Cell Culture Assays: Viability and Cytotoxicity

By: Samarendra K Singh, School of Biotechnology, BHU

In in-vitro culture the viability and measure of the same becomes important as it is the parameter
to evaluate how healthy culture is and also effect of the drug/metabolite/environment and other
various biotic and abiotic parameters.
Various xenobiotic compounds has to undergo cytotoxicity test before they are Okayed for public
use. Lot of tests are done on animals but due to ethical issues and handling expenses, cell culture
system is the best way to evaluate them.

Drugs/Xenobiotic agents may cause toxicity on cells via different mechanisms such as
destruction of cell membranes, prevention of protein synthesis, irreversible binding to
receptors, inhibition of poly-deoxynucleotide elongation, and enzymatic reactions. In vitro
cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxicity
tests of chemicals and for drug screening. Currently, these assays are also used in
oncological researches to evaluate both compound toxicity and tumor cell growth
inhibition during drug development. 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.
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. 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. However, they still have some disadvantages because they cannot replace animal
tests when comes to public administration and quality checks.

In vitro Assays can be divided into five major categories:

1. Viability: An immediate or short term response, such as increased and
uncontrolled membrane permeability.
There are mainly two types of assay to measure in-vitro viability apart from
observing the morphology and other physical parameters like adherence etc.

a) Dye Exclusion: In this assay the semi permeability factor of membrane of

living cell is explored. Dyes are added to the culture which will only
permeabilize inside the dead cells. After adding the dyes, the cells are
incubated for some time and monitored under light microscope. The cells which
hasn’t taken the dye is counted as viable cells while the cells which has taken
the dye in is considered as dead cell. The dye exclusion method is simple and
 very cost effective, but none of the dyes used in this method work with
monolayer culture, its only effective with suspension culture, hence if it has to
be used with monolayer culture first it needs to be trypsinize to dislodge and
make into suspension. A variety of such dyes have been employed, including
eosin, Congo red, erythrosine B, and trypan blue. Of the dyes listed, trypan
blue has been used the most extensively.

b) Dye Uptake: Dyes like Diacetyl fluorescein and Neutral Red are taken by cells
and retained into. Diacetyl fluorescein is permeable inside the membrane and
after going inside the cell it is hydrolyzed into fluorescein which is impermeable
and hence cannot escape the cell and live cells fluoresce green while dead
cells do not. Now one can add Propidium iodide (PI) which is taken by dead
cells and not live cell and upon intake cells will fluoresce Red, hence
combination of these dyes will give count for both live and dead cell under
fluorescent microscope.
Similarly, Neutral red is taken up by live cells and sequestered inside the
lysosomes, while non-viable cells could not retain it.

2. Survival: Here the assessment is done for cells long term self-renewal capacity.
In this category of assays, the proliferation capacity of cell is measured after
treatment (physical and chemical in nature). One of such assay is Clonogenic
Assay.
 Fig: Clonogenic Assay (for measurement of survival and proliferation). Cells are
trypsinized, counted and diluted as for monolayer dilution cloning and plating
efficiency.

Other simple and effective way to measure survival is counting the cells as they
grow and make the growth curve to see the rate of proliferation over the time.

3. Metabolic: Here metabolic changes like enzyme activity, DNA, RNA synthesis,
protein expression etc. are measured, and the nature of this assay is of
intermediate duration.
a. 5’-bromo 2- deoxy Uridine (BrdU) incorporation: This proliferation assay is
a non-isotopic immunoassay for quantification of BrdU incorporation into newly
synthesized DNA of actively proliferating cells. This assay gives direct evidence
of cell proliferation. BrdU is a Thymidine nucleoside homolog and is
incorporated in place of thymidine in newly replicating cells, hence cells
undergoing S phase toxicity will incorporate less BrdU. The measure of BrdU
incorporation could be done by FACS (fluorescent activated cell sorter)
analysis of the prepared sample after fixation.

b. Reduction of Tetrazolium to Formazen:

MTT [3-(4-5-Dimethylthiazolyl) -2-5-diphenyltetrazolium bromide] Assay:
MTT is reduced by live cells to insoluble dark purple formazan by NADH. Based
on the intensity of formazan production in the cell culture estimation of viability
could be made. This assay determines principally cell viability through
determination of mitochondrial function of cells by measuring activity of
mitochondrial enzymes such as succinate dehydrogenase. This method is
widely accepted as reliable way to examine cell proliferation.

Fig: Principle of MTT Assay

XTT assay:
A colorimetric method based on the tetrazolium salt XTT (2, 3-bis (2-methoxy-
4-nitro-5-sulphophenyl)-5-carboxanilide-2H-tetrazolium, monosodium salt) is
more convenient than the MTT assay. While MTT produces a water-insoluble
formazan compound which required dissolving the dye in order to measure its
absorbance, the XTT produces a water-soluble dye upon reduction. The
procedure of XTT is simply for measuring proliferation and is therefore an
excellent solution for quantitating cells and determining their viability. XTT is
 used to assay cell proliferation as response to different growth factors. It is also
used for assaying cytotoxicity. This assay is based on the ability to reduce
the tetrazolium salt XTT to orange-colored formazan compounds by
metabolic active cells. Orange-colored formazan is water soluble and its
intensity can be measured with a spectrophotometer. There is a linear
relationship between the intensity of the formazan and the number of viable
cells. The use of multi-well plates and a spectrophotometer (or ELISA reader)
allows for study with a large number of samples and obtaining results easily
and rapidly. The procedure of this assay includes cell cultivation in a 96-well
plate, adding the XTT reagent and incubation for 2–24 hours. During the
incubation time, an orange color is formed and the intensity of color can be
measured with a spectrophotometer.
There are various other Tetrazolium salts like MTS (5-(3-carboxymethoxyphenyl)-2-(4,5-
dimethyl-thiazoly)-3-(4-sulfophenyl) tetrazolium, inner salt assay), WST-1 (2-(4-
iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt),
WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-
2H tetrazolium, monosodium salt) which are used for cell proliferation assays.

