SLAS Discovery最新文献

{"title":"Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems","authors":"Francisca Arez ,&nbsp;Lena Preiss ,&nbsp;Isabella Ramella Gal ,&nbsp;Sofia P. Rebelo ,&nbsp;Lassina Badolo ,&nbsp;Catarina Brito ,&nbsp;Thomas Spangenberg ,&nbsp;Paula M. Alves","doi":"10.1016/j.slasd.2025.100210","DOIUrl":"10.1016/j.slasd.2025.100210","url":null,"abstract":"<div><div>Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability <em>in vitro</em> in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications.</div><div>Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100210"},"PeriodicalIF":2.7,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980832","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models","authors":"Marcus Y. Chin ,&nbsp;David A. Joy ,&nbsp;Madhuja Samaddar,&nbsp;Anil Rana,&nbsp;Johann Chow,&nbsp;Takashi Miyamoto,&nbsp;Meredith Calvert","doi":"10.1016/j.slasd.2025.100208","DOIUrl":"10.1016/j.slasd.2025.100208","url":null,"abstract":"<div><div>Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100208"},"PeriodicalIF":2.7,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960156","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1","authors":"David A. Close ,&nbsp;V. Blair Journigan ,&nbsp;Paul A. Johnston","doi":"10.1016/j.slasd.2024.100207","DOIUrl":"10.1016/j.slasd.2024.100207","url":null,"abstract":"<div><div>Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca²⁺ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca²⁺ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca²⁺ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca²⁺ mobilization kinetic data to plot curves and calculate EC₅₀ and IC₅₀ values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC₅₀ and IC₅₀ ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC₅₀ or IC₅₀ values required to distinguish compound potencies. EC₅₀ and IC₅₀ values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC₅₀ and IC₅₀ values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100207"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142924269","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drug repurposing screen for the rare disease ataxia-telangiectasia","authors":"Namrata Jayanth ,&nbsp;Gurvan Mahé ,&nbsp;Matthew Campbell ,&nbsp;Mike Lipkin ,&nbsp;Shushant Jain ,&nbsp;Rhea van de Bospoort ,&nbsp;Jennifer Thornton ,&nbsp;Brad Margus ,&nbsp;David F. Fischer","doi":"10.1016/j.slasd.2024.100200","DOIUrl":"10.1016/j.slasd.2024.100200","url":null,"abstract":"<div><div>Ataxia Telangiectasia (A-T) is a rare, autosomal recessive genetic disorder characterized by a variety of symptoms, including progressive neurodegeneration, telangiectasia, immunodeficiency, and an increased susceptibility to cancer. It is caused by bi-allelic mutations impacting a gene encoding a serine/threonine kinase ATM (Ataxia Telangiectasia Mutated), which plays a crucial role in DNA repair and maintenance of genomic stability. The disorder primarily affects the nervous system, leading to a range of neurological issues, including cerebellar ataxia. The cause of neurodegeneration due to mutations in ATM is still an area of investigation, and currently there is no known treatment to slow down or stop the progression of the neurological problems.</div><div>In this collaboration of the A-T Children's Project (ATCP) with Charles River Discovery, we successfully developed a high-throughput assay using induced pluripotent stem cells (iPSC) from A-T donors to measure DNA damage response (DDR). By measuring the changes in levels of activated phosphorylated CHK2 (p-CHK2), which is a downstream signaling event of ATM, we were able to identify compounds that restore this response in the DDR pathway in A-T derived patient cells. Over 6,000 compounds from small molecule drug repurposing libraries were subsequently screened in the assay developed, leading to identification of several promising in vitro hits.</div><div>Using the assay developed and the identified hits opens avenues to investigate potential therapeutics for A-T.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100200"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142787921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Miniaturization and characterization of patient derived hepatocellular carcinoma tumor organoid cultures for cancer drug discovery applications","authors":"David A. Close ,&nbsp;Paul A. Johnston","doi":"10.1016/j.slasd.2024.100201","DOIUrl":"10.1016/j.slasd.2024.100201","url":null,"abstract":"<div><div>Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained <em>in situ</em> with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100201"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142815215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Protocol to develop A 3D tumor model for drug testing