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C2C12 Myoblast Cells - Pioneering Muscle Biology and Regeneration Research

C2C12 is a continuous murine myoblast cell line broadly used in biomedical research studies. Mainly, it is applied to investigate various biological processes involved in skeletal muscle biology, such as muscle development, differentiation, and gene expression.

This article will review all the imperative information about C2C12 myoblasts you will look for before working with this cell line. You will learn the following:

  1. Origin and general characteristics of C2C12 cells
  2. Culturing information of C2C12 cells
  3. C2C12 cell line: Advantages & Limitations
  4. Research applications of C2C12 cell line
  5. C2C12 cells: Research publications
  6. Resources for C2C12 cell line: Protocols, Videos, and More

1.      Origin and general characteristics of C2C12 cells

Origin and general characteristics of a cell line are the primary information about a cell line that help you decide to use it in your research work. In this article section, you will know: Where the C2C12 myoblast cells come from? What is the morphology of C2C12 cells? What is the size of the C2C12 cell?

  • C2C12 is a derivative of the myoblast cell line developed by Yaffe and Saxel in 1977 at the Weizmann Institute of Science, Israel. The original C2 cell line was obtained from a 2-month-old normal C3H mouse thigh muscle after 70 hours of crush injury.
  • C2C12 muscle cells can rapidly propagate under high serum conditions and differentiate into myotubes under starvation (low serum conditions). Myotubes are the forerunners of contractile skeletal muscle cells.
  • These cells have myoblast-like morphology. They exhibit radial branching containing long fibres extending in multiple directions.
  • C2C12 myoblast cells are diploid.

Smooth muscle separated under the microscope.

2.      Culturing information of C2C12 cells

C2C12 cells are widely cultured muscle cells. Before developing C2C12 cell culture, you should know: What is the doubling time of C2C12 cells? What is C2C12 seeding density? What is C2C12 media?

Key Points for Culturing C2C12 Cells

Doubling Time:

The doubling time for C2C12 cells ranges from 12 to 24 hours.

Adherent or in Suspension:

C2C12 myoblast cells are adherent.

Seeding Density:

C2C12 seeding density is kept at 1 x 104 cells/cm2. At this seeding density, cells form a confluent monolayer in about 4 days. For seeding cells are washed with phosphate buffer saline (PBS) and incubated with passaging solution (Accutase). Afterwards, detached cells are collected through centrifugation and resuspended in a growth medium. Resuspended cells are poured into the new flask for culturing.

Growth Medium:

RPMI 1640 medium supplemented with 10% fetal bovine serum, 2.1 mM stable Glutamine, and 2.0 g/L NaHCO3 salt is used to culture C2C12 cells. Media should be renewed every 3 to 5 days.

Growth Conditions:

C2C12 mouse myoblasts are cultured in a 37°C humidified incubator connected with a 5% CO2 supply.


C2C12 muscle cells can be stored for longer terms in the vapour phase of liquid nitrogen or electric ultra-low temperature freezers at below -150°C temperature.

Freezing Process and Medium:

C2C12 cell line can be stored in CM-1 or CM-ACF freezing media. A slow freezing method is used to freeze cells. It permits a gradual 1°C drop in temperature and effectively protects cell viability.

Thawing Process:

Frozen cells are thawed in a pre-warmed water bath set at 37°C temperature for 40 to 60 seconds. Thawed cells are resuspended in fresh culture media and harvested through centrifugation. This step eliminates freezing media components. Recovered cells are then resuspended and dispensed into the new flasks containing growth medium. 

Biosafety Level:

Biosafety level 1 laboratory settings are recommended for handling C2C12 cell cultures.


Murine myoblast cell line C2C12, 20- and 10-fold magnified.

3.      C2C12 cell line: Advantages & Limitations

C2C12 mouse myoblasts cell line, developed from skeletal muscle tissue, has several advantages and limitations in biomedical research. Here are some of them:


The significant advantages of C2C12 cells are:


C2C12 is a well-characterized cell line. It is extensively studied for physiological and biological characteristics such as cell morphology, differentiation potential, behaviour and response to stimuli, etc. Therefore, its application in research ensures the reliability and reproducibility of experimental results.

Muscle differentiation

C2C12 cells can differentiate into muscle-like cells thus, are an invaluable research tool for studying muscle biology, including differentiation, muscle development, and myotube formation. C2C12 myotubes express muscle cells like contractile proteins that can help in spontaneous contraction when cells are left to differentiate for a long time [1].

Well-documented cell model

C2C12 myoblast cell line is a well-documented cell model that can help understand different cell biology processes, i.e., oxidative stress, reactive oxygen species, glucose metabolism, insulin signalling mechanism, insulin resistance, and glucose transporters at both cellular and molecular levels [2].


The limitation associated with C2C12 skeletal muscle cells are:

Non-human cell line

C2C12 is the mouse myoblast cell line. These cells are obtained from mice. Thus, they may not fully exemplify human muscle physiology and biology. Species-specific variations in gene expression and cellular metabolism may limit the direct translatability of results to humans.


