Neutral Red Uptake (NRU) Cytotoxicity Test

The experiment can be designed to quantitatively evaluate medical devices based on the current ISO 10993-5:2009 guideline “Test for in vitro cytoxicity” or to evaluate chemical compounds.

Typically, BALB/c 3T3 cells (clone 31) are seeded into 96-well plates and maintained in culture for 24 h (~ 1 doubling period) to form a semi-confluent monolayer. Cells are then exposed eight test compound concentrations covering a large range. After 24 h exposure, NRU is determined for each treatment concentration and compared to that determined in control cultures. Negative controls (blank, untreated and solvent/ZBDC) and four concentrations of positive control (sodium lauryl sulfate, H2O2 or ZDEC) are tested. The Neutral Red uptake is measured by spectrometry and, if the test substance induces a cytotoxic effect.

If the test substance induce a cytotoxic effect on the cells, the IC50 (i.e. the concentration producing 50 % reduction of viability) is calculated from the concentration-response. If achievable, the eight concentrations of each compound tested should span the range of no effect up to total inhibition of cell viability. If the relative cell viability for the highest concentration of the sample is = 70 % of the control group, then the material is considered non-cytotoxic.

The experiment will start after receipt of signed protocol and the test substance. A draft report will be ready for review 4-6 weeks after the experiment start date.

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