In Vitro Cytotoxicity and Cell Viability Assays:: Principles, Advantages, and Disadvantages
In Vitro Cytotoxicity and Cell Viability Assays:: Principles, Advantages, and Disadvantages
In Vitro Cytotoxicity and Cell Viability Assays:: Principles, Advantages, and Disadvantages
71923
Provisional chapter
Chapter 1
http://dx.doi.org/10.5772/intechopen.71923
Abstract
Cytotoxicity is one of the most important indicators for biological evaluation in vitro stud-
ies. In vitro, chemicals such as drugs and pesticides have different cytotoxicity mecha-
nisms such as destruction of cell membranes, prevention of protein synthesis, irreversible
binding to receptors etc. In order to determine the cell death caused by these damages,
there is a need for cheap, reliable and reproducible short-term cytotoxicity and cell via-
bility assays. Cytotoxicity and cell viability assays are based on various cell functions.
A broad spectrum of cytotoxicity assays is currently used in the fields of toxicology
and pharmacology. There are different classifications for these assays: (i) dye exclusion
assays; (ii) colorimetric assays; (iii) fluorometric assays; and (iv) luminometric assays.
Choosing the appropriate method among these assays is important for obtaining accu-
rate and reliable results. When selecting the cytotoxicity and cell viability assays to be
used in the study, different parameters have to be considered such as the availability in
the laboratory where the study is to be performed, test compounds, detection mecha-
nism, specificity, and sensitivity. In this chapter, information will be given about in vitro
cytotoxicity and viability assays, these assays will be classified and their advantages and
disadvantages will be emphasized. The aim of this chapter is to guide the researcher
interested in this subject to select the appropriate assay for their study.
1. Introduction
Viability levels and/or proliferation rates of cells are good indicators of cell health. Physical
and chemical agents can affect cell health and metabolism. These agents may cause toxic-
ity on cells via different mechanisms such as destruction of cell membranes, prevention
of protein synthesis, irreversible binding to receptors, inhibition of polydeoxynucleotide
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2 Genotoxicity - A Predictable Risk to Our Actual World
elongation, and enzymatic reactions [1]. In order to determine the cell death caused by these
mechanisms, there is a need for cheap, reliable and reproducible short-term cytotoxicity and
cell viability assays.
In vitro cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxic-
ity tests of chemicals and for drug screening. Application of these assays has been of increas-
ing interest over recent years. Currently, these assays are also used in oncological researches to
evaluate both compound toxicity and tumor cell growth inhibition during drug development.
Because, they are rapid, inexpensive and do not require the use of animals. Furthermore, they
are useful for testing large number of samples. Cell viability and cytotoxicity assays are based
on various cell functions such as cell membrane permeability, enzyme activity, cell adherence,
ATP production, co-enzyme production, and nucleotide uptake activity [1].
In vitro cytotoxicity and/or cell viability assays have some advantages, such as speed, reduced
cost and potential for automation, and tests using human cells may be more relevant than
some in vivo animal tests. However, they have some disadvantages because they are not tech-
nically advanced enough yet, to replace animal tests [2].
It is important to know how many viable cells are remaining and/or how many cells are dead
at the end of the experiment. A broad spectrum of cytotoxicity and cell viability assays is
currently used in the fields of toxicology and pharmacology. The choice of assay method is
crucial in the assessment of the interaction type [3].
Although there are different classifications for cytotoxicity and cell viability assays, in this
chapter, these assays are classified according to measurement types of end points (color
changes, fluorescence, luminescent etc.).
The proportion of viable cells in a cell population can be estimated in various methods. The
simplest and widely used one of the methods is dye exclusion method. In dye exclusion
method, viable cells exclude dyes, but dead cells not exclude them. Although the staining
procedure is quite simple, experimental procedure of large number of samples is difficult
and time consuming [4]. Determination of membrane integrity is possible via dye exclusion
In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages 3
http://dx.doi.org/10.5772/intechopen.71923
method. A variety of such dyes have been employed, including eosin, Congo red, erythro-
sine B, and trypan blue [5, 6]. Of the dyes listed, trypan blue has been used the most exten-
sively [7–10].
