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NIH-3T3 Cell Line - Advancing Fibroblast Studies and Applications of NIH-3T3

The NIH-3T3 cell line is a fibroblast cell line derived from a 17-19 day-old Swiss Albino mouse embryo in 1962 by Howard Green and George Todaro of the New York University School of Medicine. This cell line is highly susceptible to the propagation of leukemia virus and the formation of sarcoma virus focus. Due to its susceptibility to viral propagation, the NIH-3T3 cell line has been widely used for cell-based assays, DNA transfection, and cell cycle control studies. Additionally, NIH-3T3 cells have spontaneously immortalized and become tetraploid. "3T3" stands for "3-day transfer, inoculum 3 × 10^5 cells." It is important to consider the origin and general characteristics of the NIH-3T3 cell line before working with it in research [1,2].

  1. Characteristics of NIH-3T3 Cells
  2. Pros and Cons of Using NIH-3T3 Cell Line
  3. Applications of NIH-3T3 cell line in research
  4. Cell culture protocols

1.         Characteristics of NIH-3T3 Cells

The NIH-3T3 cell line exhibits varying morphologies depending on the confluency of the culture. Initially, single attached cells have a spindle-like appearance, but with subsequent growth, cells take on an overlapping swirling shape. The diameter of NIH-3T3 cells is approximately 18 μm.

NIH-3T3 cells at high and low confluence.

Regarding genome and ploidy, NIH-3T3 cells are hypertriploid, with 30% of cells possessing a modal chromosome number of 68. Higher ploidies may occur in 2.4% of NIH-3T3 cells. Considering these characteristics when using the NIH-3T3 cell line in research is important.

NIH-3T3 cells are easy to culture. There are no fussy requirements for maintaining their cultures. This section will focus on: What is the doubling time for NIH-3T3 cells? what growth medium is recommended for the NIH-3T3cell line? what is the seeding density of NIH-3T3?


Population Doubling Time

Approximately 20 hours

Adherent or in Suspension

Adherent cultures

Seeding Density

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

37 °C temperature, 5% CO2 supply, humidified incubator


Below -195 °C temperature in the vapor phase of liquid nitrogen

Freezing Medium and Process

CM-1 or CM-ACF freezing medium, slow freezing method (1°C drop in temperature)

Thawing Process

Rapid agitation in 37 °C water bath, centrifugation to remove freezing medium, resuspension in growth medium

Biosafety Level

Biosafety level 1 setting


2.        Pros and Cons of Using NIH-3T3 Cells

When selecting a cell line, it is important to consider its advantages and disadvantages. This article will explore the potential pros and cons of using the NIH-3T3 cell line.


  1. Transfection flexibility: NIH-3T3 cells have high transfection efficiency, making them suitable for both transient and stable gene expression. Various physical and chemical methods can be used for transfection.
  2. Feeder cells: NIH-3T3 cells are commonly used as feeder cells for co-cultured cells, such as keratinocytes and stem cells. This is due to the growth factors that are released by NIH-3T3 cells, which support the growth of other cells [3].
  3. Stem cell experiments: NIH-3T3 cells are widely used as a cellular model for stem cell-induced pluripotency experiments. By providing defined conditions and a specific growth medium, these fibroblast cells can be differentiated into various stem cell types without the need for epigenetic modification [4].


  1. Variable cell size: NIH-3T3 cells have an elongated spindle-like shape and can vary in size. This can pose a challenge during image analysis in cell-based assays.
  2. Bacterial infection: NIH-3T3 cells are susceptible to bacterial infections if aseptic conditions are compromised. Bacterial contamination can typically be detected by turbidity in the growth medium and pH changes, but mycoplasma infections are more difficult to detect. Such infections can affect gene expression and the morphology of cells.

