EN
NIH/3T3 Mouse Embryonic Fibroblast Cell Line: Complete Culture Guide and Applications
NIH/3T3 is a mouse embryonic fibroblast cell line established from NIH Swiss mouse embryos through isolation and purification. The "3T3" designation refers to its culture protocol: seeding 3×105 cells in a defined area and subculturing every 3 days. Although the original mouse embryonic fibroblasts exhibited strong contact inhibition, this property was gradually lost during prolonged culture and passaging. Nevertheless, studies on contact inhibition using NIH/3T3 cells remain highly valuable under specific experimental conditions.
Research has shown that NIH/3T3 cells are particularly sensitive to sarcoma and leukemia viruses, making them an excellent model for DNA transfection studies. This unique property has established their importance in virology and oncology research, especially for exploring viral infection mechanisms and virus-induced cellular transformation.
Get to Know NIH/3T3 Cells
Basic Cell Information
- Product Name: OriCell™ NIH/3T3 Mouse Embryonic Fibroblast Cell Line
- Catalog No.: M5-0301
- Culture Medium: OriCell™ Complete Medium For NIH/3T3 Cell Line (Catalog No.: CMM5-0301)
- Growth Characteristics: Adherent Growth; Fibroblast-like Morphology
- Culture Conditions: DMEM (high glucose) + 10% Fetal Bovine Serum
- Culture Environment: 95% air + 5% CO₂; 37°C
NIH/3T3 Cell Morphology Under Microscope
Important Note:
Monitor cell density carefully and start passaging before cells reach 80% confluence. Avoid allowing cells to become overly confluent or start to fuse. Recommended passage ratio is 1:3 to 1:4.
NIH/3T3 Cell Related Q & A
Question 1: What should I do if the cells are not visible or show very few adherent cells upon receipt of NIH/3T3 cells ?
Cause: Transportation may cause cell detachment due to shaking, leaving cells suspended in the culture medium and making them difficult to observe under the microscope.
Solution:
- Place the cells in the incubator and leave them undisturbed for 2 hours. Detached cells will reattach, making them visible.
- If cells are still scarce after this, transfer the entire culture medium from the flask to a centrifuge tube.
- Centrifuge at 265×g for 4 minutes to pellet the cells.
- Resuspend the pellet and return it to the original culture flask to continue culturing.
Question 2: Why use fetal bovine serum instead of newborn calf serum?
Answer: Fetal bovine serum contains different growth-promoting factors, attachment factors, hormones, and other bioactive substances compared to newborn calf serum. FBS provides superior nutritional support—simply control cell density to prevent excessive growth that could compromise the contact inhibition property. Our cells have undergone rigorous system validation to ensure reliable performance.
OriCell™ Complete Medium For NIH/3T3 Cell Line includes a basal medium suitable for NIH/3T3 cell growth and OriCell™ Fetal Bovine Serum (Standard quality, Uruguay source, suitable for routine cell culture).
Question 3: Difference Between 3T3-Swiss and NIH/3T3 Cell Lines
The 3T3-Swiss albino mouse embryonic fibroblast cell line was established in 1962 by G. Todaro and H. Green from disaggregated Swiss mouse embryos. These cells exhibit contact inhibition during growth.
The NIH/3T3 mouse embryonic fibroblast cell line originates from NIH Swiss mice and represents a polyclonal population with pronounced clonal variations. It contains subclones prone to spontaneous cellular transformation. Although the original cells displayed strong contact inhibition, this property has gradually diminished over time due to the factors mentioned above.
NIH/3T3 Cells in Research Applications
Application 1: Viral Envelope Protein-Induced Cell Transformation
Research Focus: Ovine pulmonary adenocarcinoma virus envelope protein and its transmembrane (TM) subunit induce NIH/3T3 cell transformation through Akt activation.
Methodology: This study used eukaryotic expression plasmids of JSRV Env and its subunits to induce transformation in NIH/3T3 cells and NIH/3T3-Hyal2-HA cells that stably express the sheep receptor Hyal-2. It was found that both Env and its transmembrane subunit (TM) can induce cell transformation and activate Akt, with the TM subunit likely playing a major role in the transformation process. Meanwhile, sheep Hyal-2 inhibits JSRV Env-induced transformation of NIH/3T3 cells. This transformation process is independent of tumor-related gene mutations.
