Laboratory Automation & Informatics 2020

Precision medicine is the paradigm to develop treatments for patients based on molecular-targets that are effective in vivo when administered.  In addition to identifying the molecular and cellular targets that are the source of disease, it is also critical to understand how these targets behave in the body based on physiological principles.  Recent developments in microfluidics have contributed to burgeoning precision medicine fields such as liquid biopsy, immunotherapy, single cell analysis, genotyping and gene sequencing, and microphysiological systems.  The fact that microfluidics bridges the scales of molecular, cellular, tissue, and can even recapitulate organ and circulatory functions of the body enables a microprocessor for a plethora of health indicators. In liquid biopsy, microfluidics can analyze biological samples such as blood for the detection of biomolecules or cells that are indicative of disease or physiological state. Our microfluidic platform, the lateral cavity acoustic transducers (LCAT) can process a drop of blood to 1) sort undiluted donor whole blood into its cellular subsets (plasma, RBCs, and WBCs), 2) enrich and retrieve spiked breast cancer cells at rare cell relevant concentrations (10 per mL), and 3) on-chip immunofluorescent label specific target cellular populations by their known marker expression patterns.  The ultimate goal is to develop a comprehensive panel that can provide a holistic representation of the body’s health status, including molecular and cellular readout that indicate infections, immune status, metabolic status, gene expressions, and cytokine levels.  Furthermore, recent developments in cell therapies requires new approaches towards engineering cells without the complications and limitations of viral vectors. I will present a five-step protein array assay in which HIV, HPV and HSV reactive antibodies from both serum and saliva were rapidly detected by an integrated, rapid and simple-to-use multiplexed microfluidic device. Another example I will present is a 3-D vascularized micro organ (VMO) system connects microfluidic channels to vascularized tissues, forming the basis of the “human body on-chip”.  Applications for this platform include microphysiological systems for screening of drugs, studying vascular malformations, and understanding immune responses. Ultimately this on-chip microcirculation platform maintains ‘microfluidic homeostasis’ for studying bottom up treatment options.

Learning Objectives:

1. Identify the potential for microfluidic devices for multi-scale processing of blood components

2. Describe the approaches for microfluidics to tap into the current cellular and immuno- therapies

3. Explain the importance of vasculature for microphysiological systems

Design and interpretation of genome sequencing assays and data in both clinical diagnostics and research labs is complicated by an inability to identify information from the medical literature and related databases quickly, comprehensively and reproducibly. This challenge is due in part to the complexity and heterogeneity of nomenclatures used by authors to describe diseases, genes, and genetic variants including single nucleotide changes, indels, and structural alterations like fusion genes and copy number variants (CNVs). Mastermind is a unique bioinformatic platform that employs a high-throughput computational technique known as Genomics Language Processing (GLP) to index more than 7.5M full-text articles and 2.5M supplemental datasets associated with such publications. It has automatically identified, disambiguated, and annotated more than 6.0M distinct genetic variants, and identified >50K disease-gene associations. In this webinar, Dr. Kiel will demonstrate how Genomenon’s approach to Big Data and Artificial Intelligence using GLP can connect single-nucleotide variants, in/dels, CNVs, and gene fusions with diseases, phenotypes, and drugs to allow the variant scientist to understand genomic associations across the entirety of the empirical evidence from the medical literature. Taken together, these results will demonstrate the suitability and superiority of GLP for evidence collection and clinical diagnostic variant interpretation for multiple genetic variant types, and highlight the potential benefit in informing both clinical practice and biomedical research.

