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Testicular Organoid Formation in Microwell Culture.
Methods in molecular biology Pub Date : 2025-03-28 DOI: 10.1007/7651_2025_624
Nathalia de Lima E Martins Lara, Anja Elsenhans, Ina Dobrinski
{"title":"Testicular Organoid Formation in Microwell Culture.","authors":"Nathalia de Lima E Martins Lara, Anja Elsenhans, Ina Dobrinski","doi":"10.1007/7651_2025_624","DOIUrl":"https://doi.org/10.1007/7651_2025_624","url":null,"abstract":"<p><p>Testicular organoids present an exciting 3D in vitro platform to bridge the gap between 2D culture and animal models in male reproduction research, allowing studies on testicular cell-cell interactions, morphogenesis, development, and the spermatogonial stem cell microenvironment in conditions that are more physiologically relevant. Therefore, research with testicular organoids offers opportunities for fertility preservation, disease modeling, and high throughput reproductive toxicity screening. Our laboratory has developed a simple and reproducible protocol using microwell plates, which facilitate the aggregation of single cells and promote the generation of thousands of homogenous organoids that recapitulate testicular cytoarchitecture and functions. In this protocol, a testicular cell suspension is obtained by enzymatic digestion of immature testes and centrifuged into pyramid-shaped microwells, where cells will aggregate and form organoids after a few days in culture. Here we detail our standard protocol for the generation of porcine testicular organoids, which can also be applied to other mammalian species.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730742","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
Organoid-Immune Cell Co-culture for Stable Live Imaging.
Methods in molecular biology Pub Date : 2025-03-28 DOI: 10.1007/7651_2025_627
Nathalia Ferreira, Frauke Alves, Andrea Markus
{"title":"Organoid-Immune Cell Co-culture for Stable Live Imaging.","authors":"Nathalia Ferreira, Frauke Alves, Andrea Markus","doi":"10.1007/7651_2025_627","DOIUrl":"https://doi.org/10.1007/7651_2025_627","url":null,"abstract":"<p><p>Patient-derived organoids (PDOs) have emerged as a promising model for personalized drug testing. Generated from human tumor samples, PDOs effectively recapitulate the genetic and phenotypic heterogeneity of patient tumors, making them an ideal ex vivo platform for studying therapeutic responses, particularly to chemotherapies. However, their lack of components of the immune system limits their use in immunotherapy testing. The following protocol facilitates the co-culture of PDOs from tumor tissue with HLA-matched peripheral blood mononuclear cells (PBMCs) in a fixed Z-plane for stable live-cell imaging. This three-dimensional co-culture method represents a significant advancement in enabling real-time assessment of immunotherapeutic effects on tumor-derived PDOs by live cell imaging.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143730741","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
Efficient Isolation and Ex Vivo Differentiation of Murine Satellite Cells from Healthy and Dystrophic Muscle. 从健康和萎缩性肌肉中高效分离和体外分化小鼠卫星细胞
Methods in molecular biology Pub Date : 2025-03-22 DOI: 10.1007/7651_2025_608
Alessio A Cusmano, Cordell A VanGenderen, Tim O Lorenz, Yafen Yang, Natasha C Chang
{"title":"Efficient Isolation and Ex Vivo Differentiation of Murine Satellite Cells from Healthy and Dystrophic Muscle.","authors":"Alessio A Cusmano, Cordell A VanGenderen, Tim O Lorenz, Yafen Yang, Natasha C Chang","doi":"10.1007/7651_2025_608","DOIUrl":"https://doi.org/10.1007/7651_2025_608","url":null,"abstract":"<p><p>Satellite cells are the stem cells of adult skeletal muscles and confer skeletal muscle with remarkable regenerative ability. Under homeostatic conditions, satellite cells reside in a quiescent state in their niche along the basal lamina of muscle fibers. Upon receiving stimuli, satellite cells activate and engage in regenerative myogenesis to repair damaged fibers. Due to the impact of satellite cell differentiation on muscle physiology, studying their differentiation is relevant both within the context of healthy and diseased muscle. Due to the abundance of cell populations within skeletal muscle, the study of satellite cells is predicated on isolating highly pure populations. Fluorescence activated cell sorting (FACS) represents the gold standard for deriving highly pure satellite cell isolates but is costly and can reduce cell viability. In addition, proliferating satellite cells in vitro invariably transition to a homogeneous myoblast population that bestows a selective advantage on fast-dividing cells, reducing satellite cell heterogeneity. In this chapter, we describe our protocol for magnetic-activated cell sorting (MACS) of satellite cells. MACS preserves cell viability to a greater degree than FACS, and our approach allows for highly pure sorted populations of satellite cells. In addition, sorted cells can enter and progress through the myogenic program immediately upon plating, avoiding the need for lengthy expansion periods.</p>","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-03-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143674220","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
Evaluation of Anti-aging Agents Using the D-Galactose-Induced Accelerated Aging Model.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_609
Serdar Bora Bayraktaroğlu, Raife Dilek Turan, Neslihan Pakize Taşlı, Fikrettin Şahin