If dye exclusion assays are used, following factors must be considered (i) lethally damaged
cells by cytotoxic agents may require several days to lose their membrane integrity, (ii) the
surviving cells may continue to proliferate during this time, and (iii) some lethally damaged
cells are not appear to be stained with dye at the end of the culture period, because they may
undergo an early disintegration. Factors (ii) and (iii) may cause an underestimate of cell death
when the results of the assay are based on percent viability expression [11–13].
Dye exclusion assays have unique advantages for chemosensitivity testing. They are compar-
atively simple, require small numbers of cells, are rapid, and are capable of detecting cell kill
in nondividing cell populations. Further investigations into the possible role of these assays
in chemosensitivity testing are warranted [11]. However, none of these dyes is recommended
for use on monolayer cell cultures but rather they are intended for cells in suspension; thus
monolayer cells must first trypsinized [6].
This dye exclusion assay is used to determine the number of viable and/or dead cells in a cell
suspension. Trypan blue is a large negatively charged molecule. Trypan blue dye exclusion
assay is based on the principle that live cells possess intact cell membranes that exclude this
dye, whereas dead cells do not. In this assay, adherent or nonadherent cells are incubated
with serial dilutions of test compounds for various times. After the compound treatment,
cells are washed and suspended. Cell suspension is mixed with dye and then visually exam-
ined to determine whether cells take up or exclude dye. Viable cells will have a clear cyto-
plasm, whereas dead cells will have a blue cytoplasm [14, 15]. Number of viable and/or dead
cells per unit volume is determined by light microscopy as a percentage of untreated control
cells [15, 16].
Advantages: This method is simple, inexpensive, and a good indicator of membrane integrity
[17], and dead cells are colored blue within seconds of exposure to the dye [18].
Disadvantages: Cell counting is generally done using a hemacytometer [19]. Therefore, count-
ing errors (~10%) could be occurred. Counting errors have been attributed to poor dispersion
of cells, cell loss during cell dispersion, inaccurate dilution of cells, improper filling of the
chamber and presence of air bubbles in the chamber [17].
While the staining procedure is quite simple, it is difficult to process large number of samples
concurrently, particularly where the exact timing of progressive cytotoxic effects is required
[4]. Furthermore, trypan blue staining cannot be used to distinguish between the healthy cells
and the cells that are alive but losing cell functions. Therefore, it is not sufficiently sensitive to
use for in vitro cytotoxicity testing. Another disadvantage of trypan blue is toxic side effect of
this dye on mammalian cells [20].
4 Genotoxicity - A Predictable Risk to Our Actual World
Erythrosine B, also known as erythrosine or Red No. 3, is primarily used as food coloring
agent [20, 21]. Erythrosine B has already been introduced as a vital dye for counting viable
cells. Principle of this dye exclusion assay is similar to trypan blue dye exclusion assay prin-
ciple. Although erythrosine B is an alternative bio-safe vital dye for cell counting; it is not
widely used to count viable or dead cells.
Advantages: It has benefits such as low cost, versatility, and bio-safety [20].
Disadvantages: Its procedure is time-consuming and labor-intensive. Moreover, potential
disadvantages include contamination of reusable cell counting chamber, variations of hemo-
cytometer filling rates, and inter-user variations [20].
Advantages: This method is far superior to the previously mentioned dye exclusion methods
because it is easy to use, safe, has a high reproducibility, and is widely used to determine both
cell viability and cytotoxicity tests [18, 25].
Disadvantages: MTT formazan is insoluble in water, and it forms purple needle-shaped crystals
in the cells. Therefore, prior to measuring the absorbance, an organic solvent such as dimethyl
sulfoxide (DMSO) or isopropanol is required to solubilize the crystals. Additionally, the cyto-
toxicity of MTT formazan makes it difficult to remove cell culture media from the plate wells
due to floating cells with MTT formazan needles, giving significant well-to-well error [18, 26].
Advantages: It is easy to use, safe, has a high reproducibility, and is widely used to determine
both cell viability and cytotoxicity tests. Furthermore, phenol red indicators in cell culture
medium do not interfere with the dye reaction. Because the colored dye which produced at
the end of experiment is water-soluble, it is not required a solvent and additional incubation
time [37].
Disadvantages: The standard incubation time of WST-1 time is 2 h. Whether one-time addi-
tion of WST-1 can reflect the effect of the testing agents at different time points on the trend of
relative cell viability is still unclear [37].
