Methods in molecular biology最新文献

{"title":"Isolation and Culture of Primary Keratinocytes from Human Skin Grafts.","authors":"Sarit Pal, Xizhao Chen, Gopika Ashokan, Annica R Stull-Lane, Jinyeon Shin, Geoffrey E Hespe, Babak J Mehrara, Raghu P Kataru","doi":"10.1007/7651_2026_701","DOIUrl":"https://doi.org/10.1007/7651_2026_701","url":null,"abstract":"<p><p>The skin, the largest organ of the human body, performs essential physiological functions including barrier protection, thermal regulation, and mechano-sensation. Despite its structural consistency, it exhibits marked regional heterogeneity, particularly under pathological conditions. Our recent investigations have revealed that in secondary lymphedema, the affected skin undergoes distinct pathomorphological changes, including hyperkeratosis, spongiosis, and altered keratinocyte polarity within the epidermis. These findings have prompted deeper studies into the molecular and epigenetic mechanisms contributing to the irreversible progression of this disease. This protocol describes a detailed, reproducible method for isolating and culturing primary keratinocytes from both full-thickness and split-thickness skin grafts obtained from lymphedematous regions. The workflow comprises tissue processing, enzymatic dissociation, selective enrichment of keratinocytes, and optimized culture conditions for downstream molecular analyses. Validation of isolated keratinocytes is performed using Western blotting, flow cytometry, and immunofluorescence histology. This approach preserves native phenotypic traits and epigenetic signatures in patient-derived keratinocytes, thereby enhancing the translational relevance of in vitro studies investigating the pathophysiology of secondary lymphedema.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775856","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}

{"title":"Modeling the Glioblastoma Microenvironment Using Stem Cell-Based 3D Organoid Platforms.","authors":"Varma Bhumika, Bandbe Tanmay, Pal Saptaparna, Mhatre Sejal, Sandbhor Puja","doi":"10.1007/7651_2026_699","DOIUrl":"https://doi.org/10.1007/7651_2026_699","url":null,"abstract":"<p><p>Glioblastoma multiforme (GBM) is sustained by glioma stem cells (GSCs), a self-renewing tumor-initiating population that contributes to cellular heterogeneity, therapeutic resistance, and disease recurrence. Reproducing the complex tumor-microenvironment interaction of GBM in vitro remains a significant challenge. Stem cell-based three-dimensional (3D) organoid systems have emerged as physiologically relevant models that recapitulate tumor architecture, stemness, and dynamic cellular interactions compared with conventional two-dimensional cultures. In this chapter, readers are provided with standardized and reproducible methodological frameworks to generate, maintain, and validate GBM microenvironment-relevant 3D organoid models. By following the step-by-step protocols described, readers will be able to establish GBM organoids from single-cell suspension and microtumor fragments, while preserving tumor-initiating capacity and intratumoral heterogeneity. The chapter further enables readers to integrate neurospheres into cerebral organoid systems to model tumor-neural interactions within a tissue relevant context and to incorporate extracellular matrix (ECM)-informed strategies, including decellularized brain ECM (BdECM), to mimic brain-specific biochemical/mechanical cues. Readers will also be able to perform long-term (up to 60 days) cortical organoid culture with defined quality control (QC) and validation, including evaluation of structural organization, stemness markers expression, differentiation status, and reproducibility across batches. The chapter further provides practical problem-solving guidance to address common technical challenges during organoid establishment and maintenance. Collectively, these protocols offer a resilient, durable, and versatile toolkit to model GBM biology, interrogate tumor-microenvironment interactions, and evaluate therapeutic efficacy using clinically relevant 3D organoid systems.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147775858","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}

{"title":"Engineering the Developmental Continuum: From Embryo-Like Structures to Functional Tissue Interfaces.","authors":"Tugba Topal","doi":"10.1007/7651_2026_700","DOIUrl":"https://doi.org/10.1007/7651_2026_700","url":null,"abstract":"<p><p>Advanced in vitro developmental models, including embryo-like structures, organoids, and organ-on-chip platforms, have emerged as powerful systems for studying human development under controlled conditions. These models are enabled by advances in stem cell biology and bioengineering, allowing multicellular self-organization that recapitulates selected aspects of embryonic patterning and tissue formation. Key engineering parameters-such as stem cell source, signaling control, geometric confinement, matrix composition, and microfluidic regulation-define the boundary conditions that guide developmental outcomes. These systems are best understood as engineered abstractions rather than complete replicas of human development, reflecting trade-offs between reproducibility, complexity, and biological fidelity. Despite challenges related to variability, standardization, and ethical considerations, these platforms provide valuable tools for mechanistic studies, disease modeling, and drug discovery and support the transition toward human-relevant, animal-free research approaches.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-04-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147674759","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}