{"title":"Evaluation of Anti-aging Agents Using the D-Galactose-Induced Accelerated Aging Model.","authors":"Serdar Bora Bayraktaroğlu, Raife Dilek Turan, Neslihan Pakize Taşlı, Fikrettin Şahin","doi":"10.1007/7651_2025_609","DOIUrl":"https://doi.org/10.1007/7651_2025_609","url":null,"abstract":"<p><p>The aging population is rapidly increasing, emphasizing the importance of understanding aging mechanisms and developing effective anti-aging therapies. This chapter investigates the efficacy of novel anti-aging agents, including exosomes and boron compounds, using the D-galactose-induced accelerated aging model. Both in vitro (skin organoid models) and in vivo (rat models) systems are employed to explore cellular, molecular, and histological changes. This comprehensive analysis provides critical insights into the potential of these agents in reversing age-associated pathologies.</p>","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":"143663720","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
Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_610
Pinar Yurdakul-Mesutoglu, Hasan Yalim Akin, Zeynep Gunaydin
{"title":"Ex Vivo Expansion of Cord Blood Hematopoietic Stem and Progenitor Cells.","authors":"Pinar Yurdakul-Mesutoglu, Hasan Yalim Akin, Zeynep Gunaydin","doi":"10.1007/7651_2025_610","DOIUrl":"https://doi.org/10.1007/7651_2025_610","url":null,"abstract":"<p><p>Umbilical cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs) hold immense potential for regenerative medicine, particularly in hematologic malignancies and immune disorders. CB offers several advantages, including easy collection and reduced risk of graft-versus-host disease compared to other sources, like bone marrow. However, the clinical application of CB is often limited due to the relatively small number of HSPCs present in CB grafts, which can be insufficient for adult patients. This limitation has prompted researchers to explore various methods to expand HSPCs ex vivo. As research continues to refine expansion techniques, the future of CB HSPC therapy appears increasingly promising, offering new hope for patients requiring stem cell transplantation. Approaches to HSPC expansion include the use of cytokines, small molecules, epigenetic modulators, and advanced culture systems that mimic the bone marrow niche as well as emerging techniques such as gene editing. Of the key CB HSPC expansion methodologies, the use of epigenetic modifiers is among the most promising strategies for inducing proliferation while maintaining the stemness of CB HSPC. This section summarizes key methodologies for CB HSPC expansion and their transformative impact on clinical practice while providing a validated protocol for ex vivo expansion of CB-derived HSPCs using valproic acid and/or nicotinamide.</p>","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":"143663733","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
Free-Floating Human Lung Organoids Derived from Induced Pluripotent Stem Cells.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_621
Bettina Budeus, Chiara Kroepel, Lisa Marie Stasch, Diana Klein
{"title":"Free-Floating Human Lung Organoids Derived from Induced Pluripotent Stem Cells.","authors":"Bettina Budeus, Chiara Kroepel, Lisa Marie Stasch, Diana Klein","doi":"10.1007/7651_2025_621","DOIUrl":"https://doi.org/10.1007/7651_2025_621","url":null,"abstract":"<p><p>Lung diseases are one of the leading causes of death worldwide, and the global burden of these respiratory diseases continues to increase. Therefore, there is a need for accurate models for basic and translational research. In addition to animal models, the development of alternative in vitro model systems is progressing rapidly, ranging from advanced lung cell cultures to complex tissue-engineered lungs. Human lung organoids have become easily transferable three-dimensional in vitro model systems for lung disease modeling. Here, we present a detailed protocol for a rather simple and therefore very practical but reliable method to generate lung organoids from induced pluripotent stem cells (iPSCs) without relying on a matrix, which would represent a step forward toward animal-origin and/or component-free in vitro modeling.</p>","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":"143663693","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
Matrix-Free Normal Human Epithelial-Fibroblast 3D Spheroid Cultures for In Vitro Lung Modeling.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_622
Lisa Marie Stasch, Maja Buchholzki, Zehra Sevindik, Bettina Budeus, Diana Klein
{"title":"Matrix-Free Normal Human Epithelial-Fibroblast 3D Spheroid Cultures for In Vitro Lung Modeling.","authors":"Lisa Marie Stasch, Maja Buchholzki, Zehra Sevindik, Bettina Budeus, Diana Klein","doi":"10.1007/7651_2025_622","DOIUrl":"https://doi.org/10.1007/7651_2025_622","url":null,"abstract":"<p><p>The cellular responses of classical 2D flat monolayer cell culture systems provide only very limited reliable predictions about possible outcomes of corresponding animal experiments and clinical studies, which is due, among other things, to the lack of (bi)directional signaling transmission between different cell types and the lack of a structural microenvironment. To study the interactive communication between different cell types in vitro, two main co-culture methods have emerged as central techniques. In the indirect co-culture method, different cells are cultured physically separately (e.g., using transwell inserts) but can communicate with each other via secreted factors (paracrine mechanism). In the direct co-culture method, the different cells have direct physical contact, which enables direct interactions. Regarding the latter method, the cultivation of cells as spherical cell aggregates, so-called spheroids, embedded in a semi-solid extracellular matrix has been established as an in vivo-related, more complex cell culture model with different functional cell states according to cell-cell and cell-ECM interactions as well as oxygen and nutrient gradients. Here, we present a matrix-free method for direct spheroidal co-cultivation of human bronchial epithelial cells and fibroblasts, which can be considered as an in vivo-approximated cultivation method, especially with regard to the cellular composition of the respective spheroids.</p>","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":"143663697","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