WST-8 assay is a colorimetric assay for the determination of viable cell numbers and can
be used for cell proliferation assays as well as cytotoxicity assays. WST-8 (2-(2-methoxy-
4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium, monosodium salt), a
highly stable and water-soluble WST, is utilized in Cell Counting Kit-8 (CCK-8). It is more
sensitive than WST-1 particularly at neutral pH [37]. Because of the electron mediator,
1-methoxy PMS in this kit is highly stable, and CCK-8 is stable for at least 6 months at the
room temperature and for 1 year at 0–5oC. Since WST-8, WST-8 formazan, and 1-methoxy
PMS have no cytotoxicity on cells in the culture media, same cells from the previous assay
may be used for additional experiments.
Advantages: WST-8 is not cell permeable, which results in low cytotoxicity. Therefore, after
the assay, it is possible to continue further experiments using the same cells. Furthermore, it
produces the water-soluble formazan upon cellular reduction, which would provide an addi-
tional advantage to the method by allowing a simpler assay procedure and not required an
extra step to dissolve the formazan [28].
Disadvantages: An important consideration is that reduction of assay substrates is impacted by
changes in intracellular metabolic activity that has no direct effect on overall cell viability [15].
In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages 7
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SRB (Sulforhodamine B) assay is a rapid and sensitive colorimetric method for measuring
the drug-induced cytotoxicity in both attached and suspension cell cultures. This assay as
first described by Skehan and colleagues was developed for use in the disease-orientated,
large-scale anticancer drug discovery program of the National Cancer Institute (NCI) that was
launched in 1985. SRB is a bright pink aminoxanthene dye with two sulfonic groups. Under
mildly acidic conditions, SRB binds to protein basic amino acid residues in TCA-fixed (tri-
chloroacetic acid) cells to provide a sensitive index of cellular protein. SRB assay is also used
to evaluate colony formation and colony extinction [43].
8 Genotoxicity - A Predictable Risk to Our Actual World
Advantages: The SRB assay is simple, fast, and sensitive. It provided good linearity with cell
number, permitted the use of saturating dye concentrations, is less sensitive to environmental
fluctuations, is independent of intermediary metabolism, and provided a fixed end point that
is not require a time-sensitive measurement of initial reaction velocity [43]. Reproducibility
of this assay is high.
Disadvantages: It is important to obtain and maintain a homogeneous cell suspension.
Cellular clumps/aggregates should be avoided for high assay performance.
The neutral red uptake (NRU) assay is also one of the most used colorimetric cytotoxicity/
cell viability assay. This assay was developed by Borenfreund and Puerner [44]. This assay
was based on the ability of viable cells to take up the supravital dye neutral red. This weakly
cationic dye penetrates cell membranes by nonionic passive diffusion and concentrates in the
lysosomes. The dye is then extracted from the viable cells using an acidified ethanol solution
and the absorbance of the dye is measured using spectrophotometer.
Neutral red uptake depends on the capacity of cells to maintain pH gradients through the ATP
production. At physiological pH, net charge of the dye is zero. This charge enables the dye to
penetrate the cell membranes. Inside the lysosomes, there is a proton gradient to maintain a
pH lower than that of the cytoplasm. Thus, the dye becomes charged and is retained inside
the lysosomes. When the cell dies or pH gradient is reduced, the dye cannot be retained. In
addition, the uptake of neutral red by viable cells can be modified by alterations in cell sur-
face or lysosomal membranes. Thus, it is possible to distinguish between viable, damaged,
or dead cells [44]. Lysosomal uptake of neutral red dye is a highly sensitive indicator of cell
viability. The assay can quantitate cell viability and measure cell replication, cytostatic effects
or cytotoxic effects depending on the seeding density [45]. Absorbance is measured at 540 nm
in multiwell plate reader spectrophotometer.
Advantages: NRR assay is a good marker of lysosomal damage. Also, speed and simple eval-
uation are some advantages of this assay.
Disadvantages: It has been reported that the NRR assay is either minimally or not at all affected
by natural factors, such as temperature and salinity, but is mainly influenced by pollutants [46].
