Cytotoxicity Testing

What Is Cytotoxicity Testing?

-Testing the effects of compounds on the viability of cells grown in culture is widely used as a predictor of potential toxic effects in whole animals. Among the several alternative assays available, measuring the levels of ATP is the most sensitive, reliable, and convenient method for monitoring active cell metabolism. However, recently developed combinations of methods have made it possible to collect more information from in vitro cytotoxicity assays using standard fluorescence and luminescence plate readers. This chapter describes two assay methods. The first utilizes beet

le luciferase for measuring the levels of ATP as a marker of viable cells. The second more recently developed multiplex method relies on selective measurement of three different protease activities as markers for viable, necrotic, and apoptotic cells. Data analysis from the measurement of three marker protease activities from the same sample provides a useful tool to help uncover the mechanism of cell death and can serve as an internal control to help identify assay artifacts.

Cytotoxicity (Tissue Culture)

 Is the quality of being toxic to cells. Examples of toxic agents are an immune cell or some types of venom, e.g. from the puff adder (Bitis arietans) or brown recluse spider (Loxosceles reclusa).

Cell culture assays are used to assess the biocompatibility of a material or extract through the use of isolated cellsin vitro. These techniques are useful in evaluating the toxicity or irritancy potential of materials and chemicals. They provide an excellent way to screen materials prior to in vivo tests.

Qualitative Cytotoxicity Tests

There are three qualitative cytotoxicity tests commonly used for medical devices. The Direct Contact procedure is recommended for low density materials, such as contact lens polymers. In this method, a piece of test material is placed directly onto cells growing on culture medium. The cells are then incubated. During incubation, leachable chemicals in the test material can diffuse into the culture medium and contact the cell layer. Reactivity of the test sample is indicated by malformation, degeneration and lysis of cells around the test material.

The Agar Diffusion assay is appropriate for high density materials, such as elastomeric closures. In this method, a thin layer of nutrient-supplemented agar is placed over the cultured cells. The test material (or an extract of the test material dried on filter paper) is placed on top of the agar layer, and the cells are incubated. A zone of malformed, degenerative or lysed cells under and around the test material indicates cytotoxicity.

The MEM Elution assay uses different extracting media and extraction conditions to test devices according to actual use conditions or to exaggerate those conditions. Extracts can be titrated to yield a semi-quantitative measurement of cytotoxicity. After preparation, the extracts are transferred onto a layer of cells and incubated. Following incubation, the cells are examined microscopically for malformation, degeneration and lysis of the cells.

Quantitative Cytotoxicity - MTT Assay

Recent regulatory additions (ANSI/AAMI/ISO 10993-5:2009) on biocompatibility for devices state that the qualitative cytotoxicity tests (direct contact, mem elution, agar diffusion

) are appropriate for screening purposes, but that quantitative evaluation is preferable.

Annex C of ISO 10993-5:2009 refers to the MTT cytotoxicity assay, which can accurately quantify as few as 950 cells. The MTT is a colorimetric method that measures the reduction of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide by mitochondial succinate dehydrogenase. Because the cellular reduction is only catalyzed by living cells, it is possible to quantify the percentage of living cells in a solution.

The MTT can be used to evaluate the cytotoxicity of:

• Extractable materials of medical devices
• Toxic compounds
• Toxins and environmental pollutants
• Potential anti-cancer drugs
• Antibodies to examine growth inhibiting potential

The major advantages of the MTT are its quantitative ability, that it can be done on either extracts or by direct contact, and that the results are not subject to analyst interpretation. Additionally, the MTT can be performed on 96-well microplates in a standard reader (such as a Bio-Tek ELx808) allowing for fast screening of multiple samples.

However, it should be noted that while the MTT is recommended, the MTT assay does not discriminate a specific cellular death mechanism - such as apopotosis vs. induced cell death. Additionally, it may underestimate cellular damage and only detect death at the last stages of the cellular dying process.

Cell-Mediated Cytotoxicity Assays

ImmunoChemistry Technologies has developed two reliable, easy, and accurate cytotoxicity assays to measure cell-mediated cytolytic activity:

• Basic Cytotoxicity Test: Measure necrotic and membrane-compromised cells in a mixed-population sample of effector and target cells.
• Total Cytotoxicity Test: Further distinguish apoptosis from necrosis in target cells, leading to a more accurate assessment of cytolytic activity.

ImmunoChemistry Technologies' fluorescence-based assay kits provide a direct measurement of cytotoxicity rather than using an indirect indicator, such as the release of LDH or ATP enzymes, and there is no need to lyse the cells. Since these assays do not use any radioisotopes, they are much safer and more cost effective to run than 51Cr assays. Both assays include a thorough flow cytometry-based protocol that provides robust data and allows for multiparametric analysis with additional probes.

Atomic Absorption Spectroscopy

What is Atomic Absorption Spectroscopy?

Atomic absorption spectroscopy (AAS) is a spectroanalytical procedure for the quantitative determination of chemical elements using the absorption of optical radiation (light) by free atoms in the gaseous state.

In analytical chemistry the technique is used for determining the concentration of a particular element (the analyte) in a sample to be analyzed. AAS can be used to determine over 70 different elements in solution or directly in solid samples used in pharmacology, biophysics and toxicology research.

Atomic absorption spectroscopy was first used as an analytical technique, and the underlying principles were established in the second half of the 19th century by Robert Wilhelm Bunsen and Gustav Robert Kirchhoff, both professors at the University of Heidelberg, Germany.

The modern form of AAS was largely developed during the 1950s by a team of Australian chemists. They were led by Sir Alan Walsh at the Commonwealth Scientific and Industrial Research Organisation (CSIRO), Division of Chemical Physics, inMelbourne, Australia.

Atomic absorption spectrometry has many uses in different areas of chemistry such as:

• Clinical analysis: Analyzing metals in biological fluids and tissues such as whole blood, plasma, urine, saliva, brain tissue, liver, muscle tissue, semen
• Pharmaceuticals: In some pharmaceutical manufacturing processes, minute quantities of a catalyst that remain in the final drug product
• Water analysis: Analyzing water for its metal content.

Principles

The technique makes use of absorption spectrometry to assess the concentration of an analyte in a sample. It requires standards with known analyte content to establish the relation between the measured absorbance and the analyte concentration and relies therefore on the Beer-Lambert Law.

In short, the electrons of the atoms in the atomizer can be promoted to h

igher orbitals (excited state) for a short period of time (nanoseconds) by absorbing a defined quantity of energy (radiation of a given wavelength). This amount of energy, i.e., wavelength, is specific to a particular electron transition in a particular element. In general, each wavelength corresponds to only one element, and the width of an absorption line is only of the order of a few picometers (pm), which gives the technique its elemental selectivity. The radiation flux without a sample and with a sample in the atomizer is measured using a detector, and the ratio between the two values (the absorbance) is converted to analyte concentration or mass using the Beer-Lambert Law.

Instrumentation

In order to analyze a sample for its atomic constituents, it has to be atomized. The atomizers most commonly used nowadays are flames and electrothermal (graphite tube) atomizers. The atoms should then be irradiated by optical radiation, and the radiation source could be an element-specific line radiati

on source or a continuum radiation source. The radiation then passes through a monochromator in order to separate the element-specific radiation from any other radiation emitted by the radiation source, which is finally measured by a detector.
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