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Society for Laboratory Automation and Screening (SLAS) conference
Expertise in automated high-throughput qPCR, stem cell and gene editing solutions, and single-cell technology
The SLAS community promotes the synthesis of new technologies, partnerships, and ideas that advance laboratory workflows. The SLAS conference is an essential hub for this community of experts, who are forging ahead to apply state-of-the-art technologies to find solutions for significant biological challenges in an interdisciplinary manner.
Adding to the body of expertise at the SLAS conference, Takara Bio offers attendees expert guidance, technologies, and services for advancing their qPCR, stem cell, gene editing, and protein discovery research. Our instruments, such as the ICELL8 Single Cell System with CELLSTUDIO Software and SmartChip Real‑Time PCR System, and our Cellartis, Guide-it, and single cell NGS portfolios, provide researchers with novel screening tools that bring unparalleled consistency to automation and screening efforts.
We are excited to help you find the best solutions for your research at SLAS 2020. In the meantime, we invite you to preview the materials we will present in San Diego, CA. You can also sign up here to meet with one of our experts or drop by Booth #408.
SLAS 2020: talk and posters
Streamlining the drug discovery workflow with human iPS-derived cells and ICELL8 cx single-cell technology
Date: Tuesday, January 28
Time: 2:00 pm–2:45 pm
Location: Room 1B
Novel human iPS-derived hepatocytes and intestinal epithelial cells for more accurate disease modeling and drug discovery
Human iPS-derived cells provide a renewable source of cells that can be used for a variety of downstream applications to study and treat disease. We have developed robust protocols for highly efficient differentiation of hiPSCs to definitive endoderm and further differentiation into hepatocytes and intestinal epithelial cells. These cells are mature and functional, providing researchers with improved models to advance their disease and drug discovery research.
Efficient, versatile assay development using the ICELL8 cx Single-Cell System with CELLSTUDIO Software
Developing new assays for high-throughput sample analysis—whether it's single cells, organoids, or nucleic acids—requires open and flexible solutions. The ICELL8 cx Single-Cell System was designed with these needs in mind and integrates cell dispensing, imaging, and processing steps on one platform. The included ICELL8 cx CELLSTUDIO v2.0 Software provides a simple graphical user interface for developers to design, test, and improve their own miniaturized protocols or next-generation sequencing assays. The versatility of the ICELL8 cx system enables assay developers to continue innovation in clinical research, pharmaceutical discovery, and applied spaces.
Detecting allele-specific genome editing outcomes using a fluorescence-based screening method
Date: Tuesday, January 28
Time: 2:00–3:00 pm
Poster number: 1082-C
One of the most powerful applications of genome editing is the ability to introduce precise changes at genomic loci of interest. However, the success rate for these types of experiments is low since it relies on endogenous repair mechanisms. Therefore, optimization of the editing protocol as well as application of sensitive screening methods for identifying successfully edited clones are essential factors for success.
To address this need, we developed a simple fluorescence-based method that enables detection of successful homology-directed repair (HDR) events independent of their length (from single-nucleotide substitutions to longer insertions) or the sequence at the targeted genomic site. The assay consists of PCR amplification of the genomic target site, followed by an enzymatic assay with a dual-color fluorescence-based readout using a standard plate reader. A positive fluorescent signal from the assay indicates the correct introduction of the desired edit.
Generating human disease models with heterozygous SNPs (one allele encoding the SNP, the other wild-type) is particularly challenging due to the low frequency of successful HDR and the propensity towards non-homologous end joining (NHEJ); as most edited clones encode the SNP in one allele and an indel in the other. Developed with these challenges in mind, the dual-color capability of our assay can be used to positively and unequivocally identify these rarely occurring heterozygous clones. As a test case, we introduced a SNP in an endogenous gene in human pluripotent stem cells (hiPSCs). To achieve a higher percentage of heterozygous clones, we used a mix of HDR templates encoding the SNP or the wild-type allele, both with mutations at the PAM site to prevent recutting by the Cas9 endonuclease. With the help of the dual-color screening assay, edited clones were successfully identified and verified by Sanger sequencing.
Additionally, for scenarios involving knockin (KI) of longer sequences, the assay allows for the simultaneous detection of seamless insertions at both 5' and 3' ends of the recombinant sequence. As a test case, we engineered hiPSCs with a fusion of a myc tag with the UGT1A9 gene (related to drug metabolism) and screened the clonal cell lines with the dual-color screening assay. We were able to discern cell lines with either partial or complete insertions due to the assay's ability to interrogate both the 5' and 3' ends of the insert.