{"title":"Optimized Protocol for Assessing Tight Junction Integrity and Claudin Expression in Human Epidermal Keratinocytes.","authors":"Shobith Rangappa, Thammanna Gowda Ss, Parimala Hanumesh, Prashantha K","doi":"10.1007/7651_2026_695","DOIUrl":"https://doi.org/10.1007/7651_2026_695","url":null,"abstract":"<p><p>Tight junctions are critical determinants of epidermal barrier integrity, with claudin proteins serving as key structural and functional regulators of paracellular permeability in human keratinocytes. Alterations in tight junction organization and claudin expression are associated with inflammatory skin diseases, defective wound healing, and barrier dysfunction; however, standardized experimental approaches for their reliable assessment in epidermal keratinocyte cultures remain limited.This chapter describes an optimized and reproducible experimental workflow for the evaluation of tight junction integrity and claudin expression in cultured human epidermal keratinocytes. The method combines transepithelial electrical resistance (TEER) measurements for functional barrier assessment, fluorescent tracer-based paracellular permeability assays, and immunofluorescence microscopy for spatial localization of tight junction proteins, including claudins, occludin, and ZO-1. Quantitative analysis of claudin expression is further achieved using Western blotting and real-time PCR. Key experimental variables such as calcium switch conditions, seeding density, keratinocyte differentiation status, and antibody validation are highlighted to ensure robustness and reproducibility. This integrated protocol provides a reliable platform for investigating epidermal tight junction dynamics in skin barrier research, disease modeling, toxicological testing, and therapeutic evaluation.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147503769","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}

{"title":"Keratinocyte Primary Isolation, Cultivation, and Air-Liquid Interface Culture.","authors":"Fei Li, Wenhua Zhu","doi":"10.1007/7651_2026_694","DOIUrl":"https://doi.org/10.1007/7651_2026_694","url":null,"abstract":"<p><p>This study establishes a stable, reproducible, and biologically relevant system for the isolation, identification, and cultivation of primary keratinocytes from mouse tails, aiming to provide reliable in vitro models for investigating psoriasis pathogenesis and drug screening. Primary cells were isolated using a sequential digestion protocol involving Dispase II and trypsin, followed by purity validation via Krt14 immunofluorescence and flow cytometry. Using primary cells and human keratinocyte cell line (HaCaT), two-dimensional (2D) and three-dimensional (3D) psoriasis-like cell models were successfully established. The 2D model was induced with the M5 cocktail, a cytokine mixture consisting of TNF-α, IL-1α, IL-6, IL-17A and IL-22 to mimic the psoriatic inflammatory microenvironment, and utilized for assessing cell proliferation and inflammatory factor expression. The 3D model was generated using the air-liquid interface (ALI) culture technique, reconstructing a physiologically stratified epidermal structure, and its features were characterized by hematoxylin-eosin (H&E) staining after M5 stimulation to evaluate epidermal hyperplasia and differentiation. This dual-model system integrates the high-throughput capacity of the 2D platform with the physiological relevance of the 3D platform, offering an important experimental tool for deciphering the pathological mechanisms of psoriasis and screening candidate therapeutic agents.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147504526","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}

{"title":"Experimental Repression Targeting Long Noncoding RNAs (lncRNAs) Using an Antisense Oligonucleotide (ASO) Technology in Primary Human Epidermal Keratinocytes.","authors":"Chloé Loyer, Tatiana Vinasco-Sandoval, Juliette Montanari, Sandra Moratille, Nicolas O Fortunel","doi":"10.1007/7651_2026_696","DOIUrl":"https://doi.org/10.1007/7651_2026_696","url":null,"abstract":"<p><p>Epidermal keratinocytes, the major cell type of the epidermis, undergo a well-orchestrated balance between proliferation and differentiation, which is essential for proper skin function. While the related control processes have been studied at the protein-coding gene level, increasing evidence highlights the importance of long noncoding RNAs (lncRNAs) in regulating gene expression and cell fate decisions. LncRNAs constitute a large proportion of the human transcriptome; however, the biological functions of most of them remain poorly characterized. Emerging studies demonstrate that lncRNAs play critical roles in cellular identity, differentiation, and tissue homeostasis. In the epidermis, several lncRNAs have been shown to regulate keratinocyte behavior, showing the importance of lncRNAs in epidermal biology and skin homeostasis. Multiple strategies have been developed to study lncRNA functions, including RNA interference (RNAi) and CRISPR-based approaches. However, these methods often show limited efficiencies to reach nuclear transcripts or may introduce confounding genomic effects. Antisense oligonucleotides (ASOs) provide a particularly effective technological option by inducing RNase H-mediated degradation of target transcripts and enabling rapid, reversible, and transcript-specific knockdown without any modification of genomic DNA. Together, these considerations highlight the use of ASOs as a powerful and appropriate approach to investigate the functional roles of lncRNAs in epidermal keratinocytes and to better understand their contribution to cell fate regulation in the skin. Here we describe an optimized protocol for ASO transfection in primary human epidermal keratinocytes cells in order to efficiently knock down specific lncRNA targets. The expression level of the transcripts was quantified using a ddPCR quantitative approach. As a proof of concept, we repressed the expression of the lncRNA LINC02154 in keratinocytes using three different ASOs. This ASO-based approach offers a reliable and versatile platform for functional interrogation of nuclear lncRNAs in epidermal keratinocytes and facilitates deeper understanding of lncRNA-mediated regulation of skin cell fate.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-03-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147504448","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}

