Cytotoxicity Testing fall into two groups:
There are three types of qualitative tests, the results of which are determined by using a microscope to look for signs of cytotoxicity, such as changes in the size or configuration of cells or inhibited growth. These are judged on a scale of 0–4, with a score of 0 indicating no reactivity to the test material and a score of 4 indicating severe reactivity.
The three qualitative tests are as follows:
Elution: The most popular qualitative testing method, elution is also known as testing by extraction. During the test, a fluid extract of the test material is placed in contact with the cultured cells, which are then incubated for three days and evaluated for any signs of cytotoxicity.
Indirect Contact: Also known as the Agar Diffusion method, this test places a layer of agar between the material being tested and the cultured cells. The layer protects the cells from the physical effects of contact with the material being tested, but allows extractables from the material to pass through to the cell cultures.
Direct Contact: During the direct contact method, the test material is placed in direct contact with the cultured cells. This method is generally used for low-density materials like contact lenses.
The quantitative method is known as the MTT Assay. The MTT method is a colorimetric assay, and it can be performed using either extracts or direct contact. This test is graded based on the percentage of cells that are inhibited or die after contact with the test material.
Generally, the tests are performed in vitro on cultured mammalian cells, such as those of mice. The human body has a number of measures in place to protect cells from cytotoxins and pH imbalances, so by performing the tests on cells existing in vitro—without these defense systems—labs ensure a high degree of sensitivity in the tests.
The current regulatory reforms on biocompatibility for devices (ANSI/AAMI/ISO 10993–5:2009) suggest that qualitative cytotoxicity tests are suitable for risk stratification, but that quantitative evaluation is favoured.
MTT Assay: This quantitative method uses a dye called 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (which is abbreviated to MTT). This dye is a yellow water soluble chemical. It is cleaved by mitochondrial succinate dehydrogenase and forms formazan which has a purple tint. An extract of the test material is used in the MTT assay. MTT is added to the cells for 24 hours, so the presence of violet formazan may be detected using a plate reader.
The MTT can be used to assess the cytotoxicity of:
- Medical Device’s Extractable Materials
- Toxic substances
- Environmental Toxins and Contaminants
- Drug sensitivity profiles for patients with haematological malignancies and in primary screening of potential chemotherapeutic drugs.
Ultimately, the type of device and the materials you use will determine which cytotoxicity testing methods you should use. However, international regulatory bodies offer guidance on cytotoxicity testing for medical device manufacturers, which brings us to our final section about the regulatory side of this topic.
Required for all types of medical devices, cytotoxicity testing is a key element of the international standards. The international standards compiled as ISO 10993, and the FDA blue book memorandum (#G95-1) that is based on 10993-1, address the critical issue of ensuring device biocompatibility by identifying several types of tests for use in selecting device materials.
Medistri’s in-house laboratory possesses an in-depth comprehension of the Cytotoxicity execution process to meet your regulatory requirements.