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Show all categories 3T3-L1 Cell Line: A Key to Understanding Obesity Back

3T3-L1 Cell Line: A Key to Understanding Obesity

The 3T3-L1 cell line, derived from mouse preadipocytes, is extensively utilized in research focusing on the fundamental cellular mechanisms implicated in obesity, diabetes, and other related health conditions. Furthermore, 3T3-L1 cells are pivotal for exploring the intricate subcellular pathways that facilitate adipogenesis, the process by which preadipocytes transform into mature adipocytes.

Background and Origins of the 3T3-L1 Cell Line

This section delves into essential details about the 3T3-L1 cell line, such as its nature, the size of 3T3-L1 adipocytes, and its derivation, which are fundamental for researchers beginning to work with this cell line.

  • Originating from mouse fibroblast cells, the 3T3-L1 line was subcloned from 3T3 cells of Swiss albino mice, selected for their capacity to accumulate lipids. The precursor 3T3 cells were derived from mouse embryos.
  • Initially, 3T3-L1 cells exhibit a fibroblast-like structure; however, under specific conditions, they undergo differentiation, adopting the characteristics of adipocytes.
  • The size of 3T3-L1 adipocytes varies through different stages of differentiation: undifferentiated cells typically have an average diameter of 15.4 μm, while upon differentiation, the average diameters at day 7 and 14 post-differentiation are approximately 18.8 μm and 20.3 μm, respectively [1].
  • 3T3-L1 cells are characterized by an unstable karyotype, with a chromosome count of 2n = 40.

3-dimensional medical animation of growing fat cells.

Culturing of 3T3-L1 cells

3T3-L1 cells are widely cultured in research laboratories. The following culturing information provided in this section might help you effectively handle and maintain 3T3-L1 cultures. Here you will know: What is the doubling time of 3T3-L1 cells? Is 3T3-L1 an adherent or suspension cell line? What is the seeding density of 3T3-L1?

Key Points for Culturing 3T3-L1 Cells

Population Doubling Time:

The approximate population doubling time for 3T3-L1 cells is 28 hours.

Adherent or in Suspension:

3T3-L1 is an adherent cell line.

Seeding Density:

3 x 103 cells/cm2 cell seeding density is recommended for 3T3-L1 cells. The cell should be passaged at 70 to 80% confluency when the cell density reaches 6 x 104 cells/cm2. For seeding, cells are washed with 1 x PBS, detached using Accutase solution, added with media, and centrifuged. Recovered cells are resuspended in a fresh medium and dispensed into a new flask.

Growth Medium:

DMEM (Dulbecco's Modified Eagle Medium) is used for the optimal growth of 3T3-L1 cells. This media is usually supplemented with, 4.0 mM L-glutamine, 3.7 g/L NaHCO3, 4.5 g/L glucose, and 10% fetal bovine serum.

Growth Conditions:

3T3-L1 cell cultures are kept in a humidified incubator at 37°C and with a 5% CO2 supply.

Storage:

3T3-L1 cells are stored at below -150°C temperature either in an electric freezer or in the vapour phase of liquid nitrogen.

Freezing Process and Medium:

CM-1 or CM-ACF media are used for 3T3-L1 adipocytes freezing through the slow freezing method. This method allows only a 1°C drop in cell temperature and protects their viability.

Thawing Process:

Frozen 3T3-L1 cells are rapidly thawed at 37°C in a water bath. Thawed cells are immediately resuspended in the culture medium and can be directly dispensed into the flask for growth. Contrary to this, the cell can be centrifuged to remove old freezing media, resuspended in fresh media, and cultured.

Biosafety Level:

Biosafety level 1 laboratory settings are recommended for the 3T3-L1 murine cell line.

Confluent monolayer of 3T3-L1 cells under 10x and 20x magnification.

3T3-L1 cell line: Advantages & Limitations

There are many pros and cons associated with this fibroblast cell line. A few important advantages and limitations of the 3T3-L1 cell line are discussed here.

Advantages

  • Easy to maintain: 3T3-L1 cells are easy to culture in laboratories, making them convenient for multiple cell-based experiments.
  • Low cost: The 3T3-L1 cell line is more affordable than freshly isolated adipocytes, providing a cost-effective alternative for studying differentiation and other cell processes.
  • Differentiation ability: Mouse fibroblast 3T3-L1 cells possess the ability to differentiate. They can acquire an adipocyte phenotype and other characteristic features when exposed to specific stimuli.