{"title":"Assessing Epidermal Permeability in Lymphedema Skin with Sulfo-NHS-Biotin Immunofluorescent Penetration Assay.","authors":"Xizhao Chen, Jung Eun Baik, Jinyeon Shin, Annica R Stull-Lane, Gopika Ashokan, Sarit Pal, Geoffrey E Hespe, Babak J Mehrara, Raghu P Kataru","doi":"10.1007/7651_2026_692","DOIUrl":"https://doi.org/10.1007/7651_2026_692","url":null,"abstract":"<p><p>Skin epidermal permeability is a critical barrier function, and its disruption is implicated in various dermatological conditions and in secondary lymphedema, leading to increased fluid loss and dermal complications. Many techniques exist to assess this permeability. However, these methods frequently require specialized instruments or lack the spatial resolution needed to identify specific cellular layers involved in barrier dysfunction. Here, we describe a step-by-step protocol for an immunofluorescence-based dye penetration assay that provides high spatial resolution for visualizing the permeation of fluorescent probes through specific epidermal strata in a mouse lymphedema model.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-02-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146181054","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}

{"title":"Differentiation of Retinal Pigment Epithelium (RPE) and Retinal Organoids from Human iPSCs.","authors":"Katy Linkens, Cécile Méjécase, Mariya Moosajee","doi":"10.1007/7651_2025_674","DOIUrl":"https://doi.org/10.1007/7651_2025_674","url":null,"abstract":"<p><p>In recent decades, human-induced pluripotent stem cell (iPSC) technology has revolutionized in vitro disease modeling and personalized medicine, enabling the generation of patient-specific \"disease-in-a-dish\" systems across a wide range of tissues, including retinal pigment epithelium (RPE) and retinal organoids. Numerous protocols have been developed for iPSC-RPE and retinal organoid differentiation, typically involving prolonged culture durations, low overall efficiency, and reliance on multiple extrinsic signaling factors. Here we describe an RPE differentiation protocol over 6 weeks, incorporating three additional factors, followed by pigment isolation to isolate differentiated RPE cells for subsequent maturation. After 4 weeks of maturation, the resulting monolayer is well polarized, exhibiting hexagonal cobblestone morphology, expressing canonical RPE markers. We also describe an effective of retinal organoid differentiation protocol, using nicotinamide and a stepwise reduction of KnockOut™ Serum Replacement (KOSR) concentration over the first 9 days, that facilitates the initiation of retinal differentiation in challenging iPSC lines.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2026-01-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146018987","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}

{"title":"Evaluating Nuclear Levels of PD-L1 in Ovarian Cancer Cells by Western Blotting.","authors":"Suprataptha U Reddy, Fatema Zohra Sadia, Tanjia Mobin, Brenden N Jung, Ivana Vancurova","doi":"10.1007/978-1-0716-4901-5_4","DOIUrl":"10.1007/978-1-0716-4901-5_4","url":null,"abstract":"<p><p>Nuclear accumulation of immune checkpoint PD-L1 has been associated with increased chemoresistance and cancer progression. Since nuclear PD-L1 is largely inaccessible to immunotherapies blocking cell surface PD-L1 signaling and to analytical methods measuring surface PD-L1 expression on cancer cells, it is important to develop convenient and reliable methods to measure the nuclear PD-L1 levels. Here, we describe a simple and efficient protocol that uses biochemical preparation of cytoplasmic and nuclear extracts, which are then analyzed by western blotting using PD-L1 monoclonal antibody that specifically recognizes endogenous PD-L1.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2983 ","pages":"33-44"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889448","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}

{"title":"Spinal Spheroids as Screening Platform for Peripheral Neuropathies.","authors":"Elizabeth H Jacobs, Jessica Medina, Christopher Yanick, Renata Maciel, Mario Saporta","doi":"10.1007/978-1-0716-4985-5_12","DOIUrl":"10.1007/978-1-0716-4985-5_12","url":null,"abstract":"<p><p>The lack of effective disease-modifying therapies for axonopathies highlights the need for novel preclinical models suitable for treatment development. Two-dimensional neuronal cultures lack the directional axonal distribution required to investigate length-dependent processes such as peripheral neuropathies. To optimize this well-established model system, we developed a robust human platform to study axonal morphology and physiology based on three-dimensional motor neuron cultures (i.e., spinal spheroids). We differentiate motor neurons from human induced pluripotent stem cells, purify them by magnetic sorting, and culture them in suspension until they form spheroids. Axons are allowed to grow out of plated spinal spheroids in a radial fashion at an average rate of 200 micrometers/day and reach up to 1 cm in length. This system is optimized for morphological analysis, including high content imaging, investigation of axonal protein expression, and time-lapse imaging of axonal transport.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":"2989 ","pages":"241-255"},"PeriodicalIF":0.0,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145889739","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}