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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":"<p><p>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.</p>","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
Establishment and Characterization of Patient-Derived Oral Cancer Organoids. 患者源性口腔癌类器官的建立与表征。
Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_594
Nadja Harnischfeger, Lili Szabo, Kai Kretzschmar
{"title":"Establishment and Characterization of Patient-Derived Oral Cancer Organoids.","authors":"Nadja Harnischfeger, Lili Szabo, Kai Kretzschmar","doi":"10.1007/7651_2024_594","DOIUrl":"https://doi.org/10.1007/7651_2024_594","url":null,"abstract":"<p><p>Oral squamous cell carcinoma (OSCC) is the most common form of head and neck cancer. The current standard for treating primary OSCC is surgical resection combined with radiotherapy and chemotherapy. Despite improved therapeutic strategies, OSCC has high rates of metastasis and mortality, with one in two patients dying of the disease. Patient-derived organoids have become promising cell culture systems for disease modeling and precision medicine. Here we describe the high-efficiency generation of organoids from OSCC patients, which can be maintained in the culture for the long term. We further provide protocols for characterizing OSCC organoids using histology and immunofluorescence staining.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142950834","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
In Silico Method for ssDNA Aptamer Binding with Aurora Kinase A Protein. ssDNA适体与极光激酶A蛋白结合的硅片法。
Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_596
Haregewoin Bezu Woldekidan, Adugna Abdi Woldesemayat
{"title":"In Silico Method for ssDNA Aptamer Binding with Aurora Kinase A Protein.","authors":"Haregewoin Bezu Woldekidan, Adugna Abdi Woldesemayat","doi":"10.1007/7651_2024_596","DOIUrl":"https://doi.org/10.1007/7651_2024_596","url":null,"abstract":"<p><p>While traditional assay methods face challenges in detecting specific proteins, aptamers, known for their high specificity and affinity, are emerging as a valuable biomarker detection tool. Aurora kinase A (AURKA) plays a role in cell division and influences stem cell reprogramming. In this study, an in silico approach method was conducted for a random ssDNA aptamer sequence selection and its binding with AURKA. The aptamer was designed based on AURKA's structure and nucleic acid sequence, obtained from PDB RCSB. Using RNAfold and RNA composer, we predicted the aptamer's secondary and tertiary structures. Protein-aptamer binding was analyzed via HDOCK and HADDOCK, with 2D interactions visualized in LIGPLOT+ v1.4. Autodock 4 and NAMD 2.3 tools were used to conduct docking and MD simulation studies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142950961","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
Mouse Intestinal Organoid Culture Protocol. 小鼠肠道类器官培养方案。
Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_591
Özüm Begüm Böke, Ezgi Bulut-Okumuş, Hazar Eren Soydan, Selinay Şenkal-Turhan, Ayşegül Doğan
{"title":"Mouse Intestinal Organoid Culture Protocol.","authors":"Özüm Begüm Böke, Ezgi Bulut-Okumuş, Hazar Eren Soydan, Selinay Şenkal-Turhan, Ayşegül Doğan","doi":"10.1007/7651_2024_591","DOIUrl":"https://doi.org/10.1007/7651_2024_591","url":null,"abstract":"<p><p>The discovery of leucine-rich-containing G-protein-coupled receptor 5 (Lgr5) as an intestinal adult stem cell marker had blazed a trail in stem cell biology and laid the foundations of modern organoid technology. Up to date, several well-established intestinal organoid protocols have been reported in the literature from different sources, including adult and induced pluripotent stem cells. Here, we demonstrate a BALB/c mouse-derived intestinal organoid culture protocol, passaging, and cryopreservation procedures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951029","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
Use of an Intramolecular Quenched Fluorescence (IQF) Cleavage Assay for Assessing Enzyme Kinetics of Gamma-Secretase in Human Skin Fibroblasts and Keratinocytes. 使用分子内淬灭荧光（IQF）切割试验评估人皮肤成纤维细胞和角质形成细胞中γ -分泌酶的酶动力学。
Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_587
Beita Badiei, Luis A Garza
{"title":"Use of an Intramolecular Quenched Fluorescence (IQF) Cleavage Assay for Assessing Enzyme Kinetics of Gamma-Secretase in Human Skin Fibroblasts and Keratinocytes.","authors":"Beita Badiei, Luis A Garza","doi":"10.1007/7651_2024_587","DOIUrl":"10.1007/7651_2024_587","url":null,"abstract":"<p><p>This study describes an intramolecular quenching assay to evaluate gamma-secretase (GS) enzyme activity in human dermal cells. The method utilizes a fluorogenic peptide substrate, mimicking a fragment of amyloid precursor protein (APP), in which a quencher suppresses the fluorescence of a fluorophore until enzymatic cleavage occurs, resulting in a measurable increase in fluorescence. This real-time, direct measurement of GS activity allows for precise kinetic analysis using Michaelis-Menten modeling to define Kd and Vmax. The assay is designed to quantify GS activity in human dermal fibroblasts and keratinocytes, enabling comparison between samples derived from hidradenitis suppurativa (HS) patients and healthy controls, as well as investigating the effects of subunit knockdown, such as nicastrin, on GS function. The method offers several advantages, including simplicity, cost-effectiveness, and adaptability for high-throughput screening for GS enzyme inhibitors.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12392851/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Ring Magnet-Guided Magnetic Manipulation for Biofabrication of 3D Cellular Structures. 环形磁体引导的三维细胞结构生物制造的磁操作。
Methods in molecular biology Pub Date : 2025-01-09 DOI: 10.1007/7651_2024_597
Muge Anil-Inevi, Engin Ozcivici