Adherent cells detach from cell culture plates during cell death. This feature can be used for
the indirect assessment of cell death and to determine differences in proliferation rate upon
stimulation with cytotoxic agents. One simple method to detect maintained adherence of cells
is crystal violet assay. In this assay, crystal violet dye binds to proteins and DNA of viable
cells, and thus, attached cells are stained with this dye. Cells lose their adherence during cell
death and are subsequently lost from the population of cells, reducing the amount of crystal
violet staining in a culture. Crystal violet assay is a quick and reliable screening method that
is suitable for the examination of the impact of chemotherapeutics or other compounds on cell
survival and growth inhibition [47].
In Vitro Cytotoxicity and Cell Viability Assays: Principles, Advantages, and Disadvantages 9
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Advantages: Crystal violet staining is a quick and versatile assay for screening cell viability
under diverse stimulation conditions [48]. However, it is potentially compromised by pro-
liferative responses that occur at the same time as cell death responses. Therefore, chemi-
cal inhibitors of caspases and/or of necroptosis may be incorporated into the assay [49, 50].
Alternatively, molecular studies (e.g., overexpression or knockdown) can be performed to
more specifically address the nature of cell death [51].
Disadvantages: Crystal violet assay is insensitive to changes in cell metabolic activity.
Therefore, this assay is not appropriate for studies used cell metabolism affected compounds.
While crystal violet assay is suitable for the examination of the impact of chemotherapeutics
or other compounds on cell survival and growth inhibition, it is not able to measure cell pro-
liferation rate [51].
Fluorometric assays of cell viability and cytotoxicity are easy to perform with the use of a fluo-
rescence microscope, fluorometer, fluorescence microplate reader or flow cytometer, and they
offer many advantages over traditional dye exclusion and colorimetric assays. Fluorometric
assays are also applicable for adherent or suspended cell lines and easy to use. These assays
are more sensitive than colorimetric assays [52–54]. Commercial kits of fluorometric assays are
available from several companies and generally experimental procedures of these assays are
available in kit packages.
alamarBlue assay is also known as resazurin reduction assay. The alamarBlue assay is based
on the conversion of the blue nonfluorescent dye resazurin, which is converted to the pink
fluorescent resorufin by mitochondrial and other enzymes such as diaphorases [53].
Resazurin is a phenoxazin-3-one dye and cell permeable redox indicator that can be used
to monitor viable cell number with protocols similar to those utilizing the tetrazolium com-
pounds [55]. It is known to act as an intermediate electron acceptor in the electron trans-
port chain between the final reduction of oxygen and cytochrome oxidase by substituting
for molecular oxygen as an electron acceptor [52]. It is a nontoxic and cell permeable com-
pound. Color of this compound is blue and it is nonfluorescent. After entering cells, resazurin
is reduced to resorufin. Resorufin is red in color and highly fluorescent compound. Viable
cells convert continuously resazurin to resofurin, increasing overall fluorescence and color of
the cell culture medium. The quantity of produced resofurin is related to the number of viable
cells. Ratio of viable cells can be quantified using a microplate reader fluorometer equipped
with a 560 nm excitation/590 nm emission filter set. Resofurin can also be measured by absor-
bance changes, but absorbance detection is not often used because absorbance detection is less
sensitive than fluorescence measurement.
The incubation period required to generate a sufficient fluorescent signal above background
is usually about 1–4 hours, depending on metabolic activity of the cells, the cell density per
well and other conditions such as the culture medium type [54].
10 Genotoxicity - A Predictable Risk to Our Actual World
Advantages: alamarBlue (resazurin reduction) assay is relatively inexpensive and more sensi-
tive than tetrazolium assays. Also, it can be multiplexed with other methods such as measur-
ing caspase activity to gather more information about the cytotoxicity mechanism.
Disadvantages: Fluorescent interference from test compounds and the often overlooked
direct toxic effects on the cells are possible [54].
Measurement of a conserved and constitutive protease enzyme activity of viable cells is used
as a good indicator of cell viability. A cell permeable fluorogenic protease substrate (gly-
cylphenylalanyl-aminofluorocoumarin; GF-AFC) has been recently developed to selectively
detect protease activity that is restricted to viable cells [59]. The GF-AFC substrate can pen-
etrate viable cells. In these cells, cytoplasmic aminopeptidase activity removes the gly and
phe amino acids to release aminofluorocoumarin (AFC) and produce a fluorescent signal pro-
portional to the number of viable cells [54].
When cells die, this protease activity is rapidly loss. Therefore, this protease activity is a selec-
tive marker of the viable cell population. The signal generated from this assay approach has
been shown to correlate well with other established methods of determining cell viability
such as an ATP assay [54].
