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{"title":"Establishing Immortalized Brown and White Preadipocyte Cell Lines from Young and Aged Mice","authors":"Xiangdong Wu, Salaheldeen Elsaid, Florian Levet, Winson Li, Sui Seng Tee","doi":"10.1002/cpz1.70072","DOIUrl":null,"url":null,"abstract":"<p>Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time consuming. Immortalization of these cells offers a solution by overcoming cellular senescence and maintaining proliferative capacity, allowing for long-term studies without the continuous need to isolate new cells from animals. Immortalized cell lines thus provide a consistent and reproducible experimental model, significantly reducing variability across different animals. However, successfully establishing immortalized preadipocyte cell lines presents challenges, including selecting appropriate adipose tissue depots, isolating primary preadipocytes, and choosing an effective immortalization strategy. In this article, we present optimized protocols and share first-hand experiences establishing immortalized brown and white preadipocyte cell lines from young and aging mice. These protocols offer a valuable resource for researchers studying adipogenesis and metabolism. © 2024 Wiley Periodicals LLC.</p><p><b>Support Protocol 1</b>: Retrovirus production</p><p><b>Basic Protocol 1</b>: Isolation and culture of primary brown and white preadipocytes from mouse interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the same region</p><p><b>Basic Protocol 2</b>: Immortalization of mouse brown and white preadipocytes</p><p><b>Basic Protocol 3</b>: Selection of immortalized preadipocytes</p><p><b>Basic Protocol 4</b>: Selection of single-cell clones of immortalized mouse preadipocytes</p><p><b>Basic Protocol 5</b>: Single-cell sorting in a 96-well plate using a flow cytometer for the selection of single-cell clones of immortalized preadipocytes</p><p><b>Support Protocol 2</b>: Cryopreservation of immortalized mouse preadipocytes</p><p><b>Support Protocol 3</b>: Thawing and culture of cryopreserved immortalized mouse preadipocytes</p><p><b>Support Protocol 4</b>: Subculture and expansion of immortalized mouse preadipocytes</p><p><b>Basic Protocol 6</b>: Differentiation of immortalized mouse brown and white preadipocytes</p><p><b>Support Protocol 5</b>: Identification of differentiated white and brown adipocytes</p>","PeriodicalId":93970,"journal":{"name":"Current protocols","volume":"4 12","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-12-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Current protocols","FirstCategoryId":"1085","ListUrlMain":"https://onlinelibrary.wiley.com/doi/10.1002/cpz1.70072","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
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研究脂肪生成和脂肪细胞生物学需要从脂肪组织中分离出原代前脂肪细胞。然而，原代前脂肪细胞的寿命有限，只能进行有限次数的分裂，而且往往在衰老之前就失去了原有的生物特性。反复分离新鲜的前脂肪细胞，尤其是从幼崽或老龄动物身上分离，既费钱又费时。这些细胞的永生化提供了一种解决方案，它克服了细胞衰老并保持增殖能力，从而可以进行长期研究，而无需不断从动物身上分离新细胞。因此，永生化细胞系提供了一致且可重复的实验模型，大大减少了不同动物之间的差异。然而，成功建立永生化前脂肪细胞系面临着各种挑战，包括选择合适的脂肪组织储库、分离原代前脂肪细胞以及选择有效的永生化策略。在本文中，我们介绍了优化方案，并分享了从幼鼠和老龄小鼠中建立永生化棕色和白色前脂肪细胞系的第一手经验。这些方案为研究脂肪生成和代谢的研究人员提供了宝贵的资源。© 2024 Wiley Periodicals LLC.支持方案 1：逆转录病毒的生产 基本方案 1：从同一区域的小鼠肩胛间棕色脂肪组织（iBAT）和皮下白色脂肪组织（sWAT）中分离和培养原代棕色和白色前脂肪细胞 基本方案 2：小鼠棕色和白色前脂肪细胞的永生化 基本方案 3：永生化前脂肪细胞的筛选 基本方案 4：永生化小鼠前脂肪细胞单细胞克隆的筛选 基本方案 5：使用流式细胞仪在 96 孔板中进行单细胞分选，以筛选永生化小鼠前脂肪细胞的单细胞克隆 支持规程 2：冷冻保存永生化小鼠前脂肪细胞 支持规程 3：解冻和培养冷冻保存的永生化小鼠前脂肪细胞 支持规程 4：基本程序 6：永生化小鼠棕色和白色前脂肪细胞的分化 支持程序 5：已分化的白色和棕色脂肪细胞的鉴定。
本文章由计算机程序翻译，如有差异，请以英文原文为准。
Establishing Immortalized Brown and White Preadipocyte Cell Lines from Young and Aged Mice
Studying adipogenesis and adipocyte biology requires the isolation of primary preadipocytes from adipose tissues. However, primary preadipocytes have a limited lifespan, can only undergo a finite number of divisions, and often lose their original biological characteristics before becoming senescent. The repeated isolation of fresh preadipocytes, particularly from young pups or aged animals, is costly and time consuming. Immortalization of these cells offers a solution by overcoming cellular senescence and maintaining proliferative capacity, allowing for long-term studies without the continuous need to isolate new cells from animals. Immortalized cell lines thus provide a consistent and reproducible experimental model, significantly reducing variability across different animals. However, successfully establishing immortalized preadipocyte cell lines presents challenges, including selecting appropriate adipose tissue depots, isolating primary preadipocytes, and choosing an effective immortalization strategy. In this article, we present optimized protocols and share first-hand experiences establishing immortalized brown and white preadipocyte cell lines from young and aging mice. These protocols offer a valuable resource for researchers studying adipogenesis and metabolism. © 2024 Wiley Periodicals LLC.
Support Protocol 1 : Retrovirus production
Basic Protocol 1 : Isolation and culture of primary brown and white preadipocytes from mouse interscapular brown adipose tissue (iBAT) and subcutaneous white adipose tissue (sWAT) in the same region
Basic Protocol 2 : Immortalization of mouse brown and white preadipocytes
Basic Protocol 3 : Selection of immortalized preadipocytes
Basic Protocol 4 : Selection of single-cell clones of immortalized mouse preadipocytes
Basic Protocol 5 : Single-cell sorting in a 96-well plate using a flow cytometer for the selection of single-cell clones of immortalized preadipocytes
Support Protocol 2 : Cryopreservation of immortalized mouse preadipocytes
Support Protocol 3 : Thawing and culture of cryopreserved immortalized mouse preadipocytes
Support Protocol 4 : Subculture and expansion of immortalized mouse preadipocytes
Basic Protocol 6 : Differentiation of immortalized mouse brown and white preadipocytes
Support Protocol 5 : Identification of differentiated white and brown adipocytes
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