Wu Xiangdong, Elsaid Salaheldeen, Levet Florian, Li Winson, Tee Sui Seng
Diagnostic Radiology and Nuclear Medicine, University of Maryland School of Medicine, Baltimore, Maryland.
Interdisciplinary Institute for Neuroscience, University of Bordeaux, Bordeaux, France.
Curr Protoc. 2024 Dec;4(12):e70072. doi: 10.1002/cpz1.70072.
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.
研究脂肪生成和脂肪细胞生物学需要从脂肪组织中分离原代前脂肪细胞。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，并且在衰老之前常常会失去其原始生物学特性。反复分离新鲜的前脂肪细胞，尤其是从小鼠幼崽或老年动物中分离，成本高昂且耗时。使这些细胞永生化提供了一种解决方案，即克服细胞衰老并维持增殖能力，从而无需持续从动物中分离新细胞即可进行长期研究。永生化细胞系因此提供了一个一致且可重复的实验模型，显著降低了不同动物之间的变异性。然而，成功建立永生化前脂肪细胞系面临挑战，包括选择合适的脂肪组织库、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，我们展示了优化的方案，并分享了从年轻和衰老小鼠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威利期刊有限责任公司。支持方案1：逆转录病毒生产 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）中分离并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前脂肪细胞的单细胞克隆 支持方案2：永生化小鼠前脂肪细胞的冷冻保存 支持方案3：冷冻保存的永生化小鼠前脂肪细胞的解冻和培养 支持方案4：永生化小鼠前脂肪细胞的传代培养和扩增 基本方案6：永生化小鼠棕色和白色前脂肪细胞的分化 支持方案5：分化的白色和棕色脂肪细胞的鉴定
