Quantitative assessment of cellular metabolic activity

Evaluation of cell viability based on mitochondrial activity.

The MTT test is an in vitro cytotoxicity assay based on the cleavage of the yellow tetrazolium salt MTT to form a violet formazan product. A decrease in the number of viable cells results in reduced metabolic activity within the test culture. This reduction directly correlates with the amount of violet formazan formed, as determined by absorbance measurements.

Using the MTT assay, cell proliferation and cell viability following treatment with the test item are determined colorimetrically.

Assessment of cell viability by extraction method and MTT dye

  • The test item is extracted under agitation for a defined period in DMEM supplemented with 10% FBS at 37 ± 1°C, and L929 cells are incubated with the extract.
  • At the end of the incubation period, cells are evaluated microscopically and the MTT reagent is added.
  • Cells are incubated for approximately 2 hours. Thereafter, solubilisation solution (HCl) is added to each well, and absorbance is determined at 570 nm (reference wavelength 650 nm).
  • Cell viability below 70% compared with untreated control cultures (solvent control) is considered a clear cytotoxic effect according to ISO 10993-5.

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MTT test protocol

Protocol

Cell line

L929 cells (ATCC No. CCL1, NCTC clone 929; connective tissue mouse cell line, clone of strain L, DSMZ)

Analysis

The MTT test is based on the cleavage of the yellow tetrazolium salt MTT to form a violet formazan product.

Concentrations

4 concentrations of the test extract: 29.6%, 44.4%, 66.7% and 100%

Extraction time

4 - 72 h at 37 ± 1°C

- 4 h (short-term contact and intact skin or mucosa)
24 ± 2 h (short-term and limited contact to patient)
72 ± 2 h (prolonged and permanent contact)

Incubation time

24 - 72 h at 37 ± 1°C

Quality controls

Solvent Control: DMEM 10% FBS

Negative control: Polypropylene extracted in DMEM 10% FBS

Positive control: Latex extracted in DMEM 10% FBS

Data delivery

Cell viability is determined through the absorbance at 570 nm (reference wavelength 650 nm).

Positive prediction

Cell viability below 70% compared with untreated control cultures (solvent control) is considered a clear cytotoxic effect.

 

References

  1. Scudiero, P. A.; Shoemaker, R. H.; Paull, K. D.; Monks, A.; Thierney, S.; Nofziger, T. H.; Currens, M. J.; Seniff, D.; Boyd, M. R.: 1988, “Evaluation of a soluble Tetrazolium/Formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines“. Cancer Res., 48, 4827 – 4833
  2. ISO 10993-5: 2009, “Biological evaluation of medical devices – Part 5: Tests for in vitro cytotoxicity“
  3. ISO 10993-12: 2012, “Biological evaluation of medical devices – Part 12: Sample preparation and reference materials“