Spheroid Invasion Assay of Melanoma Cells by Hanging Drop Technique.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_615
Asiye Busra Boz Er, Ceren Sumer
{"title":"Spheroid Invasion Assay of Melanoma Cells by Hanging Drop Technique.","authors":"Asiye Busra Boz Er, Ceren Sumer","doi":"10.1007/7651_2025_615","DOIUrl":"https://doi.org/10.1007/7651_2025_615","url":null,"abstract":"<p><p>The development of more physiologically relevant cancer models has led to the increased adoption of three-dimensional (3D) cell culture systems, such as tumor spheroids, which more accurately replicate the complex tumor microenvironment compared to traditional two-dimensional (2D) cultures. This study utilizes the hanging drop technique to generate melanoma spheroids from A375 and SK-MEL28 cell lines, including both parental and drug-resistant variants. These spheroids were embedded in Matrigel and treated with vemurafenib, cilengitide, or their combination to evaluate the effects on invasion. The combination therapy significantly reduced invasion, particularly in resistant SK-MEL28 spheroids. These findings underscore the importance of 3D spheroid models in studying drug efficacy and resistance mechanisms in cancer.</p>","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":"143663740","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
Fusion of Human Synovium-Derived Mesenchymal Stem/Stromal Cells with Primary Human Chondrocytes Using the Modified Adherence Method (MAM).
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_620
Tilen Burnik, Janja Zupan, Matjaž Jeras, Maša Kandušer
{"title":"Fusion of Human Synovium-Derived Mesenchymal Stem/Stromal Cells with Primary Human Chondrocytes Using the Modified Adherence Method (MAM).","authors":"Tilen Burnik, Janja Zupan, Matjaž Jeras, Maša Kandušer","doi":"10.1007/7651_2025_620","DOIUrl":"https://doi.org/10.1007/7651_2025_620","url":null,"abstract":"<p><p>Cell fusion is a complex phenomenon that is key in maintaining tissue homeostasis, particularly in aiding tissue regeneration processes. Studies show that mesenchymal stem/stromal cells (MSCs) are capable of restoring damaged tissue by adopting the phenotype of various cell types via cell fusion. As cell fusion of MSCs with cells of different origin remains poorly researched, we have developed a protocol that allows successful electrofusion between human synovium-derived MSCs and human chondrocytes. Building on from our protocol can help researchers study the cell fusion processes in the in vitro environment and could set basis for development of fusion cell-based advanced therapy medicinal products (ATMPs). In our protocol, we provide a detailed description on how to culture both of the fusion partner cells, how to carry out the modified adherence method (MAM) to achieve a high yield of successfully fused cells, and how to determine the yield of cell fusion using either methyl violet or fluorescent cell trackers.</p>","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":"143663695","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 of 3D Tooth Organoid Culture from Early-Postnatal Mouse Molar and Incisor.
Methods in molecular biology Pub Date : 2025-03-20 DOI: 10.1007/7651_2025_623
Florian Hermans, Hugo Vankelecom, Annelies Bronckaers, Ivo Lambrichts
{"title":"Establishment of 3D Tooth Organoid Culture from Early-Postnatal Mouse Molar and Incisor.","authors":"Florian Hermans, Hugo Vankelecom, Annelies Bronckaers, Ivo Lambrichts","doi":"10.1007/7651_2025_623","DOIUrl":"https://doi.org/10.1007/7651_2025_623","url":null,"abstract":"<p><p>Organoid models are a powerful 3D stem cell technology to explore tissue (patho-)biology and development. Tissue-derived (i.e., from tissue biopsies) organoids are long-term and stably expandable while more closely recapitulating key phenotypical and functional characteristics of the tissue-of-origin than traditional 2D culture systems. Additionally, organoids can differentiate into tissue-specific cell types, for instance, following exposure to defined differentiation cues. Although prevailing in vitro cell models have deepened our understanding of mouse tooth development and biology, in vitro representations of the dental epithelium lack (the combination of) these benefits of tissue-derived organoids and are at most derived from one tooth type. Here, we describe the protocol to establish, propagate, and differentiate mouse tooth organoids from both early postnatal molar and incisor teeth. The established organoids display a dental epithelial stemness phenotype and acquire a maturation-stage ameloblast-like phenotype following differentiation.</p>","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":"143663719","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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