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 study, 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, metabolism, and adipocyte biology.

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 preadipocytes

Support Protocol 2 : Cryopreservation of immortalized preadipocytes

Support Protocol 3 : Wake up and culture of immortalized preadipocytes

Support Protocol 4 : Subculture and expansion of immortalized preadipocytes

Basic Protocol 5 : Differentiation of immortalized mouse brown and white preadipocytes

Support Protocol 5 : Lipid droplet staining and nucleus counterstaining

Support Protocol 6 : Mitochondria staining and nucleus counterstaining