Methods in molecular biology最新文献_第5页

Application of Cerium Oxide Nanozymes (CeONZs) in Human Pluripotent Stem Cell-Derived Cardiomyocytes. 氧化铈纳米酶（CeONZs）在人多能干细胞衍生心肌细胞中的应用。

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_606

Chengwen Hang, Huixin Guo, Mohamed S Moawad, Christie M Sayes, Yi-Han Chen, Jian Yang

{"title":"Application of Cerium Oxide Nanozymes (CeONZs) in Human Pluripotent Stem Cell-Derived Cardiomyocytes.","authors":"Chengwen Hang, Huixin Guo, Mohamed S Moawad, Christie M Sayes, Yi-Han Chen, Jian Yang","doi":"10.1007/7651_2025_606","DOIUrl":"https://doi.org/10.1007/7651_2025_606","url":null,"abstract":"Cardiovascular diseases (CVDs) are a leading cause of death globally. Excessive production of reactive oxygen species (ROS) is detrimental to cardiomyocytes (CMs), triggering inflammation, inducing cell death, disrupting calcium homeostasis, and leading to arrhythmia. Thus, ROS is considered a common pathological factor in CVDs. Although the efficacy of antioxidants targeting ROS is currently limited, nanotechnology offers opportunities to develop antioxidants with improved selectivity and bioavailability, which can effectively prevent or treat oxidative stress-related CVDs. Cerium oxide nanozymes (CeONZs) can efficiently scavenge excessive ROS by mimicking the activity of endogenous antioxidant enzymes. However, their nanosafety and efficacy in human CMs remain unclear, posing a critical issue to be addressed before clinical applications. Due to the scarcity of primary human CMs, human pluripotent stem cells (hPSCs) and their derived cardiomyocytes (hPSC-CMs) provide a valuable source for modeling CVDs and their therapeutic interventions. This chapter presents a preparation method for CeONZs and outlines the assessment of their biosafety and antioxidant efficacy in hPSC-CMs.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143662481","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes. CRISPR/Cas9敲除HaCaT角质形成细胞中抗炎蛋白TNIP1的方案开发

Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_616

Liam E Carman, Michael L Samulevich, Brian J Aneskievich

{"title":"Protocol Development for CRISPR/Cas9 Knockout of the Anti-inflammatory Protein TNIP1 in HaCaT Keratinocytes.","authors":"Liam E Carman, Michael L Samulevich, Brian J Aneskievich","doi":"10.1007/7651_2025_616","DOIUrl":"https://doi.org/10.1007/7651_2025_616","url":null,"abstract":"Gene editing in cultured cells, including the intent of sequence disruption to achieve a functional knockout of the targeted gene, has been greatly facilitated with the advent of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) technology. Primary cell strains and immortalized cell lines from diverse tissue types have been successfully targeted both for basic research examining the effects of loss of the correlating protein and for modeling select loss-of-function disorders. Such accomplishments have extended to cutaneous cells, especially epidermal keratinocytes given their key structural and functional role in barrier formation and surveillance of and response to surface events such as triggering and processing inflammatory responses. Here we describe disruption of the Tumor Necrosis factor-induced protein 3-Interacting Protein 1 (TNIP1) gene in human HaCaT keratinocytes to generate an ongoing loss of expression as a parallel system to transient knockdown we had previously achieved with siRNA transfection. The TNIP1 protein restricts cytoplasmic progression of inflammatory signals. We cover our CRISPR/Cas9 vector choice, enrichment of transfected cells via positive selection for puromycin resistance, their subsequent cloning, and gene disruption and expression analysis. We also emphasize prior keratinocyte-CRISPR/Cas9 literature as a springboard for other investigators and to illustrate the widespread relevance of such editing to the diverse, yet highly consequentially different, genes expressed in keratinocytes.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143663738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation of Functional Brain Region-Specific Neural Spheroids for High Throughput Screening. 用于高通量筛选的功能性脑区域特异性神经球体的生成。

