3T3-L1 Cells - Expert Overview of Adipocyte Differentiation and Metabolic Research

3T3-L1 is a preadipocyte cell line of murine origin. It is commonly used to study the essential cellular processes related to obesity, diabetes, and other diseases or disorders. In addition, 3T3-L1 cells are employed to investigate subcellular signaling pathways involved in adipogenesis (differentiation of preadipocytes to mature adipocytes).

This article will go through all the basics of the 3T3-L1 cell line. Mainly it will discuss:

General information and origin of the 3T3-L1 cell line
Culturing of 3T3-L1 cells
3T3-L1 cell line: Advantages & Limitations
Applications of 3T3-L1 cells
Research Publications Featuring 3T3-L1 Cells
Resources for 3T3-L1 Cell line: Protocols, Videos, and More

1. General information and origin of the 3T3-L1 cell line

This section will cover some primary questions about the 3T3-L1 cell line: What are 3T3-L1 cells? What is the size of 3T3-L1 adipocytes? Where do 3T3-L1 cells come from? This information is necessary before you start working with a cell line.

3T3-L1 is a mouse fibroblast cell line subcloned from 3T3 Swiss albino cells based on their lipid accumulation ability. The original 3T3 cells were extracted from the embryo of a mouse.
These adipocytic 3T3-L1 cells possess a fibroblast-like morphology. However, these cells differentiate under appropriate conditions and acquire an adipocyte-like appearance.
3T3-L1 adipocytes possess varying cell sizes at different stages. Such as undifferentiated cells exhibit a mean cell diameter of 15.4 μm, whereas differentiated cells possess a mean diameter of 18.8 μm and 20.3 μm on days 7 and 14, respectively [1].
The 3T3-L1 cells possess an unstable karyotype with 2n = 40 chromosomes.

3-dimensional medical animation of growing fat cells.

2. 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.

3. 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

The main advantages of 3T3-L1 cells are mentioned here:

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

The limitations of 3T3-L1 cells are:

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.

4. Applications of 3T3-L1 cells

3T3-L1 cells are commonly used for studying molecular mechanisms related to adipogenesis, diabetes, obesity, and related disorders. Some of the research applications of 3T3-L1 cells are:

Adipocyte differentiation

The 3T3-L1 cell line is commonly used to study adipocyte biology, the differentiation process, and related cellular and molecular mechanisms. Several studies have been conducted on 3T3-L1 cells, such as a study investigating the functional role of microRNa-16-5p in differentiating preadipocyte 3T3-L1 cells to mature adipocytes. The study found that overexpressed miRNA-16-5p facilitates differentiation by encouraging the expression of mature adipocyte-specific genes and accumulation of fat droplets in vitro and in vivo [2].

Similar to this, another study evaluated the effect of Diisononyl phthalate (DINP) on adipogenesis using the 3T3-L1 cell line. Researchers observed that DINP, a high molecular weight phthalate, extensively induced adipogenesis in 3T3-L1 cells in 10 days [3].

Diabetes and obesity

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 [4].

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 [5].

5. 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.

6. 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.

Differentiation of 3T3-L1 cells: This link provides a detailed protocol for differentiating 3T3-L1 preadipocytes.
Transfection of 3T3-L1 cells: This video is a tutorial guide for the transfection of 3T3-L1 cells.
Passaging of 3t3 cells: This video is explaining the procedure for passaging 3T3 mouse fibroblast cells.

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

Culturing 3T3-L1 cells: This link contains a detailed step-by-step guide for splitting 3T3-L1 cells. Moreover, it has a protocol for cell freezing and differentiation.
3T3-L1 cell culture and differentiation: This link will provide a protocol for the culturing and differentiation of 3T3-L1 cells.

References

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.
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.
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.
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.
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.