a machine learning based image analysis enabled us to track interaction and killing events, viability status and apoptotic body formation in high throughput. The label free phase contrast assays are currently utilized in therapy development.
Improving Multiple Displacement Amplification for Single-Cell Sequencing using Droplet Microarrays
Karlsruhe Institute of Technology --- GERMANY
We present a commercially available droplet microarray (DMA) chip to perform ultra-high-throughput multiple displacement amplification (MDA) reactions in nanoliter droplets to overcome MDA's high cost and amplification bias when amplifying single bacterial cells. The chip consists of hydrophilic spots separated by superhydrophobic barriers to separate individual reactions and reduce contamination risk. Initial tests with single-cell levels of E. coli DNA show that DNA can be successfully amplified on the DMA. This technology will advance the field of microbial single-cell genomics by providing a high-throughput method to obtain cheaper and less biased amplification of single-cell genomes.
Blood Cell Sorting with SAW-based Acoustofluidics
Leibniz-IFW Dresden --- GERMANY
The sorting of specific cells from body fluids, e.g. blood, urine or saliva, is critical for personalized therapies and diagnostics. Current methods often employ a stepwise procedure combining several techniques, whereby a cell fractionation without additional sample treatment is desired. Surface acoustic wave (SAW)-based acoustofluidics can fulfil this demand, whereby the complexity of fabricating microchannels on a wafer substrate is a current limitation to mass produce acoustofluidic chips. Here, we demonstrate an acoustofluidic cell sorter with lithographically defined channels enabling a precise and reliable large-scale production, and its usage in the on-chip separation of blood components.
SC1: A Tool for Interactive Web-Based Single Cell RNA-Seq Data Analysis
University of Connecticut, School of Medicine --- USA
We present SC1 a web-based highly interactive scRNA-Seq data analysis tool publicly accessible at https://sc1.engr.uconn.edu. The tool presents an integrated workflow for scRNA-Seq analysis, implements a novel method of selecting informative genes based on Term- Frequency Inverse-Document-Frequency (TF-IDF) scores, and provides a broad range of methods for clustering, differential expression analysis, gene enrichment, interactive visualization, and cell cycle analysis. The tool integrates other single cell omics data modalities like TCR-Seq and supports several single cell sequencing technologies. In just a few steps, researchers can generate a comprehensive analysis and gain powerful insights from their scRNA-Seq data.
Imaging-based analysis in droplet-microfluidics for high-throughput single-cell experiments
University of Ulm --- GERMANY
Microliter-sized droplets are an isolated reaction chamber for encapsulated cells. They can be produced in large numbers and allow further manipulation like the injection of reactants. However, encapsulation of single cells is a statistical process and has a relatively low yield. Therefore, we developed an imaging-based sensor system that analyses the droplets and their contents at frequencies of up to 10000 Hz in real-time and allows sorting in a microfluidic system based on various object properties like droplets containing a single cell.
CloneSeq: A Highly Sensitive Analysis Platform for the Characterization of 3D cultured single cell derived Clones
The hebrew university of jerusalem --- ISRAEL
Single-cell assays have revealed the scope and importance of heterogeneity in many biological systems. However, single-cell limited sensitivity is a major hurdle for uncovering the full range of cellular variation. To overcome this limitation, we developed "CloneSeq" for 3D hydrogel clone culturing and droplet-based RNA sequencing. We show that clonal cells maintain cell states and share similar transcriptional profiles. Moreover, "CloneSeq" revealed the presence of novel cancer-specific subpopulations, including cancer-stem-like-cells while single-cell RNA-seq assays failed to identify. Our results demonstrate that CloneSeq leveraging the broader expression space within clones to achieve better sensitivity
DIscBIO: A User-Friendly Pipeline for Biomarker Discovery in Single-Cell Transcriptomics
Oslo University --- NORGE
The growing attention toward the benefits of single-cell RNA sequencing (scRNA-seq) is leading to a myriad of computational packages for the analysis of different aspects of scRNA-seq data. For researchers without advanced programing skills, it is very challenging to combine several packages in order to perform the desired analysis in a simple and reproducible way. Here we present DIscBIO, an open-source, multi-algorithmic pipeline for easy, efficient and reproducible analysis of cellular sub-populations at the transcriptomic level. The pipeline integrates multiple scRNA-seq packages and allows biomarker discovery with decision trees and gene enrichment analysis in a network context using single-cell sequencing read counts through clustering and differential analysis.