Learning Objectives:

1. How GLP can improve the sensitivity and specificity of genetic variant searches for single nucleotide and indel variants compared with conventional approaches

2. How GLP can improve the efficiency and accuracy of evidence search for structural variants like CNVs and fusion genes

3. How the evidence resulting from GLP can be used to increase the speed and diagnostic utility of genetic variant interpretation

The current gold standard for COVID-19 diagnosis is based on the detection of SARS-CoV-2 nucleic acid using real-time quantitative PCR (RT-qPCR). Recent studies suggest limitations of RT-qPCR in the detection of SARS-CoV-2, possibly leading to false-negative results. Development of robust and sensitive laboratory tests is of primary importance. Digital PCR is a promising approach to address this challenge, in particular the Crystal Digital™ PCR workflow from Stilla Technologies. To confirm this hypothesis, a SARS-CoV-2 detection kit was developed on the Naica™ System. The assay exploits the system’s unique 3-color 3-channel detection capability to provide a highly sensitive and internally controlled SARS-CoV-2 assay. The targets are two distinct regions (Nucleocapside (N) and ORF1ab genes) of the SARS-CoV-2 positive-strand RNA genome, and an endogenous PCR reference detecting a human housekeeping gene. On-going studies performed in China and France confirm that Crystal Digital PCR allows to 1) reliably classify non conclusive high Ct results obtained in RT-qPCR and 2) uncover false negative results in RT-qPCR. The assay is therefore a promising complementary tool to improve Covid-19 testing and our response to the pandemic.

Learning Objectives:

1. Discover how a unique droplet microfluidic technology automates the digital PCR workflow

2. Understand how Crystal Digital PCR addresses key limitations of RT-PCR for SARS-CoV-2 detection and quantification

Techniques to analyze and sort single cells based on secreted products have the potential to transform our understanding of cellular biology as well as accelerate the development of next generation cell and antibody therapies. However, secretions are rapidly transported away from cells, such that specialized equipment and expertise has been required to compartmentalize cells and capture their secretions. In this presentation I will explain how cavity-containing hydrogel microparticles can be leveraged to perform functional single-cell secretion analysis and sorting in high-throughput using only commonly accessible lab infrastructure. These microparticles act as a solid support which facilitates cell attachment, templates formation of uniform sub-nanoliter aqueous compartments which prevent cross-talk between cells, and captures secreted proteins. Using this “lab on a particle” platform, we demonstrate high-throughput analysis and sorting of Chinese Hamster Ovary cells based on their relative production of human IgG using commercially available flow sorters. We successfully screen over 100,000 cells in a single day and isolate a subpopulation of cells from a production grade cell line that maintained ~50% increased levels of antibody production after 2 weeks of culture. Microparticles can be fabricated in bulk and are easily distributed and used, democratizing access to high-throughput functional cell screening. Ultimately, we believe the ability to rapidly create millions of uniform compartments using lab on a particle technology can extend across fields, providing a fundamental tool that can enable any researcher to perform single-cell and single-molecule assays in the future.

Learning Objectives:

1. Define current approaches to single-cell secretion analysis and identify limitations

2. Explain how microparticles can be leveraged to perform assays with standard lab equipment

The basic units of biological structure and function are cells, which exhibit wide variation in regard to both type and state.  We assess such variation by simultaneously profiling the transcriptomes of thousands of single mammalian cells or nuclei, using high-throughput emulsion microfluidics and DNA barcodes. When extracting intact cells from tissues and organs is technically challenging or impossible, single-nucleus RNA sequencing can provide an alternative way to obtain transcriptome profiles of such tissues. To systematically assess the differences between single cell and single-nuclei RNA-seq, we compared Drop-seq (single cell) and DroNc-seq (single nucleus) using a time course experiment on human induced pluripotent stem cells (iPSCs) differentiating into cardiomyocytes. We then applied DroNc-seq to postmortem heart tissue which is not accessible by Drop-seq. Our data confirm that DroNc-seq yields similar results to Drop-seq on matched samples and can be successfully used to generate reference maps for the human cell atlas. We are using Drop-seq and DroNc-seq to generate cellular maps and their transcriptional profiles of the gut (ileum and proximal colon in health and disease), female reproductive system, and heart. In addition to Drop-seq and DroNc-seq, we are also benchmarking and using single cell ATAC-seq and organoid models to generate these cellular, molecular and functional maps for the global Human Cell Atlas initiative.

Learning Objectives:

1. Describe and benchmark two microfluidic droplet based single cell and single nucleus RNA seq techniques on human iPSC derived cardiomyocytes

2. Recent applications of the above techniques in generating molecular and functional maps of in different complex tissues in context of health and disease