LDH (lactate dehydrogenase) assay:

LDH (lactate dehydrogenase) cytotoxicity assay is a colorimetric method of

assaying cellular cytotoxicity. LDH Cytotoxicity Assay can be used with
different cell types not only for assaying cell-mediated cytotoxicity but also
for assessment of cytotoxicity mediated by toxic chemicals and other test
compounds. The assay measures the stable, cytosolic, lactate dehydrogenase
(LDH) enzyme quantitatively. This enzyme releases from damaged cells. LDH
is an enzyme that is normally found within the cell cytoplasm. When cell
viability reduced leakiness of the plasma membrane increase and therefore
LDH enzyme is released into the cell culture medium. The released LDH is
measured with a coupled enzymatic reaction that results in the conversion of
a tetrazolium salt (iodonitrotetrazolium (INT)) into a red color formazan by
diaphorase. In the first step, LDH catalyze conversion of lactate to pyruvate
and thus NAD is reduced to NADH/H+. In a second step, catalyst (diaphorase)
transfers H/H+ from NADH/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-(4-
nitrophenyl)-5-phenyltetrazolium chloride (INT), which is reduced to red
formazan. The LDH activity is determined as NADH oxidation or INT reduction
over a defined time period. The resulting red formazan absorbs maximally at
492 nm and can be measured quantitatively at 490 nm.

Acid Phosphatase (ACP) Assay:

The acid phosphatase (ACP) method, developed by Bellavite et al., is based on acid
phosphatase activity of the sample. Acid phosphatase is a stable enzyme present
in many organ cells. This method is similar to the LDH method. On addition of p-
nitrophenyl phosphate (PNP) substrate, acid phosphatase converts the substrate
into p-nitrophenol, which can be easily measured by a photo-spectrometer at a
wavelength of 405 nm. Side-by-side comparison of both DCH and ACP assays suggests
that the ACP assay is better than the LDH assay in terms of reproducibility.

Alamar Blue Assay:

Dye Alamar blue has active ingredient Resazurin which is permeable to living cells but
non fluorescent. When it enters the cells it gets reduced to Resorufin which is
fluorescent (bright red). Viable cells continuously converts resazurin to resorufin,
hence generating quantitative measure of viability and cytotoxicity.

4. Genotoxicity and Transformation: Here, changes in genetic make-up which could

transform the cells are measure or monitored. It could be of short as well as long term
duration.

a. Apoptotic Assays:
These assays are based on effect of drugs or molecules which can induce
apoptosis and measure various parameters of genotoxicity. The various
parameters which could be assessed for this assay are;

1. Fragmentation of DNA:
DNA fragmentation could be assessed by either flow cytometry or by
analyzing on agarose gel electrophoresis. FACS analysis will reveal low
molecular weight DNA after propidium iodide incorporation. A
population of cells will be shown way earlier (before “n” DNA content) on X
axis.
On Agarose gel electrophoreses it will show a DNA ladder pattern
because of fragmentation of the genomic DNA [as shown below in the
figure].
 Fig. Apoptotic DNA fragmentation, visualized by the DNA ladder assay (left). A 1 kb
mrke (middle) and control DNA (genomic DNA, right) are included for comparison.

2. Alteration in membrane permeability:

For example, during apoptosis, an increase in the permeability of the outer
mitochondrial membrane is crucial and is regulated by multidomain pro-
apoptotic members of the Bcl-2 family (Bax and Bak), resulting in the release of
several apoptogenic factors into the cytoplasm.

3. Activation of apoptotic caspases:

Caspases are proteases that initiate and execute apoptotic cell death. These
caspase-dependent events are caused by cleavage of specific substrates that
propagate the proapoptotic signal. A number of techniques have been developed
to follow caspase activity in vitro and from apoptotic cellular extracts. Many of these
techniques use molecules that are based on optimal peptide motifs for each
caspase and on our understanding of caspase cleavage events that occur during
apoptosis. Although these approaches are useful, there are several drawbacks
associated with them. The optimal peptide motifs are not unique recognition sites
for each caspase, so techniques that use them may yield information about more
than one caspase. Furthermore, caspase cleavage does not take into account the
different caspase activation mechanisms. Recently, probes having greater
specificity for individual caspases have been developed and are being used
successfully. Hence various substrates are being developed to give calorimetric
assay to access individual caspase activity.
 Fig. Induction of Cytochrome C mediated Caspase activation during Apoptosis

4. Release of Cytochrome C Oxidase into cytoplasm by mitochondria:

Selective permeabilization of the mitochondrial outer membrane is an integral
event in apoptosis induced by numerous stimuli. As a result, several proapoptotic
proteins including cytochrome c oxidase are released from the mitochondrial
intermembrane space to the cytoplasm. Cytochrome c oxidase then triggers
activation of caspase proteases via the formation of a complex known as
apoptosome. By using ferricytochrome C as substrate samples prepared from
opoptotic cells when subjected to the substrate gives a calorimetric assessment.

4. Irritancy: Response such as allergic or inflammatory or irritancy are quite hard to

develop in-vitro but other parameters of those conditions are measured like
release of cytokine and various other signaling factors.