Research Significance: Due to their rapid growth and ease of transfection with exogenous genes, these cells are important tools in genetic engineering and gene therapy research. Scientists can use NIH/3T3 cells to study the effects of specific genes on cell growth, differentiation, and apoptosis, providing new insights and approaches for disease treatment.
Application 2: GFP Recombinant Adeno-Associated Virus Construction
Research Objective: Construction of GFP recombinant adeno-associated virus reporter systems and expression analysis in NIH/3T3 cells.
Methodology: Using DNA recombination techniques, a recombinant adeno-associated virus (AAV) vector encoding green fluorescent protein (GFP) as a reporter gene was constructed. After transfection into NIH/3T3 cells, efficient GFP expression was observed, providing a reference for mediating gene transfer into eukaryotic cells for gene therapy applications.
Application 3: Human Lung Cancer Genomic DNA Transformation Studies
Research Design: Genomic DNA extracted from non-small cell lung cancer patients (treatment-naive) was used to transfect NIH/3T3 mouse fibroblasts.
Key Findings: Second-round transformed cells exhibited tumor cell characteristics, including:
-
Survival and growth in soft agar
-
Tumor formation in nude mice
-
Identification of ha-ras oncogene as one of the transforming genes
Clinical Implications:
Results suggest that smoking may play a key role in the development of human lung squamous cell carcinoma and the activation of ha-ras.
Cyagen OriCell™ NIH/3T3 Products and Services
1. NIH/3T3 Cell Stock and Supporting Reagents
Cyagen OriCell™ provides reliable, high-quality NIH/3T3 cell line (Catalog No: M5-0301) with comprehensive support. All cells undergo rigorous testing for bacteria, fungi, mycoplasma, and endotoxin contamination, plus STR authentication. We also supply various culture reagents for optimal cell maintenance.
Available Products:
- OriCell™ NIH/3T3 Mouse Embryonic Fibroblast Cell Line, Catalog No.: M5-0301, 1×106 cells/vial
- OriCell™ Complete Medium For NIH/3T3 Cell Line, Catalog No.: CMM5-0301 (500mL)
- NIH/3T3 Knockout Cell Lines, Gene knockout for Smo, 4933427D06Rik, and other genes - contact for details
- OriCell™ NCR Cryopreservation Medium For General Use, Catalog No.: NCRC-10001, 20mL/50mL
- OriCell™ NCR Protein-free Cryopreservation Medium For General Use, Catalog No.: NCPF-10001, 20mL/50mL
2. NIH/3T3 Cell Gene Editing Services
NIH/3T3 cells demonstrate excellent transfection efficiency, making them ideal for laboratory transfection and gene expression studies. Leveraging our mature and stable cell technology service platform, Cyagen offers comprehensive NIH/3T3 cell line genetic modifications including:
-
Routine cellular function and characterization assays
References
- Jainchill JL, et al. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4: 549-553, 1969. PubMed: 4311790
- Copeland NG, Cooper GM. Transfection by exogenous and endogenous murine retrovirus DNAs. Cell 16: 347-356, 1979. PubMed: 222457
- Loffler S, et al. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus. J. Virol. 71: 42-49, 1997. PubMed: 8985321
About Cyagen OriCell™
Cyagen OriCell™ is a Cyagen brand focused on the research and development of cell biology products, including stem cells, primary cells, and cell lines, as well as cell culture reagents and technical services. Serving universities, research institutes, hospitals, CROs, and CDMOs worldwide, Cyagen OriCell™ has accumulated extensive expertise in cell isolation and culture. The team has developed “spatial replication” culture technology to rapidly establish growth‑supportive environments, and runs an Antibiotic‑Free process grounded in strict environmental, materials, and personnel controls. Cyagen OriCell™ provides end‑to‑end solutions—from MSC isolation and identification to directed differentiation and assay services.
Cyagen OriCell™’s offerings are cited in over 10,000 publications, with a cumulative impact factor exceeding 90,000 and more than 160,000 citations, and the team has supported more than 3,000 research groups. Products are used by tens of thousands of customers across dozens of countries and regions.
