Methods in molecular biology最新文献

Human Stem Cell-Based Embryo Model Research: Ethics and Regulations. 基于人类干细胞的胚胎模型研究：伦理与规范。

Methods in molecular biology Pub Date : 2025-10-04 DOI: 10.1007/7651_2025_668

Chie Kobayashi, Shu Ishida, Tsutomu Sawai

引用次数: 0

2D and 3D Differentiation of Human Mammary Basal Cells Long-Term Expanded in YDAC-Supplemented F Medium. 人乳腺基底细胞在ydac - F培养基中长期扩增的二维和三维分化

Methods in molecular biology Pub Date : 2025-09-29 DOI: 10.1007/7651_2025_665

Akihito Inoko

{"title":"2D and 3D Differentiation of Human Mammary Basal Cells Long-Term Expanded in YDAC-Supplemented F Medium.","authors":"Akihito Inoko","doi":"10.1007/7651_2025_665","DOIUrl":"https://doi.org/10.1007/7651_2025_665","url":null,"abstract":"The mammary epithelium comprises basal and luminal cell layers, with the basal compartment harboring stem/progenitor cells that can exhibit bipotent potential to generate both myoepithelial and luminal lineages under appropriate conditions. We present a robust two-dimensional (2D) culture protocol for the long-term expansion of primary human mammary basal cells using F medium supplemented with a defined chemical cocktail, \"YDAC.\" This system preserves basal cell identity in commercially available primary human mammary epithelial cells (HMECs) during extended culture, providing a renewable source of undifferentiated basal cells. Upon YDAC withdrawal, the expanded 2D basal cells undergo synchronized epithelial differentiation, accompanied by lineage marker shifts and characteristic changes in Claudin expression. Prolonged culture in YDAC-containing medium beyond confluence induces the formation of bilayered 3D epithelial structures, while embedding in Matrigel yields heterogeneous organoids. This combination of scalable basal cell expansion and controllable differentiation enables comprehensive molecular and functional analyses. It also facilitates integration of 2D and 3D epithelial models to ensure reproducible, physiologically relevant insights.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145186299","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}

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Isolation, Culture, and Analyses of Keratinocytes, Fibroblasts, and Hair Follicles from Normal Human Skin and Cancer-Associated Fibroblasts from Skin Tumors. 正常人类皮肤中角质形成细胞、成纤维细胞和毛囊的分离、培养和分析，以及皮肤肿瘤中癌症相关成纤维细胞的分离、培养和分析。

Methods in molecular biology Pub Date : 2025-09-25 DOI: 10.1007/7651_2025_663

Verena Natalie Lorenz, Anja Uhmann

{"title":"Isolation, Culture, and Analyses of Keratinocytes, Fibroblasts, and Hair Follicles from Normal Human Skin and Cancer-Associated Fibroblasts from Skin Tumors.","authors":"Verena Natalie Lorenz, Anja Uhmann","doi":"10.1007/7651_2025_663","DOIUrl":"https://doi.org/10.1007/7651_2025_663","url":null,"abstract":"The human skin is a complex organ composed of multiple layers and specialized cell types. At its core, the skin's function is maintained by the interplay between two key compartments: the epidermis, that is composed of suprabasal and basal keratinocytes, and the dermis, which is inhabited by a heterogeneous population of fibroblasts. Additionally, the skin contains stem cell-bearing hair follicles that span across both the epidermis and dermis, providing a vital source of cellular renewal. Each of these niches-the epidermis, dermis, and hair follicles-individually and collectively contribute to skin integrity, homeostasis, and regeneration. However, dermal fibroblasts can also undergo malignant transformation in pathological conditions such as skin cancer development, giving rise to cancer-associated fibroblasts (CAFs) that promote tumor progression and may mediate therapeutic resistance.To study key aspects of skin cell populations/niches, in vitro culture systems offer a controlled, convenient platform. We here summarize standardized protocols for the isolation of primary human epidermal keratinocytes, dermal fibroblasts, and hair follicles from healthy skin, as well as CAFs from cancerous human skin. We describe methods for feeder-free keratinocyte culture as well as the culture of dermal fibroblasts and CAFs. In addition, we provide a detailed description of functional assays, including three-dimensional (3D) culture, flow cytometry, and migration analysis of primarily isolated skin cells and immunofluorescent staining of individually isolated hair follicles. These protocols provide a versatile toolkit to investigate skin biology wound healing, epithelial-mesenchymal interactions, and tumor microenvironment dynamics under physiologically relevant conditions.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145131222","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