Methods in molecular biology Pub Date : 2025-02-19 DOI: 10.1007/7651_2024_593

Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee

{"title":"Generation of Functional Brain Region-Specific Neural Spheroids for High Throughput Screening.","authors":"Jiajing Zhang, Angelica Medina, Marc Ferrer, Emily M Lee","doi":"10.1007/7651_2024_593","DOIUrl":"https://doi.org/10.1007/7651_2024_593","url":null,"abstract":"Therapeutic development and research in the neurodegenerative disease field encounters many challenges such as availability of reproducible and scalable cellular model systems that are biologically, physiologically, and pharmacologically relevant. These cellular models must be informative of cellular mechanisms of diseases and predictive for therapeutics efficacy and toxicity testing during drug discovery and development. Neural spheroids fill the gap of cellular models of the brain that are functional, versatile in neural cell type composition, robust, and scalable for high-throughput screening (HTS). We have previously developed a protocol to aggregate pre-determined ratios of differentiated human-induced pluripotent stem cell (hiPSC)-derived neurons and astrocytes in a scaffold-free environment to form 3D brain-region specific spheroids. By mixing different neuronal types, neural spheroids can be used to simulate the neuronal-type heterogeneity of distinct brain regions in vivo, including the prefrontal cortex (PFC) and ventral tegmental area (VTA). Here, we present a detailed description of a method for generating functional brain region-specific spheroids with HTS-compatible assay readout that monitors changes in neural network activity by measuring calcium oscillations. The versatility of the platform is such that these neural spheroids cellular assays are applicable for a wide range of disease modeling, compound validation, and screening and are limited only by the availability of input cells, including neural subtype, disease cells, and immune cells such as microglia.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143441335","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Generation and Characterization of a New Aging Skin Human Dermal Extracellular Matrix Scaffold. 一种新型老化皮肤人真皮细胞外基质支架的制备与表征。

Methods in molecular biology Pub Date : 2025-01-18 DOI: 10.1007/7651_2024_579

Estibaliz Fernández-Carro, Jesús Ciriza

引用次数: 0

Reproducible, Scale-Up Production of Human Brain Organoids (HBOs) on a Pillar Plate Platform via Spheroid Transfer. 通过球体转移在柱板平台上可重复的，大规模生产人脑类器官（HBOs）。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_604

Pranav Joshi, Prabha Acharya, Mona Zolfaghar, Manav Goud Vanga, Sunil Shrestha, Moo-Yeal Lee

{"title":"Reproducible, Scale-Up Production of Human Brain Organoids (HBOs) on a Pillar Plate Platform via Spheroid Transfer.","authors":"Pranav Joshi, Prabha Acharya, Mona Zolfaghar, Manav Goud Vanga, Sunil Shrestha, Moo-Yeal Lee","doi":"10.1007/7651_2024_604","DOIUrl":"https://doi.org/10.1007/7651_2024_604","url":null,"abstract":"Human brain organoids (HBOs) derived from pluripotent stem cells hold great potential for disease modeling and high-throughput compound screening, given their structural and functional resemblance to fetal brain tissues. These organoids can mimic early stages of brain development, offering a valuable in vitro model to study both normal and disordered neurodevelopment. However, current methods of generating HBOs are often low throughput and variable in organoid differentiation and involve lengthy, labor-intensive processes, limiting their broader application in both academic and industrial research. Key challenges include high costs of growth factors, variability in organoid size and function, suboptimal maturation, and manual handling that reduces throughput. Here, we present a standard operating procedure (SOP) for the scalable production of HBOs using a novel pillar plate system that simplifies the spheroid transfer process and allows miniature organoid culture. This method enables the reproducible generation of HBOs without the need for extensive manual intervention, providing a streamlined solution for high-throughput screening (HTS). The resulting assay-ready pillar plate with HBOs is optimized for compound testing, in situ staining, and analysis, offering an efficient platform to advance neurodevelopmental research and therapeutic screening.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Biosynthesis of Zinc Oxide Nanoparticles Using Dried Leaves of Camellia sinensis: Methods to Characterize and Assess Their Effects on Mesenchymal Stem Cell Viability. 利用茶树干叶生物合成氧化锌纳米颗粒：表征和评估其对间充质干细胞活力影响的方法

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_598

Serap Yeşilkır Baydar, Tuba Akgül Çağlar, Fatma Ebru Koç

{"title":"Biosynthesis of Zinc Oxide Nanoparticles Using Dried Leaves of Camellia sinensis: Methods to Characterize and Assess Their Effects on Mesenchymal Stem Cell Viability.","authors":"Serap Yeşilkır Baydar, Tuba Akgül Çağlar, Fatma Ebru Koç","doi":"10.1007/7651_2024_598","DOIUrl":"https://doi.org/10.1007/7651_2024_598","url":null,"abstract":"Stem cell nanotechnology (SCN) is an important scientific field to guide stem cell-based research of nanoparticles. Currently, nanoparticles (NPs) have a rich spectrum regarding the sources from which they are obtained (metallic, polymeric, etc.), the methods of obtaining them (physical, chemical, biological), and their shape, size, electrical charge, etc. properties. It is also essential to expand green synthesis applications for the use of NPs in the field of biomedical sciences. For this purpose, there is a need to produce NPs using biological sources (plant, microorganism, algae, yeast etc.…), characterization and investigation of their effects on biological activities of stem cells. This process involves long and laborious procedures, and there may be differences in methods between individual laboratories.In this protocol, biofabrication and characterization of ZnO NPs using dried leaves of Camellia sinensis is described. This experimental setup includes conventional and novel methods that can be applied to biofabricate and characterize the NPs and to examine the viability, apoptotic, and necrotic effects on human adipose tissue-derived mesenchymal stem cells (ADMSCs) in vitro.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Protocol for the Generation and 3D Culture of Fluorescently Labeled Multicellular Spheroids. 荧光标记的多细胞球体的生成和三维培养方案。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_592