4.      Research applications of C2C12 cell line

Here, you will learn about some significant C2C12 mouse cell line research applications.

  • Study of muscle biology: C2C12 mouse myoblast cells are frequently used as an in vitro model to study muscle development, metabolism, and differentiation. Researchers can induce the differentiation of C2C12 cells into muscle-like cells to explore the cell and molecular mechanisms involved in myotube formation, myogenesis, and muscle regeneration. Such as a study investigated that transforming growth factor beta 1 (TGF-β1) is involved in regulating C2C12 cell proliferation and differentiation. Further, findings revealed that microRNA-22 also regulates these muscle cell functions by targeting TGFβR1 [3].
  • Drug screening and toxicity testing: C2C12 muscle-like cells are utilized for evaluating potential therapeutic agents, i.e., compounds or drugs for combating muscle-related disorders. Several studies have been conducted to assess the effects of drugs on muscle cell metabolism, proliferation, and differentiation using the C2C12 cell line. Research conducted in 2023 investigated and proposed that Cnidoscolus aconitifolius (chaya) leaf extract can positively regulate fatty acid oxidation and mitochondrial bioenergetics in C2C12 cells [4]. Similarly, a study stated that Moringa oleifera leaf extract protects C2C12 myotubes against hydrogen peroxide-induced oxidative stress damage [5].

5.      C2C12 cells: Research publications

Some significant and frequently cited publications featuring C2C12 cells are mentioned here:

Interleukin-6 induces myogenic differentiation via JAK2-STAT3 signaling in mouse C2C12 myoblast cell line and primary human myoblasts

This paper was published in 2019 in the International Journal of Molecular Sciences. The study proposed that interleukin-6 (IL-6) induces differentiation in primary human muscle cells and the C2C12 myoblast cell line via regulating JAK2/STAT3 signalling.

Effect of Rubus anatolicus Leaf Extract on Glucose Metabolism in HepG2, CRI-D2 and C2C12 Cell Lines

This research was published in Diabetes, Metabolic Syndrome, and Obesity (2023). The study explored the effect of Rubus anatolicus on the glucose metabolism of C2C12 and the other two cell lines. The extract probably activates glycogenesis to facilitate glucose uptake and cellular glycogen synthesis.

Differentiation of murine C2C12 myoblasts strongly reduces the effects of myostatin on intracellular signaling

This research proposed that C2C12 cell differentiation markedly inhibits the effect of myostatin, an important negative regulator of muscle size, on intracellular signalling. This paper was published in Biomolecules in 2020.

Effects of genistein on insulin pathway-related genes in mouse differentiated myoblast C2C12 cell line: evidence for two independent modes of action

This study was published in Folia Histochemica et Cytobiologica (2018). This study used a differentiated mouse myoblast cell line, C2C12, to evaluate the potential effect of genistein on genes related to the insulin pathway

Moringa oleifera leaf extract influences oxidative metabolism in C2C12 myotubes through SIRT1-PPARα pathway

This research published in Phytomedicine Plus (2021) proposed that a medicinal plant, Moringa Oleifera, leaf extract favours mitochondrial biogenesis and oxidative energy metabolism via regulating the SIRT1-PPARα pathway.

6.      Resources for C2C12 cell line: Protocols, Videos, and More

The following are a few online resources featuring C2C12 cells:

The cell culture protocols for C2C12 cells are listed here:

  • C2C12 Myoblasts: This document provides protocols for passaging and transfecting C2C12 muscle cells.
  • C2C12 culture: This link contains useful information for culturing and differentiating C2C12 cells.
  • C2C12 differentiation: This document explains how to grow frozen C2C12 cell cultures. Moreover, it also has a protocol for the differentiation of C2C12 cells.


  1. Denes, L.T., et al., Culturing C2C12 myotubes on micromolded gelatin hydrogels accelerates myotube maturation. Skeletal muscle, 2019. 9(1): p. 1-10.
  2. Wong, C.Y., H. Al-Salami, and C.R. Dass, C2C12 cell model: its role in understanding of insulin resistance at the molecular level and pharmaceutical development at the preclinical stage. J Pharm Pharmacol, 2020. 72(12): p. 1667-1693.
  3. Wang, H., et al., miR-22 regulates C2C12 myoblast proliferation and differentiation by targeting TGFBR1. European Journal of Cell Biology, 2018. 97(4): p. 257-268.
  4. Avila-Nava, A., et al., Chaya (Cnidoscolus aconitifolius (Mill.) IM Johnst) leaf extracts regulate mitochondrial bioenergetics and fatty acid oxidation in C2C12 myotubes and primary hepatocytes. Journal of Ethnopharmacology, 2023. 312: p. 116522.
  5. Ceci, R., et al., Moringa oleifera leaf extract protects C2C12 myotubes against H2O2-induced oxidative stress. Antioxidants, 2022. 11(8): p. 1435.