Limitations

  • Lack of physiological relevance: The 3T3-L1 adipocytic cells derived from mice lack physiological relevance to human adipocytes and adipose tissue. They do not fully represent the heterogeneity and complexity of adipose tissue in vivo, limiting the direct applicability of experimental outcomes to humans.


Applications of 3T3-L1 cells

Differentiation of 3T3-L1 Adipocytes

The 3T3-L1 cell line is commonly used to study adipocyte biology, adipocyte cell differentiation, and related cellular and molecular mechanisms. The differentiation of 3T3-L1 cells into adipocytes is closely mimics the in vivo differentiation pathway of adipocytes. In adipose tissue, precursor cells residing within the stromal vascular fraction have the potential to differentiate into mature adipocytes in response to various physiological cues, including nutritional status and hormonal signals. The 3T3-L1 model allows for the detailed study of the adipocyte precursor differentiation pathways, providing insights into the molecular mechanisms that govern adipogenesis and its regulation by external factors.

The differentiation process can be induced in culture by exposing confluent 3T3-L1 preadipocytes to a specific cocktail of inducers typically containing insulin, dexamethasone, and isobutylmethylxanthine (IBMX). The induction triggers a series of transcriptional and cellular events that lead to the acquisition of an adipocyte phenotype characterized by lipid droplet accumulation, insulin sensitivity, and the expression of adipocyte-specific proteins such as peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (C/EBPα).

Functional Characteristics of Mature 3T3-L1 Adipocytes

Differentiated 3T3-L1 adipocytes express adipogenic genes and exhibit many functional characteristics of mature adipocytes, the ability to store and mobilize lipids, secrete adipokines, and respond to insulin. These cells become capable of synthesizing and breaking down triglycerides, thereby playing a role in energy homeostasis. The study of 3T3-L1 adipocytes has also shed light on the endocrine functions of adipose tissue, highlighting the secretion of various bioactive peptides and proteins that influence systemic metabolism.

Diabetes and obesity research

3T3-L1 preadipocytes are used as an in vitro model to study molecular pathways involved in diabetes and obesity. Moreover, it may help screen drugs or other therapeutic agents to combat these diseases. For instance, research conducted in 2022 explored the anti-diabetic effects of a traditional herb, Ocimum forskolei Benth, using 3T3-L1 cells. They evaluated glucose uptake, adipogenic markers, and transcription markers i.e., DGAT1, CEBP/α, and PPARγ in treated cells. Accordingly, a study assessed the anti-obesity effects of a plant compound, kaempferol using 3T3-L1 cells. Researchers explored that compound showed anti-obesity potential by inhibiting adipogenesis and promoting lipolysis in these preadipocytes.


Research Publications Featuring 3T3-L1 Cells

Here are prominent and some most cited recent publications featuring 3T3-L1 cells.

Apigetrin inhibits adipogenesis in 3T3-L1 cells by downregulating PPARγ and CEBP-α

This publication in Lipids in Health and Disease (2018) proposed that apigetrin, a flavonoid, suppresses adipogenesis by reducing transcription factor levels, i.e., CEBP-α and PPARγ, in 3T3-L1 cells.

Antiadipogenic effects of loganic acid in 3T3-L1 preadipocytes and ovariectomized mice

This study was published in the Molecules journal in 2018. It proposed that a compound loganic acid in Gentiana lutea L. (GL) root possesses anti-obesity potential as it exerts adipogenic effects in 3T3-L1 cells.

A dose-dependent effect of dimethyl sulfoxide on lipid content, cell viability and oxidative stress in 3T3-L1 adipocytes

This paper in the Toxicology reports (2018) explored the potential effect of dimethyl sulfoxide on 3T3-L1 cell’s lipid content, oxidative stress, and viability in a dose-dependent manner.

Effects of adropin on proliferation and differentiation of 3T3-L1 cells and rat primary preadipocytes

This article was published in the Molecular and Cellular Endocrinology journal in 2019. In this study, researchers evaluated the possible effects of adropin protein on 3T3-L1 cell proliferation and differentiation and primary adipocytes of the rat.

Fucoidan from Undaria pinnatifida has anti-diabetic effects by stimulation of glucose uptake and reduction of basal lipolysis in 3T3-L1 adipocytes

This Nutrition Research (2019) study investigated the anti-diabetic potential of a sulfated polysaccharide, Fucoidan obtained from Undaria pinnatifida. The results revealed that fucoidan stimulates glucose uptake, reduces basal lipolysis in preadipocyte 3T-L1 cells, and exerts these effects.