An Optimized Protocol for the Extended Culture of Gastruloids from Mouse Embryonic Stem Cells. 小鼠胚胎干细胞扩展培养原肠样细胞的优化方案。

Methods in molecular biology Pub Date : 2025-09-20 DOI: 10.1007/7651_2025_666

Marloes Blotenburg, Shruthi Buddharaju, Beril Esin, Peter Zeller

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Patient-Derived Bladder Tumor Organoids Isolation and Culture: Conventional and Cost-Reduction Strategy. 患者来源的膀胱肿瘤类器官分离和培养：传统和降低成本的策略。

Methods in molecular biology Pub Date : 2025-09-13 DOI: 10.1007/7651_2025_664

Mahsa Mollapour Sisakht, Shirin Hekmatirad

{"title":"Patient-Derived Bladder Tumor Organoids Isolation and Culture: Conventional and Cost-Reduction Strategy.","authors":"Mahsa Mollapour Sisakht, Shirin Hekmatirad","doi":"10.1007/7651_2025_664","DOIUrl":"https://doi.org/10.1007/7651_2025_664","url":null,"abstract":"Organoids are three-dimensional structures generated in vitro from tissue samples, induced pluripotent stem cells (iPSCs), and/or adult stem cells. Patient-derived organoids (PDOs) represent one of the most physiologically relevant culture systems, closely recapitulating the histological and functional features of the original tissue. They can be established in the laboratory for various applications, including regenerative medicine, drug screening, personalized medicine, and targeted therapy. Since 2009, organoid isolation and culture protocols have been reported for multiple organs, such as the colon, stomach, liver, lung, brain, breast, and bladder. Here, we describe a protocol for the isolation and culture of bladder tumor organoids derived from patients undergoing cystectomy or transurethral resection of bladder tumor (TUR). In addition to the conventional methodology, we introduce a cost-effective alternative approach utilizing sodium alginate hydrogel and fibroblast-conditioned medium (FCM). This strategy offers a reproducible, xeno-free, and low-cost platform that is well suited for both clinical research and resource-limited laboratory settings.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145054770","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

Live Cell Imaging for Keratinocyte Lineage Tracing. 角质形成细胞谱系追踪的活细胞成像。

Methods in molecular biology Pub Date : 2025-08-06 DOI: 10.1007/7651_2025_656

Brook Abegaze, Brooke Vittimberga, Nwamaka Ijeh, Giselle Vitcov, Richard Kim, Ruby Ghadially

引用次数: 0

Use of miR-200-Mediated Reprogramming and Mechanical Cues to Generate Human Blastocyst Models. 使用mir -200介导的重编程和机械线索生成人囊胚模型

Methods in molecular biology Pub Date : 2025-08-05 DOI: 10.1007/7651_2025_662

Sharon Arcuri, Georgia Pennarossa, Fulvio Gandolfi, Tiziana A L Brevini

{"title":"Use of miR-200-Mediated Reprogramming and Mechanical Cues to Generate Human Blastocyst Models.","authors":"Sharon Arcuri, Georgia Pennarossa, Fulvio Gandolfi, Tiziana A L Brevini","doi":"10.1007/7651_2025_662","DOIUrl":"https://doi.org/10.1007/7651_2025_662","url":null,"abstract":"Several early developmental events remain unclear due to ethical and technical limitations related to the use of natural embryos. To overcome this problem, over the last decade, different approaches aimed at the generation of in vitro blastocyst-like models have been developed.Here, we describe a protocol that combines miR-200-mediated cell reprogramming and mechanical stimuli to create 3D spheroids phenotypically similar to natural embryos. Specifically, dermal fibroblasts are reprogrammed exploiting the miR-200 family property to induce a high plasticity state in somatic cells. Subsequently, miR-200-reprogrammed cells are either differentiated into trophoblast (TR)-like cells, using an ad hoc induction protocol, or encapsulated into polytetrafluoroethylene (PTFE) micro-bioreactors to generate inner cell mass (ICM)-like spheroids. Subsequently, TR-like cells and ICM-like spheroids are encapsulated and co-cultured in the same micro-bioreactor and then transferred to microwells to allow further differentiation and boost blastoid development.The generated 3D artificial blastoids, phenotypically similar to natural embryos, could find a useful application in the field of assisted reproduction technologies to further characterize the events taking place during early embryogenesis as well as to study embryo disorders.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784649","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

Extracellular Vesicle Production from Human Blood Vessel Organoids in a Vertical Wheel Bioreactor. 在立式轮式生物反应器中制备人血管类器官的细胞外囊泡。

