Viability of the cells represents the capability of their existence and survival and development. Study on cell cytotoxicity broadly involves the metabolic alternations of the cells, including the death of cells as a result of toxic effects of the cytotoxic metabolic compounds.
Assays for Cell Viability and Cell Cytotoxicity
There are several assays developed in the laboratory for quantifying the cell viability and cytotoxicity. They are broadly categorized in the following types:
1. Cytotoxicity and Viability Assays
2. Survival Assays
3. Metabolic Assays
4. Transformation Assays
5. Inflammation Assays
Cytotoxicity and Viability Assays
A majority of the cytotoxicity and viability assays are based on the detection on membranous integrity, cellular respiration, radioisotope incorporation, colorimetric assays and luminescence based assays.
Assays Based on Membrane Integrity
The most common determination of cell viability is based on membrane integrity. The damage to membrane may occur due to cell disaggregation and cell separation or freezing or cell thawing. Membrane integrity can be determined by the uptake of dyes to which viable cells are impermeable such as naphthalene black, trypan blue, erythrosine or by release of dyes normally taken up and retained by viable cells such as neutral red, diacetyl flurescein.
The other assays for membrane integrity are release of labeled chromium 51Cr. Enzymes are use fluorescent probes. Cell viability measurements, based on membrane integrity can give the results within few hours. However, these measurements cannot predict the ultimate survival of cells. The several assays are explained in brief below.
Dye Exclusion Assays
The principle of the assay is based on the fact that viable cells are impermeable to several dyes such as naphthalene black, trypan blue, eosin Y, nigrosin blue and erythrosin B. the percentage of unstained cells represents the viable cells.
Dye Uptake Assay
The viable cells can take up the dyes such as diacetyl flurescein and hydrolyze it to flurescein. The latter is held up by the viable cells as it is impermeable to membrane. The viable cells therefore emit flurescein green while the dead cells do not. Thus the viable cells can be identified.
Chromium Uptake Assay
Labeled chromium 51Cr binds to intracellular proteins to basic amino acids. When the cell membrane is damaged the labeled proteins leak out of the cell and the degree of leakage is proportional to the amount of damage.
Enzymes Release Assays
The membrane integrity of cells can also be assessed by estimating the enzymes released. Lactate dehydrogenase or LDH has been the most widely used enzyme for this purpose.
Assays Based on Cellular Respiration
Respiration of the cells measured by oxygen utilization or carbon dioxide production is used to assess cell viability. This is usually done by using Warburg Manometer.
Assays Based on Radioisotope Incorporation
By using radio labeled substrates or metabolites the radiation level in products formed can be detected. This method is particularly useful for measuring cytotoxicity assays. Some of the radioisotopes incorporation methods are briefly described below.
Incorporation of deuterium containing thymidine and deuterium uridine into RNA are widely used for the measurements of drug toxicity.
The cells are labeled with 32P.When the damage occurs to cells they release labeled phosphates which can be measured. The efficiency of the drugs can be evaluated by this approach.
Assays Based on Colorimetric Assays
The recent developments in the colorimetric assays by using sophisticated micro plate readers are fruitfully utilized for quantification of cells. A good correlation between the colorimetric assay and the cell numbers is observed. Protein content, DNA, lysosomal activity, Golgi body activity, enzyme activity are quantified by the use of assays or enzymes.
Assays Based on Luminescence Test
The viability of cells can be measured with good sensitivity by estimating ATP levels by luminescence based tests. The principal is based on the reaction between ATP and luciferin in presence of oxygen and luciferase enzyme. The reaction produces AMP, 2Pi, carbon dioxide and light. The sensitivity of the test quantifies for the cell number in the range of 20 to 2 X 10^7 cells/ml.
Assays Based on Apoptosis
Most of the anticancer drugs kill cells by apoptosis which can be measured for the assessments of cytotoxicity. Apoptosis can be detected by the following ways.
• Changes in morphology
• Detection of phosphatidyl serine in the membrane by using annexin V conjugated to
fluorescein isothiocyanate or biotin
• DNA laddering
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