Laboratory Automation & Informatics 2020

Many questions at the forefront of biology depend on the interactions of millions of single cells. My lab develops technologies for studying large numbers of single cells. In this talk, I will describe our approaches for sorting cells based on genomic and transcriptomic markers, and performing multi-omics analysis of single cells that allow simultaneous characterization of genomic, transcriptomic, and proteomic signatures. I will also describe how we are adapting these techniques to integrate genomics with other single cell measurement approaches, including imaging, mass spectrometry, and atomic force microscopy. Finally, I will describe how we are using these techniques to build cells into controlled consortia for microbiological studies and bottom-up tissue synthesis.

Learning Objectives:

1. Familiarization with microfluidic methods for single-cell multi-omics analysis

2. Application of microfluidics to bottom-up cell community assembly

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

We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI) MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision and accuracy; subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro and in vivo adsorption, distribution, metabolism, elimination, pharmacokinetic (PK) and biomarker analysis, and HT parallel medicinal chemistry.

Learning Objectives:

1. Explain the front-end separation bottleneck to high-throughput mass spectrometry

2. How to achieve 50X faster mass spectrometry sample analysis with no compromise in data quality

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

If you have ever worked in the field of gene-editing, then you must know the powerful gene-editing technology known as CRISPR.  It has the potential to alter science and biotechnology at an inconceivable rapid rate.  But what you may not know is the laborious and manual intensive work that is needed to edit cells.  Gene editing of course has incredible promise in fields like synthetic biology and medicine, but without automated technologies to expedite their process, these could just be figments of our imagination.  In my talk, I will describe how my lab has created an automated microfluidic benchtop platform that will edit cells on-demand using a user-activated sequence.  I will also describe the use of our system for rapid editing of mammalian cells and how we can carry out manipulations at the single cell level with near instant generation of clonal, isogenic cell lines (and comparing to gold-standard techniques).  Finally, I will describe our ongoing efforts to make this technology accessible to all users (in collaboration with our startup DropGenie).

Learning Objectives:

1. Define current paradigm of gene editing

2. Identify bottlenecks in the process of gene editing

3. Explain automation approaches to gene edit cells

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

The digital lab is knocking at your door. In the age of digitalization and Industry 4.0, the laboratory is also on its way to the future. Lab 4.0 includes robotic systems, fully automated workflows, and complete integration of instruments and software systems. The software products work hand in hand and manual input from the laboratory employee is reduced to a minimum. Enterprise installations such as ERP, MES, PLS and LIMS exchange data fully automatically with each other and communicate bi-directionally with technical equipment such as balances, HPLC, GC, Bioanalyzers, PCR and many more. The laboratory employee is completely focused on his scientific work and is supported by the supporting software. The employee will work on the lab equipment and as if by magic the software will automatically create lab orders, samples, results and enter the measured values including its meta data. Raw data like AniML Files will be stored in a save place inside a database for later review on demand. Since QA/QC laboratories, clinical diagnostic and routine laboratories work according to high regulatory requirements such as 21 CFR Part 11, GAMP 5, GxP, DIN EN ISO 17025, DIN EN ISO 15189, a highly integrated and fully automatic operation saves & supports the modern laboratory as risks are reduced significantly and data is not handled manually. The example of the integration of a LIMS Middle Ware with SAP will show how such a solution can work and which special conditions should be considered. SAP represents other ERP solutions that can be integrated using the same procedure. In addition to the technical implementation, strategic and commercial aspects are also discussed, because the best technical solution is not helpful if it is not cost-neutrally feasible on a long term.

Learning Objectives:

1. Explaining the benefit of an end to end workflow (Example SAP-LIMS Middleware-SAP)

2. Understanding LAB 4.0 data structure in a modern lab (Instrument Integration & RAW data)

3. Learning about strategic & commercial key criteria for a LAB 4.0 solution

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

Proteins are the functional blocks of any biological entity from 50 nm sized viruses to the 100 ft long mammals (blue whale). They perform intra cellular, tissue, organ, system, or organism level essential functions. As one of the most diverse, the human proteome consists of millions of different proteins serving even a greater number of functions. Most diseases are viewed and targeted from a proteomic perspective via 1) a result of functional change of a protein, 2) up or down regulation of a protein 3) or introduction of intruder proteins to serve intruder functions (ie infection). In this perspective, most drugs developed in pharma (along with the ones in the pipeline) are either a small molecule targeting a protein, or they are proteins themselves, or involves a genomic approach again targeting to fix a proteomic problem at the functional level. While in the early stage discovery phase, mass spec is utilized; in most downstream stages of R&D, immunoassays are the work horse. Every day, the relevance of proteomic data for actionable outcomes is increasing.  Despite this interest, from the early days since the technology was introduced (1950s), immunoassays have not seen much of a change.  The principle underlying biophysics still relies on diffusion limited interaction of a capture molecule (usually an antibody) and the target molecule. Expanding on the above discussion, we will talk about the biophysical challenges prohibiting the ubiquitous adoption of immunoassays and general shortcomings. Later we will discuss how Correlia’s ANDI gel technology coupled with advanced automation offers complete workflow solutions, speeds up assays, and reduces sample use.  We will also discuss how these value offerings increase decision making prover, open up new possibilities in precision medicine, and enable more data driven biology. The talk will end with reflections over the role of efficient immunoassays in the fight with COVID-19.

Learning Objectives:

1. How microfluidics advancements coupled with automation offer solutions to overcome challenges with 70-year-old ELISA technology

2. How above solutions can be applied to precision medicine, pharma drug development, and the fight with COVID-19

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

The amount of data that is produced each day is mind boggling. Over 2.5 quintillion bytes of data are created every single day, and this number continues to grow every single day. This year (2020) it is estimated that 1.7MB of data will be created every second for every person on Earth according to the Digital Information World Blog. In the analytical testing laboratory, we have entered a new age in the scale and complexity of data as well as the pace at which the data is generated. Today laboratories require secure, compliant Laboratory Information Management Systems and powerful tools for analysis and to extract both laboratory and business intelligence from their data. More and more organizations will begin feeding their LIMS as well as their unstructured data into a Data Lake. The key to making good decisions is having high quality data. This presentation will examine the importance of data integrity, along with the role of LIMS and other tools that can be used to help analytical laboratory professionals to produce, manage, analyze and maintain high quality data. We will also examine the impacts of poor data integrity with a few examples and understand what it means to the laboratory.

Learning Objectives:

1. Understanding the importance of Data Integrity, what it entails and how to achieve it

2. Learning how LIMS and laboratory automation tools can facilitate regulatory compliance and enforce data integrity

3. Understanding the potentially devastating ramifications when data integrity is not a priority and is not managed, maintained and supported.

LIMS systems are used extensively in laboratories everywhere. As a tool in the lab we need to be able to trust the system to have the necessary data integrity. Just like the analytical methods, also the tool LIMS must be validated. Otherwise: How can you trust the system? Validation of tools like LIMS is a part of all lab standards; the GxP and ISO 17025 to mention a couple. This short presentation will cover the approach to validation of LIMS. The same approach can be used for all other IT systems including the instrument systems in the lab. Tools in the process are also mentioned.

Learning Objectives:

1. Approach to validation and which tools to use

2. Content of the validation plan and its parts, including how to create test plans and document the testing

3. On-going validation

Analytical Method Validation work is based on a variety of guidelines and requirements hence being time-consuming and stressful. Reducing this work and improving efficiency while ensuring the highest level of data integrity is welcome by both QA and laboratory – but how can this be achieved? Doing method validation is much more than just calculating results – it’s setting up your testing workload, both simple and flexible, with final documentation matching customer expectations as well as regulatory and industry requirements. The key for an optimized workflow is predefining all aspects of typical method validation work by means of powerful software workflow templates that reduce new projects to a matter of a few clicks. Such templates follow your SOPs and structure your tasks to optimize efficiency and compliance. Communication with your laboratory equipment (CDS, LIMS, ECM, ERP, DMS) should be automatic and easy with manual intervention limited to a minimum. While templates define routine work, it’s critical to allow flexibility for each new project – additional calculations, graphics and report extensions should be added easily. Since your data is worth being protected and comprehensible all this must be accompanied by a maximum level of data integrity, which means strict access control, full traceability, version history and full 21 CFR Part 11 compliance. This presentation will provide guidance and show optimized software workflows saving at least 70% of your time compared to performing these tasks manually.

Learning Objectives:

1. Analytical method validation – requirements and data integrity

2. Establishing an optimized workflow with minimum effort

3. Integrate and embed your method validation workflow into your lab environment