Luminometric assays provide fast and simple determination of cell proliferation and cyto-
toxicity in mammalian cells. These assays can be performed in a convenient 96-well and 384-
well microtiter plate format and detection by luminometric microplate reader [54, 60, 61]. A
remarkable feature of the luminometric assays is the persistent and stable glow-type signal
produced after reagent addition. This attribute can be harnessed to produce both viability
and cytotoxicity values from the same well [59]. Commercial kits of luminometric assays are
available from several companies and generally experimental procedures of these assays are
available in kit packages.
ATP (adenosine tri-phosphate) represents the most important chemical energy reservoir
in cells and is used for biological synthesis, signaling, transport, and movement processes.
Therefore, cellular ATP is one of the most sensitive end points in measuring cell viability [62].
When cells damaged lethally and lose membrane integrity, they lose the ability to synthetize
ATP and the ATP level of cells decreases dramatically [54, 63]. The ATP assay is based on the
reaction of luciferin to oxyluciferin. Enzyme luciferase catalyzes this reaction in the presence
of Mg2+ ions and ATP yielding a luminescent signal. There is a linear relationship between the
intensity of luminescent signal and ATP concentration [61] or cell number [64].
The ATP assay chemistry can typically detect fewer than 10 cells per well, and therefore, it has
been widely used 1536-well plate format.
Advantages: ATP assay is the fastest cell viability assay to use, the most sensitive, and is less
prone to artifacts than other viability assays. The luminescent signal reaches steady state
and stabilizes within 10 min after addition of reagent. It does not have an incubation step
for conversion of substrate into colored compound. This also eliminates a plate handling
step [54].
Disadvantages: The ATP assay sensitivity is usually limited by reproducibility of pipetting
replicate samples rather than a result of the assay chemistry [54].
Recently, a new approach is developed to measure viable cell number in real time [60]. In this
assay, an engineered luciferase derived from a marine shrimp and a small molecule prosub-
strate is used. The pro-substrate and luciferase are added directly to the cell culture medium
as a reagent. The pro-substrate is not a substrate of luciferase. Viable cells with an active
metabolism reduce the pro-substrate into a substrate, which used by luciferase, to generate
a luminescent signal. The assay can be performed in two formats: continuous read and end-
point measurement. In the continuous read format, the luminescent signal can be repeatedly
recorded from the sample wells over an extended period to measure the number of cells in
“real time” [54, 60].
12 Genotoxicity - A Predictable Risk to Our Actual World
Advantages: This assay is the only assay which allows to real-time measurement of cell viability/
cytotoxicity. The rapid decrease in luminescent signal following cell death enables multiplexing
this assay with other luminescent assays that contain a lysis step that will kill cells. The decrease
in luminescence following cell death is important to eliminate interference with subsequent
luminescent assays [54, 60].
Disadvantages: A limitation of the real time assay results from the eventual depletion of
pro-substrate by metabolically active cells. Generally, the luminescent signal generated cor-
relates with the number of metabolically active cells. However, the length of the time the
luminescent signal will be linear with cell number will depend on the number of cells per
well and their metabolic activity. Therefore, it is recommended that the maximum incubation
time to maintain linearity should be empirically determined for each cell type and seeding
density [54, 60].
3. Conclusions
A broad spectrum of cytotoxicity and cell viability assays is currently used in the fields of
toxicology and pharmacology. An ideal assay for in vitro viability and/or cytotoxicity deter-
mination should be a rapid, safe, reliable, efficient, and time- and cost-effective. It should
not interfere with test compound. The choice of assay method is crucial in the assessment of
the interaction type. The assay may change the interpretation of the compound interaction.
Therefore, the assay method should be chosen with caution, considering the mechanism of
action of the test compound [3]. Tissue or cell type used in the study also affects the perfor-
mance of cytotoxicity and/or cell viability assays. Therefore, before choosing an assay for
study, different methods should be tried and compared. If it is possible, more than one assay
should be used to determine cytotoxicity and/or cell viability in in vitro studies. Thus, reliabil-
ity of the obtained results would increase.
Acknowledgements
The author would like to thank Asst. Prof. Tülay AŞKIN ÇELİK for her helpful advice in this
chapter.
Author details
Özlem Sultan Aslantürk
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