Cytotoxicity measurement in cultured primary rat hepatocytes

Commonly used acronym: MTT assay

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Scope of the method

The Method relates to
  • Human health
The Method is situated in
  • Basic Research
Type of method
  • In vitro - Ex vivo
This method makes use of
  • Animal derived cells / tissues / organs
Species from which cells/tissues/organs are derived
rat
Type of cells/tissues/organs
primary hepatocytes

Description

Method keywords
  • cell viability test
  • MTT cytotoxicity assay
  • cytotoxicity of chemicals
  • mitochondria
  • Succinate dehydrogenase
  • Formazan
Scientific area keywords
  • Cell culture
  • cell viability
  • Primary hepatocytes
  • rat
  • liver
Method description

The MTT test is performed to determine the in vitro cytotoxicity of selected chemicals. The mitochondrial enzyme succinate deydrogenase is responsible for the biotransformation of toxic agents and MTT. The ability of cells to reduce MTT provides an indication of the mitochondrial integrity and activity, which in turn may be interpreted as a measure of viability and/or cell number. When chemical compounds are induced in primary rat hepatocytes, their cell viability and their possibility to transform xenobiotical substances decreases. In this respect, when the MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is added to the exposed cells, the possibility to transform this pale yellow salt into dark blue formazan crystals decreases. The formazan crystals formed in the cells are solubilized in DMSO and can be measured colorimetrically.

Lab equipment
  • Multiplate reader.
Method status
  • History of use

Pros, cons & Future potential

Advantages
  • Easy to apply;
  • Not so many extra materials or solutions needed;
  • The method itself is very fast (4h).
Challenges
  • The density of the cells should be even in each well. Make sure that the cell suspension is homogenous and devoid large cell aggregates.
  • There are possible interferences between the tested chemical and the MTT as substrate. MTT can be directly reduced by test substances and give artifacts. Therefore, before initiating experiments, a special procedure that allows quantification of the ''true'' MTT mitochondrial reduction from the ''false'' chemical MTT reduction should be performed.
  • Considerable cell death is observed shortly after isolation of hepatocytes from a freshly removed liver and during the early phases of cultivation. To reduce the effects of this experimentally-induced cell injury and thus to avoid false positive results, experiments should be initiated at earliest 24h after cells seeding.

References, associated documents and other information

References

Mosmann T. (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. Journal of Immunological Methods 65: 55-63

Xu G., Yan Z., Wang N. and Liu Z. (2011) Synthesis and cytotoxicity of cis-dichloroplatinum (II) complexes of (1S,3S)-1,2,3,4-tetrahydroisoquinolines. European Journal Medicinal Chemistry 46(1): 356-363

Porto I.C., Oliveira D.C., Raele R.A., Ribas K.H., Montes M.A. and De Castro C.M. (2011) Cytotoxicity of current adhesive systems: In vitro testing on cell cultures of primary murine macrophages. Dental Materials 27(3): 221-228

Vinken M., Decrock E., De Vuyst E., Leybaert L., Vanhaecke T. and Rogiers V. (2009) Biochemical characterisation of an in vitro model of hepatocellular apoptotic cell death. Alternatives to Laboratory Animals 37: 209-218

Contact person

Axelle Cooreman

Organisations

Vrije Universiteit Brussel (VUB)
Pharmaceutical and Pharmacological Sciences
In Vitro Toxicology and Dermato-Cosmetology
Belgium
Brussels Region