Society for Laboratory Automation and Screening (SLAS) conference
Expertise in automated, high-throughput qPCR, stem cell and gene editing solutions, and protein purification
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 SmartChip Real‑Time PCR System, and our Cellartis, Guide-it, and Capturem product 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 SLAS2020. In the meantime, we invite you to review the materials we presented at past SLAS meetings and reach out to our scientists with any questions or requests via the "talk to a scientist" link below.
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.
SLAS 2018: talks and posters
Flexible, high-throughput qPCR for genotyping and gene expression analysis using the SmartChip Real‑Time PCR System
In this Exhibitor Tutorial, we present multiple gene expression and genotyping studies that demonstrate the reliability and flexibility of the SmartChip Real‑Time PCR System. This quantitative PCR platform combines the high-throughput nature of microarrays with the sensitivity, precision, and dynamic range of quantitative real-time PCR. The power of the SmartChip system is derived from the 5,184 individual nanowells included in each chip provided in the SmartChip MyDesign Kit. The chips can be configured with 14 different assay and sample arrangements, allowing you to run the experiments you want instead of limiting them to rigid sample and assay formats. The SmartChip system utilizes 100-nl reactions with only 3–10 ng/µl of input required per reaction, which provides: 1) the sensitivity needed to eliminate the preamplification step, and 2) significant reagent and cost savings over 25‑µl reactions in 384‑well plates. With the SmartChip system, you can seamlessly switch between dispensing assay reagents and samples into blank chips and dispensing samples into custom, preprinted chips—without the need for revalidation. Run experiments the way you want, while getting the accurate and consistent results you expect.
If you'd like to learn more, request a copy of the technical brochure.
Innovative CRISPR/Cas9 gene knockin and SNP-detection tools for establishing human iPSC-derived disease model lines for drug screening
The unique combination of precise, footprint-free editing using CRISPR/Cas9 and human induced pluripotent stem (hiPS) cells facilitates a new level of sophistication in generating disease models which allow for rapid advancement in the development of new therapeutics. While CRISPR/Cas9-based gene editing is an effective technique to obtain knockout mutations with high efficiency, knocking in longer genes or sequences (>200 bp) via homology directed repair (HDR) is difficult to complete successfully. Therefore, more sophisticated screening tools are required for these low-efficiency knockins so that researchers can easily identify the edited clonal cell lines containing the engineered sequence.
One of the most powerful applications of genome editing is the introduction of base changes at specific genomic sites, resulting in sequences that mimic single-nucleotide polymorphisms (SNPs) related to human diseases or contain stop codons which generate gene knockouts. However, screening large numbers of clones to identify edited clonal cell lines containing the engineered base-of-interest is still a bottleneck, especially in the absence of a phenotypic readout.
To address this need, we developed a simple, high-throughput SNP-detection method that allows for rapid screening of clones from 96‑well plates and detection of edited clonal cell lines independent of the engineered nucleotide substitution and the surrounding targeted genomic loci. As a proof-of-concept, we applied this method to successfully detect all of the possible transitions in several human gene loci using genomic DNA as template or performed directly in cultured human fibroblasts. This screening method was then successfully used to screen hiPSCs clonal cell lines for SNPs related to tyrosinemia that were generated using CRISPR/Cas9.
A novel maintenance medium extends the lifespan and enables long-term applications for both human primary hepatocytes and human pluripotent stem cell-derived hepatocytes in conventional 2D cultures
Human primary hepatocytes are considered the gold standard for in vitro model systems of liver function for drug development, toxicity assessment, and metabolic disease research; however, their rapid loss of cell viability in conventional 2D culture limits their utility in these applications. Human induced pluripotent stem (hiPS) cell-derived hepatocytes have potential as a better in vitro model if they possess a relevant usage window and functionality—but this is challenging to accomplish.
Addressing these problems, our newly developed hepatocyte maintenance medium enables the culture of cryopreserved human primary hepatocytes or hiPS cell-derived hepatocytes for four or two weeks, respectively, with maintained viability and stable activities of several key cytochrome P450 enzymes (CYPs). Multiple analyses on cryopreserved hiPS cell-derived hepatocytes, including RT-qPCR, immunostainings, functional assays such as albumin secretion, and CYP activity assays demonstrate mature features and high functionality. Importantly, the hiPS cell-derived hepatocytes show expression of the essential genes of the drug-metabolizing machinery, such as CYPs, phase II enzymes, and transporters.
An extended in vitro culture time for hepatocytes enables chronic toxicity testing. We show that hiPS cell-derived hepatocytes can be exposed to known hepatotoxins for up to 14 days. Cells respond as expected to these toxic compounds, demonstrating their utility for chronic toxicity studies. The hiPS cell-derived hepatocytes also respond to insulin, and they can take up and store low-density lipoproteins and fatty acids.
The novel maintenance medium presented here maintains the viability and functionality of cryopreserved human primary hepatocytes and hiPS cell-derived hepatocytes from multiple lines for a much longer time than existing commercially available hepatocyte maintenance media. We hope that the increased assay window of functional hepatocytes in 2D cultures will empower new areas of liver research and applications.
A fast and reliable method for detecting base editing in clonal cell lines
One of the most powerful applications of genome editing is the introduction of base changes in specific genomic sites to mimic single-nucleotide polymorphisms (SNPs) related to human diseases or introducing stop codons to generate precise gene knockouts. However, screening a large number of clones to identify edited clonal cell lines containing the engineered base of interest is still a bottleneck, especially if no phenotypic readout is applicable. Sanger sequencing is a potential approach to detect SNPs, but it is not easy to apply in a high-throughput manner; next-generation sequencing, in contrast, allows researchers to screen 96-well plates but at a far higher cost.
To address this need, we developed a simple SNP detection method that allows for rapid screening of clones from 96-well plates. Our assay comprises PCR amplification of the target site, followed by an enzymatic assay and a fluorescence-based readout using a standard plate reader. No additional special instrumentation is required. The overall workflow takes approximately four hours and any positive fluorescent signal is highly correlated with the successful introduction of the desired SNP. This method allows for the detection of edited clonal cell lines independent of the engineered nucleotide substitution and the surrounding targeted genomic loci. As a proof of concept, we have applied this method to successfully detect all possible transitions in several human loci using genomic DNA as template. As a final test, several nucleotide exchanges have been detected directly in cultured human fibroblasts.
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