CLS will be called Cytion
Fastest deliveries on the market
> 800 well characterized cell lines
Worldwide service – one hand, one partner
Visit for your cell line needs

NIH-3T3 Cells: Advancing Fibroblast Studies and Applications of NIH-3T3

The NIH-3T3 cell line, established from the tissue of a 17-day-old Swiss Albino mouse embryo in 1962 by Howard Green and George Todaro at the New York University School of Medicine, has become a fundamental resource in biomedical research. Recognized for its high receptiveness to leukemia virus and sarcoma virus focus formation, NIH-3T3 cells serve as a critical tool for a plethora of scientific inquiries, including viral oncology studies, gene expression analysis, and exploration of cellular growth dynamics. The "3T3" nomenclature reflects the cell culture method, denoting a "3-day transfer" interval with an initial seeding density of 3 × 10^5 cells, highlighting the standardized conditions under which these cells were first cultured and expanded.

Diverse Morphologies and Applications of NIH-3T3 Cells

One of the hallmark characteristics of NIH-3T3 cells is their morphological adaptability, which varies significantly with culture confluency. At lower densities, these fibroblasts display a spindle-shaped, solitary cell structure, evolving into dense, swirling patterns as the population reaches confluence. With an average diameter of about 18 μm, NIH-3T3 cells offer a versatile model for in-depth cell biology studies, ranging from tissue repair mechanisms to the intricate pathways of cell cycle regulation.

NIH-3T3 cells at high and low confluence.

Culturing information

  • Key Culturing Details:

    • Population Doubling Time: Roughly 20 hours.

    • Growth Type: Adherent cultures.

    • Seeding Density: Recommended: 3 to 4 x 10^4 cells/cm^2.

    • Growth medium: DMEM or Ham's F12, supplemented with 5% FBS and 2.5 mM L-glutamine.

    • Growth Conditions: Maintain at 37 °C in a humidified incubator with 5% CO2.

    • Storage: Keep at temperatures below -195 °C in the vapor phase of liquid nitrogen.

    • Freezing Method: Use CM-1 or CM-ACF medium; employ a slow freezing method (1°C temperature drop).

    • Thawing Protocol: rapid warming in a 37 °C water bath, followed by centrifugation to remove freezing medium, then resuspension in growth medium.

    • Biosafety Level: Culturing requires a biosafety level 1 setting.

Swiss Albino mouse in a laboratory.

Pros and Cons of Using NIH 3T3 Cells


  • Transfection Efficiency: Known for their high transfection rates, NIH-3T3 cells are excellent for both transient and stable gene expression studies, accommodating a variety of transfection techniques.

  • Feeder Layer Utility: These cells often serve as a supportive feeder layer for co-cultures with cells like keratinocytes and stem cells, thanks to their release of growth factors that promote co-cultured cell growth.

  • Stem Cell Research: NIH-3T3 cells are a preferred choice in stem cell research for inducing pluripotency without genetic modification and providing a conducive environment for stem cell differentiation.

  • Culture stability: NIH-3T3 cells are known for their stability and low frequency of spontaneous transformation. However, under certain conditions or after exposure to specific oncogenes or mutagens, NIH-3T3 cells can undergo spontaneous transformation. This transformation can lead to the acquisition of cancerous properties such as uncontrolled growth, loss of contact inhibition, and the ability to form tumors when injected into susceptible hosts. 


  • Inconsistent Cell Size: The elongated, spindle-like morphology of NIH-3T3 cells can vary, complicating image analyses in assays.

  • Infection Susceptibility: These cells are prone to bacterial and mycoplasma infections if not maintained in stringent aseptic conditions, potentially impacting experimental integrity.

Research Applications of NIH-3T3 Cells

  • DNA Transfection Studies: NIH-3T3 cells' robustness makes them ideal for introducing and studying the function of various genes, demonstrated in research examining proteins like NAB2-STAT6 and their roles in cellular processes.

  • Cell-Based Assays: Their reliability extends to various assays, including viability, apoptosis, and focus formation assays, offering insights into cellular responses under different experimental conditions.

  • Cell Cycle Research: The cell line's straightforward cell cycle manipulation via serum levels makes it a potent model for studying cell cycle regulation and its aberrations in disease contexts.

Elevate Your Research with NIH-3T3 Cells

Highlighting Key Studies Involving the fibroblast cell line NIH 3T3 

The NIH-3T3 cell line has been pivotal in numerous research projects, spanning various facets of cellular biology. Below are some significant studies utilizing these cells:

Essential Resources for NIH-3T3 Cell Research

For researchers interested in working with NIH-3T3 cells, a variety of resources are available to guide culturing and experimental protocols:

  • Spheroid Formation in NIH-3T3 Cells: This video provides a detailed walkthrough of forming spheroids, a 3D cell culture technique that aggregates NIH-3T3 cells into clusters, offering a more physiologically relevant model for studies.
  • Monitoring NIH-3T3 Cell Growth: Through the JuLI Br live cell imaging system, this video captures the growth dynamics of NIH-3T3 cells over 65 hours, showcasing real-time cell proliferation.