Ginsenoside Rg2 inhibits adipogenesis in 3T3-L1 preadipocytes and suppresses obesity in high-fat-diet-induced obese mice through the AMPK pathway

This research article was published in 2019 in the Food and Function journal. It proposed that a natural product, ginsenoside Rg2 exerts anti-obesity effects by inhibiting adipogenesis in 3T3-L1 cells and obese mice by regulating the AMPK cascade.


Resources for 3T3-L1 Cell line: Protocols, Videos, and More

3T3-L1 is a famous mouse fibroblast cell line. Multiple resources are available on this cell line's culturing, transfection, freezing, and thawing protocols.

A few resources are mentioned here.

Here you can find some protocols for culturing of 3T3-L1 cell line.

3T3-L1 Adipocytes: FAQ on Their Role in Adipose Tissue Biology and Metabolic Research

3T3-L1 cells, derived from mouse embryonic fibroblasts, are extensively utilized as a model for white adipocytes. They are pivotal for researching adipocyte differentiation, metabolic functions, and the role of adipocytes in obesity and insulin resistance due to their ability to closely mimic the behavior of natural adipose tissue.

Cultivating 3T3-L1 cells in a 3D agarose culture provides a more physiologically relevant environment than traditional 2D cultures. This method allows researchers to observe adipocytes in a configuration that more closely resembles their natural state in tissues, which may affect adipokine secretion and cell interactions.

Adipokines are critical signaling molecules secreted by adipocytes that influence metabolic regulation, inflammation, and insulin sensitivity. Studying the secretion profiles of these adipokines in 3T3-L1 cells sheds light on the endocrine functions of adipose tissue and its systemic metabolic impact.

This technique is employed to examine protein-protein interactions within 3T3-L1 cells, providing insights into the complex signaling networks involved in adipocyte differentiation, lipid metabolism, and insulin signaling pathways.
Biochemical markers, particularly those related to lipid and glucose metabolism, are crucial for assessing the metabolic state of 3T3-L1 cells and the impact of various treatments. Preparing extracts from these cells allows for a detailed examination of these markers, offering a deeper understanding of adipocyte functionality and potential dysfunctions.
Differentiation is induced chemically, typically through a combination of dexamethasone, IBMX, and insulin. This process is vital for investigating the underlying molecular and cellular mechanisms of adipogenesis and the metabolic properties of mature adipocytes.
Researching how glucose levels influence glucose metabolism in 3T3-L1 adipocytes, including aspects like uptake, storage, and oxidation, is key to understanding adipose tissue's role in maintaining systemic glucose balance and insulin sensitivity.
3T3-L1 cells can be manipulated to model insulin resistance and the metabolic conditions associated with obesity. This aids in uncovering the cellular and molecular factors that contribute to insulin resistance.
Lactate production, influenced by factors like hypoxia and changes in glucose metabolism, serves not just as an energy source but also as a signaling molecule that affects adipocyte function and insulin sensitivity.
Analyzing lipid accumulation in 3T3-L1 adipocytes provides insights into their storage capacity and lipid metabolism dynamics, which are crucial for understanding obesity and related metabolic disorders.
These adipocytes have the ability to metabolize glucose and possibly export glucose carbon in various forms, such as lactate, highlighting adipose tissue's integral role in systemic energy and glucose regulation.

References

  1. Rapid Analysis of Human Adipose- Derived Stem Cells and 3T3-L1 Differentiation Toward Adipocytes Using the Scepter™ 2.0 Cell Counter. BioTechniques, 2012. 53(2): p. 109-111.
  2. Xu, J., et al., microRNA-16–5p promotes 3T3-L1 adipocyte differentiation through regulating EPT1. Biochemical and biophysical research communications, 2019. 514(4): p. 1251-1256.
  3. Zhang, L., et al., Promoting differentiation and lipid metabolism are the primary effects for DINP exposure on 3T3-L1 preadipocytes. Environmental pollution, 2019. 255: p. 113154.
  4. Khalil, H.E., et al., Ameliorative Effect of Ocimum forskolei Benth on Diabetic, Apoptotic, and Adipogenic Biomarkers of Diabetic Rats and 3T3-L1 Fibroblasts Assisted by In Silico Approach. Molecules, 2022. 27(9): p. 2800.
  5. Torres-Villarreal, D., et al., Anti-obesity effects of kaempferol by inhibiting adipogenesis and increasing lipolysis in 3T3-L1 cells. Journal of physiology and biochemistry, 2019. 75: p. 83-88.

 

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