{"title":"Ring Magnet-Guided Magnetic Manipulation for Biofabrication of 3D Cellular Structures.","authors":"Muge Anil-Inevi, Engin Ozcivici","doi":"10.1007/7651_2024_597","DOIUrl":"https://doi.org/10.1007/7651_2024_597","url":null,"abstract":"<p><p>Negative magnetophoresis is employed to levitate cells in a paramagnetic medium without the need for magnetic labeling, preserving their natural state and minimizing toxicity. The single-ring magnet configuration that provides an open space in the levitation chamber enhances culture accessibility and scalability, enabling the formation of millimeter-sized 3D structures through cellular self-assembly. This system provides a versatile and cost-effective approach for diverse applications, including tissue engineering and biofabrication. This protocol outlines a method for biofabrication and maintenance of 3D cellular structures using magnetic levitation with a ring magnet-based setup.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142951204","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
Enzymatically Ligated Nucleic Acid Nanocapsules for the Delivery of Therapeutic Nucleic Acids and Small Molecule Drugs. 用于输送治疗性核酸和小分子药物的酶联核酸纳米胶囊。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4402-7_4
Jenna N Cannata, Jessica L Rouge
{"title":"Enzymatically Ligated Nucleic Acid Nanocapsules for the Delivery of Therapeutic Nucleic Acids and Small Molecule Drugs.","authors":"Jenna N Cannata, Jessica L Rouge","doi":"10.1007/978-1-0716-4402-7_4","DOIUrl":"10.1007/978-1-0716-4402-7_4","url":null,"abstract":"<p><p>Spherical nucleic acids (SNAs) offer intriguing properties for cellular uptake and stability. A novel SNA-like structure known as the nucleic acid nanocapsule (NAN) combines the benefits of SNAs with the added properties of nucleic acid functionalization and drug cargo release. Herein, we will discuss various ways NANs can be adapted to allow for gene targeting via the delivery of nucleic acids, as well as the delivery of small molecule drugs for combination therapies.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"55-67"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542583","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
Precision Cell-Cell Assembly Through Light-Mediated DNA Interactions. 精确细胞-细胞组装通过光介导的DNA相互作用。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4402-7_11
Katelyn Mathis, Brian Meckes
{"title":"Precision Cell-Cell Assembly Through Light-Mediated DNA Interactions.","authors":"Katelyn Mathis, Brian Meckes","doi":"10.1007/978-1-0716-4402-7_11","DOIUrl":"10.1007/978-1-0716-4402-7_11","url":null,"abstract":"<p><p>Complex interactions between diverse cell populations influence processes ranging from cell division to programmed cell death. Replicating this complexity with reproducibility remains challenging. We introduce an innovative approach that combines DNA-mediated interactions with photolithography to achieve controlled cell-cell interactions. By coating cells with DNA sequences responsive to light, we enable meticulous spatial organization through light activation, facilitating rapid and programmable construction of intricate cell structures. This method not only addresses the challenges of orchestrating cell arrangements in vitro, particularly in three-dimensional settings but also offers a new avenue for the on-demand creation of complex cellular architectures.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"173-182"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542675","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
Surface Functionalization of Elastomers with Biopolymers. 弹性体与生物聚合物的表面功能化。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4402-7_13
Emilie Morin, Elana Muzzy, Andrea S Carlini
{"title":"Surface Functionalization of Elastomers with Biopolymers.","authors":"Emilie Morin, Elana Muzzy, Andrea S Carlini","doi":"10.1007/978-1-0716-4402-7_13","DOIUrl":"10.1007/978-1-0716-4402-7_13","url":null,"abstract":"<p><p>Biopolymer coatings on elastomeric surfaces have significant impact for advancements in biomedicine as they combine flexible devices with complex biological functionality. Biopolymers offer increased ability for antimicrobial coatings, sensing of relevant biological markers, and controlled drug delivery. The methodologies available to conjugate these important biopolymers to flexible elastomeric substrates are vast and rapidly evolving. This chapter aims to compile methodologies across the application space of biopolymer conjugation to elastomers. We present a guide to the field and methods ranging from surface activation and functionalization, grafting-to and grafting-from of biopolymers, and characterization of the resulting substrates.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2902 ","pages":"197-227"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143542676","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
Nonhematopoietic Stem Cell Identification and Sorting in Adult Retina. 成人视网膜非造血干细胞的鉴定与分选。
Methods in molecular biology Pub Date : 2025-01-01 DOI: 10.1007/978-1-0716-4386-0_5
Ana Flores, Laura Fernández-Sánchez, Oksana Kutsyr, Alberto Yáñez, María Luisa Gil, Daniel Gozalbo, Victoria Maneu, Pedro Lax
{"title":"Nonhematopoietic Stem Cell Identification and Sorting in Adult Retina.","authors":"Ana Flores, Laura Fernández-Sánchez, Oksana Kutsyr, Alberto Yáñez, María Luisa Gil, Daniel Gozalbo, Victoria Maneu, Pedro Lax","doi":"10.1007/978-1-0716-4386-0_5","DOIUrl":"10.1007/978-1-0716-4386-0_5","url":null,"abstract":"<p><p>Flow cytometry enables the detection and characterization of specific cell populations, and proper identification of particular cell types can be assessed by immunohistochemistry. Here, we describe the identification and sorting of nonhematopoietic stem cells expressing stem cell antigen-1 from adult murine retinas using flow cytometry and immunohistochemistry strategies. These sorted cells can be further used for regenerative purposes.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2899 ","pages":"59-65"},"PeriodicalIF":0.0,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143605437","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
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