Methods in molecular biology Pub Date : 2025-08-05 DOI: 10.1007/7651_2025_661

Justice Ene, Falak Syed, Shaoyang Ma, Shaoxuan Ma, Sailesti Joshi, Yan Li

{"title":"Extracellular Vesicle Production from Human Blood Vessel Organoids in a Vertical Wheel Bioreactor.","authors":"Justice Ene, Falak Syed, Shaoyang Ma, Shaoxuan Ma, Sailesti Joshi, Yan Li","doi":"10.1007/7651_2025_661","DOIUrl":"10.1007/7651_2025_661","url":null,"abstract":"Although distinguished for their differentiation capacity, human-induced pluripotent stem cells (iPSCs)-derived extracellular vesicles (EVs) have been shown to contribute to functional recovery in the treatment of various traumatic and degenerative diseases. This promising role in therapeutic applications has resulted in considerable attention aimed toward their effective bio-manufacturing. However, traditional culture systems face various insufficiencies. Planar 2D culture results in a lack of scalability, with difficulty in manufacturing clinically relevant doses. Additionally, planar 2D culture lacks the complexity of in vivo biological systems. Although organoids have been proposed to fit this gap by better mimicking in vivo conditions, the traditional generation method of using static culture results in inefficient nutrient and waste transfer. Earlier bioreactor systems, which aim to resolve these issues, also face limitations of homogeneity and stress. Thus, vertical wheel bioreactors (VWBRs) with low shear stress profiles have recently emerged for stem cell organoid cultures, resulting in a more efficient and true-to-form manufacturing process for the secreted EVs. In this chapter, we describe an approach to generate and quantify EVs secreted by iPSC-differentiated human blood vessel organoids (iBVOs) grown in a scalable VWBR. iPSCs are expanded and then differentiated into iBVOs with differentiation media in VWBRs. Their produced EVs are subsequently isolated from the media and quantified using nanoparticle tracking analysis. This culture system should be able to produce a large quantity of the iBVO-derived EVs for the subsequent preclinical and clinical applications.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12497411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144784647","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

Derivation of Lung Organoids from Human-Induced Pluripotent Stem Cells for Respiratory Infection Studies. 人诱导多能干细胞衍生肺类器官用于呼吸道感染研究。

Methods in molecular biology Pub Date : 2025-07-31 DOI: 10.1007/7651_2025_660

Sandra L Leibel

引用次数: 0

Studying Hair Growth in Mice: Synchronization of Hair Follicle Growth by Depilation. 小鼠毛发生长研究：脱毛对毛囊生长的同步作用。

Methods in molecular biology Pub Date : 2025-07-31 DOI: 10.1007/7651_2025_653

Wei-Hung Wang, Renzhi Hou, Tyng-Shiuan Hsieh, Maksim V Plikus, Sung-Jan Lin

{"title":"Studying Hair Growth in Mice: Synchronization of Hair Follicle Growth by Depilation.","authors":"Wei-Hung Wang, Renzhi Hou, Tyng-Shiuan Hsieh, Maksim V Plikus, Sung-Jan Lin","doi":"10.1007/7651_2025_653","DOIUrl":"https://doi.org/10.1007/7651_2025_653","url":null,"abstract":"Hair follicles manifest distinct morphological, cellular, and molecular features as they progress through active growth (anagen), regression (catagen), and rest (telogen) phases of regenerative cycles. Since hair growth stalls in vitro and because numerous skin-specific murine genetic tools are readily available, studies on hair growth are commonly performed in mice in vivo. In such murine studies, it is often necessary to determine accurate hair cycle stages and to obtain large numbers of synchronized hair follicles at predefined experimental time points. These goals are hindered by the fact that natural hair growth in mice is temporally and spatially asynchronous. Thus, artificial hair growth synchronization by means of easy-to-perform hair depilation is a commonly used technique. Hair depilation rapidly resets hair cycle, such that skin with uniform anagen, catagen, or telogen hair follicles can be reliably collected from mice at specific post-depilation experimental time points. Further, progression of hair growth cycle after depilation can be monitored non-invasively in mice and compared between mutant and control mice. This is achieved through observing and recording hair pigmentation-driven changes in skin color tone. In this chapter, we discuss technical aspects of performing hair depilation procedure, commonly used experimental means for post-depilation hair growth analyses, as well as the limitations of the depilation method.","PeriodicalId":18490,"journal":{"name":"Methods in molecular biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144742602","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