Emily C Liu, Amy L Ryan, Sinem Koc-Günel

引用次数: 0

RNA Interference Approaches to Study Epidermal Cell Adhesion. 研究表皮细胞粘附的RNA干扰方法。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_584

Anamika Dutta, Michele Calder, Lina Dagnino

引用次数: 0

Reproducible, Scale-Up Production of Human Liver Organoids (HLOs) on a Pillar Plate Platform via Microarray 3D Bioprinting. 通过微阵列3D生物打印技术在柱板平台上可重复、大规模生产人类肝脏类器官（HLOs）。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_603

Sunil Shrestha, Manav Goud Vanga, Charishma Jonnadula, Prabha Acharya, Minseong Lee, Moo-Yeal Lee

{"title":"Reproducible, Scale-Up Production of Human Liver Organoids (HLOs) on a Pillar Plate Platform via Microarray 3D Bioprinting.","authors":"Sunil Shrestha, Manav Goud Vanga, Charishma Jonnadula, Prabha Acharya, Minseong Lee, Moo-Yeal Lee","doi":"10.1007/7651_2024_603","DOIUrl":"https://doi.org/10.1007/7651_2024_603","url":null,"abstract":"Human liver organoids (HLOs) derived from pluripotent stem cells hold potential for disease modeling and high-throughput compound screening due to their architectural and functional resemblance to human liver tissues. However, reproducible, scale-up production of HLOs for high-throughput screening (HTS) presents challenges. These include the high costs of additives and growth factors required for cell differentiation, variability in organoid size and function from batch to batch, suboptimal maturity of HLOs compared to primary hepatocytes, and low assay throughput due to excessive manual processes and the absence of assay-ready plates with HLOs. To address some of these issues, here we present standard operating procedures (SOPs) for the scale-up production of HLOs using a pillar plate through microarray 3D bioprinting. This technology facilitates the rapid, uniform seeding of foregut cells onto the pillar plate, maintaining cell viability and enabling the scale-up generation of HLOs. The assay-ready pillar plate with HLOs is suitable for compound testing, as well as in situ organoid staining and analysis.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008152","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Polarized Calu-3 Cells Serve as an Intermediary Model for SARS-CoV-2 Infection. 极化Calu-3细胞作为SARS-CoV-2感染的中介模型

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_602

Sarah L Harbach, Bang M Tran, Georgios Kastrappis, Hoanh Tran, Samantha L Grimley, Julie L McAuley, Abderrahman Hachani, Elizabeth Vincan

{"title":"Polarized Calu-3 Cells Serve as an Intermediary Model for SARS-CoV-2 Infection.","authors":"Sarah L Harbach, Bang M Tran, Georgios Kastrappis, Hoanh Tran, Samantha L Grimley, Julie L McAuley, Abderrahman Hachani, Elizabeth Vincan","doi":"10.1007/7651_2024_602","DOIUrl":"https://doi.org/10.1007/7651_2024_602","url":null,"abstract":"Human nasal epithelium (HNE) organoid models of SARS-CoV-2 infection were adopted globally during the COVID-19 pandemic once it was recognized that the Vero cell line commonly used by virologists did not recapitulate human infection. However, the widespread use of HNE organoid infection models was hindered by the high cost of media and consumables, and the inherent limitation of basal cells as a scalable continuous source of cells. The human Calu-3 cell line, generated from a lung adenocarcinoma, was shown to largely recapitulate infection of the human epithelium and to preserve the SARS-CoV-2 genomic fidelity. We have previously shown that continuous cancer cell lines can polarize along the apical-basal axis when embedded in matrix and to more closely mimic infection of human cells when compared to their non-polarized, simple monolayer state. We have established and demonstrated that polarized Calu-3 cells constitute a robust SARS-CoV-2 infection model. The polarized Calu-3 cells are implemented in our respiratory virus isolation and amplification pipeline as an inexpensive, scalable, intermediary culture system to complement the HNE organoid model against which all respiratory culture models are benchmarked.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143008203","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0