Development of novel hiPSC-derived hepatocytes and intestinal epithelial cells to advance disease modeling and drug discovery
Date: Tuesday, January 28
Time: 5:00–6:00 pm
Poster number: 1088-D
Reliable and relevant in vitro cell models are crucial for advancing disease modeling and drug discovery. Primary cells and immortalized cell lines have long been the gold standard models, but they have several shortcomings. Primary cells mimic the functionalities of cells in vivo, but their utility is significantly limited by their rapid loss of function when cultured in vitro, the large variation between donors, and the finite number of cells harvestable from each donor. Immortalized cell lines provide an inexpensive and readily available source of cells but, as they are often derived from tumors, they may not provide physiologically relevant results and/or accurately reflect in vivo cell function.
Human induced pluripotent stem (hiPS) cell-derived cells address several limitations of these other models. hiPS-derived cells provide a renewable source of cells that can be used for a variety of downstream applications to study and treat disease. To leverage this, we have developed a robust protocol for the highly efficient differentiation of hiPS cells into definitive endoderm and further differentiation into disease-relevant cell types. We have used this protocol to generate mature and functional hiPS cell-derived hepatocytes that express genes important to drug-metabolizing machinery such as CYPs, phase II enzymes, and transporters for the entire culture time. Next, we exposed these hiPS cell-derived hepatocytes to known hepatotoxins for up to 14 days and found they respond appropriately to these toxic compounds, demonstrating their utility for chronic toxicology studies. The hiPS cell-derived hepatocytes also respond to insulin and can take up and store low-density lipoproteins and fatty acids.
We also developed a novel protocol for the differentiation of definitive endoderm cells into small intestinal epithelial cells (IECs). These cells express key IEC markers, as well as important enzymes and transporters that are involved in drug metabolism, at levels similar to primary intestinal cells. Additionally, the hiPS cell-derived IECs form a functional barrier for intestinal permeability and absorption studies.
Taken together, we have developed differentiation protocols to produce mature, functional hiPS-cell derived hepatocytes and intestinal epithelial cells with improved functionality and relevance compared to the current gold standards. These cells provide researchers with a readily available and more accurate model to advance their disease and drug discovery research.
Development of custom single-cell assay protocols using the ICELL8 cx system and open architecture
Date: Tuesday, January 28
Time: 2:00–3:00 pm
Poster number: 1087-C
The use of next-generation sequencing in clinical research and applied spaces requires accurate, parallel processing of large numbers of samples and the availability of chemistries that enable robust library preparation from the desired targets. Given the rapid advancements in single-cell and nucleic acid research, one of the challenges is finding an instrument platform that can support such dynamic fields. In this poster, we will present a diversity of single-cell assay workflows developed successfully using the ICELL8 cx Single-Cell System's nanowell technology, imaging capabilities, and software solutions.
Developing new assays for high-throughput analysis of single cells requires an open and flexible platform. The ICELL8 cx system was designed with these features in mind, and the new ICELL8 cx CELLSTUDIOTM v2.0 Software (CELLSTUDIO Software) provides a simple graphical user interface for developers to design, test, and improve their own miniaturized protocols. The types and number of samples that can be tested are configurable and utilizing multiple reagent source wells allows for multiple reaction conditions to be tested in parallel. Along with high numbers of replicates for each reaction condition, positive and negative controls can be included with each test condition, giving researchers confidence in their results. The ICELL8 cx system is also a high-throughput method to isolate nuclei or single cells of any size—even accommodating cardiomyocytes and organoids—while providing control over the selection of the isolated cells out of all available 5,184 nanowells. Furthermore, the system enables the flexibility to analyze multiple parameters per experiment and uses the power of imaging to distinguish and select desired cells based on viability or phenotype—allowing meaningful conclusions to be drawn from the data. CELLSTUDIO Software and the ICELL8 cx system together provide an ideal system for the development of miniaturized, high-throughput single-cell assays.
