Methods in molecular biology最新文献

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

A Protocol for Detecting DNA Methylation Changes at CpG Sites of Stemness-Related Genes in Aging Stem Cells. 检测衰老干细胞中干细胞相关基因CpG位点DNA甲基化变化的方案。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_590

Mahshid Hodjat, Seyed Mojtaba Daghighi, Mohammad Abdollahi

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

Functional Imaging Methods for Investigating 3D Choroid Plexus Organoids. 三维脉络膜丛类器官的功能成像方法研究。

Methods in molecular biology Pub Date : 2025-01-17 DOI: 10.1007/7651_2024_601

See Swee Tang, Elizabeth J Apsley, Laura Pellegrini

{"title":"Functional Imaging Methods for Investigating 3D Choroid Plexus Organoids.","authors":"See Swee Tang, Elizabeth J Apsley, Laura Pellegrini","doi":"10.1007/7651_2024_601","DOIUrl":"https://doi.org/10.1007/7651_2024_601","url":null,"abstract":"The choroid plexus (ChP) is a vital brain structure that produces cerebrospinal fluid (CSF) and forms a selective barrier between the blood and CSF, essential for brain homeostasis. Composed of secretory epithelial cells, connective stroma, and a fenestrated vascular network, the ChP supports nutrient transport, immune surveillance, and the clearance of toxic by-products. Despite its significance in maintaining cerebral function, the mechanisms underlying its development and maturation remain poorly understood. Recent advancements, such as the creation of stem cell-derived three-dimensional (3D) ChP organoid model, provide a promising platform for studying these processes. The ChP organoid model replicates key developmental stages and functions of the ChP, including CSF secretion and barrier formation. Additionally, they offer unique opportunities to investigate the impacts of drugs, pathogens, and toxins on the blood-CSF barrier. This study highlights imaging techniques critical for the characterization and utilization of ChP organoids, illustrating their value in advancing our understanding of ChP biology and its role in health and disease.","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":"143008192","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

Analyzing Muscle Stem Cell Function Ex Vivo. 体外分析肌肉干细胞功能。

Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_589

Julia von Maltzahn

引用次数: 0