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Hepatocarcinoma Cytotoxicity Assay (MTT and LDH)

The Hepatocarcinoma Cytotoxicity Assay tests the cytotoxicity of nanoparticle formulations in human hepatocarcinoma cells (Hep G2). The protocol utilizes two methods for estimation of cytotoxicity, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release (1, 2).

The MTT assay is a colorimetric assay that can assess the viability of cells by quantitation of the reduction of the yellow substrate MTT (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to a product that has a purple color. This assay can measure the cytotoxicity of a chemical or drug by determining the affect of treatment on cell viability. The Assay File represents an example for a MTT cytoxicity assay (MTT) performed on three nanoparticle samples, where 9 dilutions of each nanoparticle sample are exposed to porcine proximal tubule cells for three different times of exposure (6h, 2h, and 48 h) (3).

LDH is a cytoplasmic enzyme that is released into the cytoplasm upon cell lysis. The LDH assay is a measure of membrane integrity. The basis of the LDH assay: (1) LDH oxidizes lactate to pyruvate, (2) Pyruvate reacts with the tetrazolium salt INT to form formazan, and (3) the water-soluble formazan dye is detected spectrophotometrically (4,5).

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References

1. ISO 10993-5 Biological evaluation of medical devices: Part 5 Tests for in vitro  cytotoxicity.
2. F1903 – 98 Standard Practice for Testing for Biological Responses to Particles in vitro .
3. Alley, et al. (1988) Cancer Res.  48:589-601.
4. Decker, T. & Lohmann-Matthes, M.L. (1988) J. Immunol Methods  15:61-69.
5. Korzeniewski, C. & Callewaert, D.M. (1983) J. Immunol Methods  64:313-320.