SLAS 2019: talk and posters
Advances in Industrial-Scale Generation of Human Hepatocytes for Liver-Disease and Drug Development Studies
To realize the full potential of human pluripotent stem cells (hPSC) in regenerative medicine, disease modeling, and drug discovery, optimized culture conditions are required that allow homogenous populations of undifferentiated stem cells to be generated, followed by directed differentiation into preferred cell types of interest in a robust and predictable manner. We have previously developed an optimized hPSC culture system, called the Cellartis DEF-CS 500 Culture System, which enables non-colony, monolayer culture of hPSCs and results in highly pluripotent cells that exhibit low spontaneous differentiation and stable karyotypes. These cells provide a rapidly renewable source of hPSCs that are highly amenable to downstream differentiation into a variety of disease-relevant cell types. Using an optimized protocol that combines DEF-CS media, supplements, and coating reagents optimized for 2D monolayer culture, this tutorial will describe a standardized workflow that mimics embryonic development, allowing for highly efficient differentiation of hPSCs to definitive endoderm and further differentiation into hepatocytes. A case study will be presented that highlights the application of this novel endodermal differentiation system to drug metabolism/safety toxicology studies and disease modeling, which includes the creation of large panels of industrial-scale hPSC-derived hepatocytes with specific genotypes and phenotypes. We will also demonstrate that the novel hepatocyte maintenance medium developed for our cryopreserved hPSC-derived hepatocytes maintains the viability of cryopreserved human primary hepatocytes for over four weeks in culture, which is in sharp contrast to existing hepatocyte maintenance media available on the market today. The increased assay window for both functional hPSC-derived and human primary hepatocytes in 2D cultures represents an important step toward advancing the discovery of new treatments for metabolic disease, reducing the incidence of drug-induced liver injury, and developing new strategies for liver regeneration and transplantation.
High-capacity system for rapid purification of antibodies using Protein A and Protein G membranes
Antibody engineering, production, and purification are critical in a wide range of research settings, such as academic research institutions and biopharmaceutical organizations. There is a constant need for better, faster, and more efficient processes for antibody purification at various scales. Protein A has historically been one of the most widely used methods for affinity purification of immunoglobulins (IgG) and allows the opportunity for several-fold enrichment in fewer steps, along with high recovery rates. Agarose resins with immobilized Protein A are typically used for this process, with capacities ranging from 18 to 35 mg/ml. Resin-based purification requires a significant amount of work and may take up to a few hours to complete, due to long column equilibration/binding times and slow diffusion of large macromolecules through the resin bed. These longer times, in turn, increase the possibility of antibody aggregation or degradation or loss of activity due to unfolding or denaturation. Membrane-based affinity systems have rapid, flow-induced mass transport and short residence times; however, traditionally they have been plagued with low capacity, due to small internal surface areas. Here we describe a novel, membrane-based system with Protein A or Protein G affinity chemistry in which the pore surface area has been chemically enhanced, leading to a protein binding capacity better than that of resins at 75 mg or more per cm3 of membrane. However, unlike traditional resin-based systems, the entire purification process—from loading the sample to eluting pure antibody—can be completed at room temperature in less than five minutes. We have assembled these high-capacity membranes into spin columns and filtration devices, such as 96-well plates, and demonstrate that they can purify antibodies from a variety of samples, such as animal sera, cell culture supernatants, etc. We further characterize the binding properties of these Protein A membranes and demonstrate their utility in immunoprecipitation (IP) and co-immunoprecipitation (Co-IP) experiments. We have compared our Protein A and Protein G membranes with commercially available resins and show that Capturem membranes result in more concentrated antibodies in significantly less time. These novel membrane-based affinity columns are extremely useful for purification and characterization of various antibody isotypes for a variety of applications.
Streamlined production, application, and analysis of pooled, genome-wide sgRNA lentiviral libraries
Genome-wide loss-of-function genetic screens are a powerful way to identify novel protein functions and biological processes within a cell. A common approach for in vitro loss-of-function screens is to knock out genes in a population of cells, apply selective pressure, and then identify mutations that are either enriched or depleted in the selected population relative to a control. The easy programmability and high knockout efficiency of the CRISPR/Cas9 system have helped researchers maximize the potential of this in vitro screening method to identify genes responsible for a given phenotype of interest. Current methods using pooled sgRNAs in loss-of-function screens rely on lentiviral-vector-based delivery followed by next-generation sequencing (NGS) to analyze the resulting distribution of sgRNA sequences in screened cell populations. Inherent challenges include maintaining sgRNA representation in lentiviral plasmids, achieving optimal titers upon scale-up of lentivirus production, and preparing high-quality NGS libraries that accurately reflect the distribution of sgRNA sequences.
We present a streamlined approach for producing Cas9+/sgRNA+ cell populations in sufficient quantities for a genome-wide screen and for generating NGS libraries used to assess changes in sgRNA representation, using the Guide-It CRISPR Genome-Wide sgRNA Library System. Our methods enable even novice users to perform genome-wide phenotypic screens without concerns for sgRNA representation, low virus titer, or NGS library preparation.
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