These resources aim to support your research endeavors with NIH-3T3 cells, providing a foundation for successful experiments and discoveries.

Frequently Asked Questions about NIH-3T3 cells

3T3 mouse cells are a fibroblast cell line derived from embryonic mouse tissue, specifically from NIH Swiss mouse embryos. They are used extensively in biological research for studying cell behaviors like division, migration, and transformation.
NIH3T3 cells are used extensively in biological and medical research for various purposes, including studying cell behavior (such as growth, division, and differentiation), understanding cancer biology and cellular transformation mechanisms, gene expression and regulation studies, and drug discovery and development. Their ease of culture and transfection also make them ideal for studying the effects of gene overexpression or silencing.
The "3T3" in NIH3T3 stands for "3-day transfer, inoculum 3 × 10^5 cells." This nomenclature refers to the original culturing protocol developed for these cells, which involved transferring the cells every three days with a set inoculum density to maintain optimal growth conditions and prevent contact inhibition.
3T3 cells, including NIH3T3, are not inherently cancerous. They are immortalized fibroblast cells derived from mouse embryos. However, they can undergo transformation into a cancerous state when exposed to certain oncogenes or under specific experimental conditions, making them useful for studying cancer biology and tumorigenesis.
Through techniques such as NIH3T3 transfection, researchers can manipulate the NIH 3T3 cell line to study specific molecular pathways involved in cell growth, differentiation, and disease processes, like cancer.
NIH 3T3 cells serve as a model to study various fibroblast subtypes, including myofibroblasts, by providing a system to observe the characteristics of cell differentiation and the cellular composition's role in tissue repair and fibrosis.
Yes, NIH 3T3 cells are immortalized. They were derived from mouse embryonic fibroblast cells and have acquired the ability to proliferate indefinitely in vitro, making them a valuable tool for long-term biological studies.
The growth rate of NIH3T3 cells can vary depending on the culture conditions, but under optimal conditions, they typically have a doubling time of about 18 to 24 hours. This means the population size of the NIH3T3 cells can double in approximately one day, making them suitable for experiments requiring rapid cell proliferation.
The NIH3T3 cell line was established in the early 1960s from mouse embryonic fibroblast cells. Specifically, they were derived from the tissue of 17- to 19-day-old embryos of NIH Swiss mice. This cell line was developed to provide a consistent and reproducible cell model for scientific research.
While both 3T3 and 3T3-L1 cells originate from mouse embryonic fibroblast cells, the key difference lies in their specialization and application. NIH3T3 cells are used broadly in cell biology and oncology research. In contrast, 3T3-L1 cells are a subline of 3T3 cells that have been specifically selected for their ability to differentiate into adipocytes (fat cells) under the right conditions. This makes 3T3-L1 cells particularly valuable for studies related to adipogenesis, metabolism, and obesity.


  1. Rahimi, A.M., M. Cai, and S. Hoyer-Fender, Heterogeneity of the NIH3T3 Fibroblast Cell Line. Cells, 2022. 11(17): p. 2677.
  2. Leibiger, C., et al., First molecular cytogenetic high resolution characterization of the NIH 3T3 cell line by murine multicolor banding. Journal of Histochemistry & Cytochemistry, 2013. 61(4): p. 306-312.
  3. Wang, H.-X., et al., Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells. International Journal of Ophthalmology, 2017. 10(7): p. 1021.
  4. Wang, Z., et al., Differentiation of neuronal cells from NIH/3T3 fibroblasts under defined conditions. Development, growth & differentiation, 2011. 53(3): p. 357-365.
  5. Park, Y.-S., et al., NAB2-STAT6 fusion protein mediates cell proliferation and oncogenic progression via EGR-1 regulation. Biochemical and Biophysical Research Communications, 2020. 526(2): p. 287-292.
  6. Mattsson, M., Expression of the Sloppymerase™ in NIH/3T3 Cells: Exploring the Versatility of an Error Prone Fusion Polymerase. 2021.
  7. Sahinturk, V., et al., Acrylamide exerts its cytotoxicity in NIH/3T3 fibroblast cells by apoptosis. Toxicology and Industrial Health, 2018. 34(7): p. 481-489.
  8. Lusi, E.A. and F. Caicci, Discovery of the First Human Retro-Giant Virus: Description of its morphology, retroviral kinase and ability to induce tumours in mice. bioRxiv, 2019: p. 851063.
  9. Endo, M., et al., E2F1Ror2 signaling mediates coordinated transcriptional regulation to promote G1/S phase transition in bFGFstimulated NIH/3T3 fibroblasts. The FASEB Journal, 2020. 34(2): p. 3413-3428.
  10. Long, L., et al., Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle–Related Gene Transcription. The Journal of Nutrition, 2018. 148(6): p. 834-843.


We have detected that you are in a different country or are using a different browser language than currently selected. Would you like to accept the suggested settings?