3.        Applications of NIH-3T3 cell line in research

The NIH-3T3 cell line is commonly used in cell and molecular biology research due to its low-maintenance culture requirements. Here are some examples of its applications:

  • DNA transfection studies: NIH-3T3 cells are frequently used for transfection studies, where genes are introduced to mouse fibroblast cells to investigate their functions. The cell line has been used for both stable and transient transfection, such as a study in 2020 that used NIH-3T3 cells to transfect a fusion protein NAB2-STAT6 to study its role in cell migration and proliferation [5]. Another study used NIH-3T3 cells for stable transfection of an enzyme, sloppymerase [6].
  • Cell-based assays: The NIH-3T3 cell line is commonly used for various cell-based assays. For example, a study used NIH-3T3 cells to determine cell viability and apoptosis in response to acrylamide through MTT assay, caspase 3/7, and annexin V assays [7]. The virus-infected NIH-3T3 cell line is also utilized for the focus formation assay [8].
  • Cell cycle control studies: The NIH-3T3 cell line is frequently used in cell cycle control studies because its cell cycle mechanism can be easily controlled by serum deprivation or stimulation through serum or growth factors [9]. Researchers have cultured the mouse fibroblast cell line in a riboflavin-deficient medium and observed tumorigenesis in NIH-3T3 cells. These cells showed an increased proliferation rate and dysregulation in cell cycle-related genes [10].

Swiss Albino mouse in a laboratory.

Notable Research Studies on NIH-3T3 Cells

NIH-3T3 cells have been the subject of numerous research studies. Here are a few notable examples:

  1. NAB2-STAT6 fusion protein mediates cell proliferation and oncogenic progression via EGR-1 regulation:  This study, published in Biochemical and Biophysical Research Communications, utilized NIH-3T3 cells to express the NAB2-STAT6 fusion protein. The findings suggest that this fusion protein enhances cell growth and migration by regulating EGR-1.
  2. Mouse APOBEC3 expression in NIH 3T3 cells mediates hypermutation of AKV murine leukemia virus: The Virology journal published this study, which proposed that the expression of the mouse APOBEC3 gene in NIH-3T3 cells facilitates hypermutation of the AKV murine leukemia virus.
  3. Antimetastatic effect of epigenetic drugs, hydralazine and valproic acid, in Ras-transformed NIH 3T3 cells:In this study published in Oncotargets and Therapy, researchers used RAS-transformed NIH-3T3 cells to evaluate the anti-metastatic potential of epigenetic drugs, such as valproic acid and hydralazine.
  4. Baicalein inhibits proliferation and collagen synthesis of mice fibroblast cell line NIH/3T3 by regulation of miR-9/insulin-like growth factor-1 axis: This study used the mouse fibroblast cell model NIH-3T3 to investigate the inhibitory effect of Baicalein, a natural compound, on cell proliferation and collagen synthesis.
  5. Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle–Related Gene Transcription:This study proposed that the depletion of riboflavin from the growth medium of the NIH-3T3 cell line promotes tumorigenesis. The research found that deprivation of vitamin B2 increases cell proliferation and causes dysregulation of cell cycle-related genes.

NIH-3T3 is a popular mouse fibroblast cell line, and multiple online resources are available on it. Here, we will enlist a few resources explaining the culturing and transfection protocols of NIH-3T3 cells.

4.       Cell culture protocols

Spheroid Formation of NIH-3T3 Cells: This video showcases the process of spheroid formation in NIH-3T3 cells. The spheroid formation is a three-dimensional (3D) cell culture technique where cells aggregate to a cluster. This technique provides a more physiologically relevant model for in vitro studies. The video demonstrates the aggregation and compaction of NIH-3T3 cells over time, resulting in the formation of spheroids.

NIH 3T3 cells growth by using JuLI Br 65 hrs:The video demonstrates the growth of NIH 3T3 cells over 65 hours, captured using the JuLI Br live cell imaging system. The imaging shows the proliferation of cells in real time, allowing for a detailed understanding of their growth patterns. The video highlights the importance of live cell imaging in cell biology research, allowing for the continuous observation of cell behavior over extended periods.

We hope the data in this article will help you start working with this cell line. If you are interested in using this cell line, order from us.


  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.


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