applications","authors":"Prasiddha Guragain,&nbsp;Sunil Singh,&nbsp;Hossein Tavana","doi":"10.1016/j.slasd.2024.100206","DOIUrl":"10.1016/j.slasd.2024.100206","url":null,"abstract":"<div><div>Three-dimensional (3D) tumor models provide physiologically relevant tumor environments and have become a major tool in cancer research and drug discovery. This article presents a protocol for creating a 3D organotypic tumor model by embedding a cancer cell spheroid within a collagen matrix containing dispersed fibroblasts. This model offers significant advantages over the conventional monolayer cell culture, monoculture spheroids of cancer cells, and intermixed co-culture of cancer and stromal cells by mimicking the spatial organization and mechanical properties of a solid tumor. Compatible with robotic automation, our protocol significantly enhances reproducibility and scalability of creating a tumor model to study tumor-stromal interactions and test therapeutic compounds</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100206"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866355","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Absolute quantification of Neuron-specific enolase based on surface plasmon resonance","authors":"Cui Lin ,&nbsp;Yijie Wang ,&nbsp;Tao Peng ,&nbsp;Pengpeng Liu ,&nbsp;Yuanyuan Liang ,&nbsp;Wencheng Kang ,&nbsp;Xiaoping Yu ,&nbsp;Yang Song ,&nbsp;Xuping Shentu","doi":"10.1016/j.slasd.2024.100205","DOIUrl":"10.1016/j.slasd.2024.100205","url":null,"abstract":"<div><div>Neuron-specific enolase (NSE) is currently the most reliable biomarker for small cell lung cancer (SCLC), which is important for disease monitoring, clinical evaluation and diagnosis. However, traditional methods suffer from various disadvantages, including instability, complexity, time-consuming operations, and the necessity for standards. In this study, we developed a calibration-free concentration analysis (CFCA) method based on surface plasmon resonance (SPR) technology, to accurately quantify the active concentration of NSE without relying on any standards. Based on the principle of CFCA, the active concentration of NSE can be calculated by observing binding rate variations at two flow rates under partial mass transport limitation and combining it with the known diffusion coefficient of the NSE. Using the method of CFCA, the active concentration of NSE was determined was only 0.48 mg/mL with an intra-day repeatability of 4.75%. The method has the advantages of simplicity, rapidity, realistic analysis and ease of implementation of high-throughput automated detection. Therefore, the method is expected to become the main measurement method for protein active concentration, which will be beneficial for the development of active protein standards.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100205"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866347","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel platform for oral epithelia sheet biofabrication via magnetic 3D bioprinting","authors":"Tien T.T. Truong ,&nbsp;Toan V. Phan ,&nbsp;Yamin Oo ,&nbsp;Oranart Matangkasombut ,&nbsp;João N. Ferreira","doi":"10.1016/j.slasd.2024.100203","DOIUrl":"10.1016/j.slasd.2024.100203","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100203"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142831069","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of a DNA-encoded library screening method “DEL Zipper” to empower the study of RNA-targeted chemical matter","authors":"Zhongyao Ma ,&nbsp;Bin Zou ,&nbsp;Jiannan Zhao ,&nbsp;Rui Zhang ,&nbsp;Qiaoqiao Zhu ,&nbsp;Xiaofeng Wang ,&nbsp;Linan Xu ,&nbsp;Xiang Gao ,&nbsp;Xinyue Hu ,&nbsp;Wei Feng ,&nbsp;Wen Luo ,&nbsp;Min Wang ,&nbsp;Yunyun He ,&nbsp;Zhifeng Yu ,&nbsp;Weiren Cui ,&nbsp;Qi Zhang ,&nbsp;Letian Kuai ,&nbsp;Wenji Su","doi":"10.1016/j.slasd.2024.100204","DOIUrl":"10.1016/j.slasd.2024.100204","url":null,"abstract":"<div><div>To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called “DEL Zipper”, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the “DEL Zipper” method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100204"},"PeriodicalIF":2.7,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883990","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “Varieties of Interactions of anti-CD133 Aptamers with Cell Cultures from Patient Glioblastoma” [SLAS Discov. 2024 Nov 16;29(8):100195]","authors":"Olga Antipova ,&nbsp;Valeria Moiseenko ,&nbsp;Fatima Dzarieva ,&nbsp;Ekaterina Savchenko ,&nbsp;Igor Pronin ,&nbsp;Galina Pavlova ,&nbsp;Alexey Kopylov","doi":"10.1016/j.slasd.2024.100199","DOIUrl":"10.1016/j.slasd.2024.100199","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100199"},"PeriodicalIF":2.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142792961","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}