COURSE ABT 604 ANIMAL CELL CULTURE: PRINCIPLES AND

APPLICATIONS
CYTOTOXICITY ASSAYS

Term paper submitted by

P.S.Vinod
I year M.V.Sc.,
I semester
Animal Biotechnology

Toxicity is a complex event in vivo where there may be

direct cellular damage (cytotoxic anticancer drug)
Physiological effects (membrane transport in kidney,
neurotoxicity )
Inflammatory effects
Systemic effects
Definition of cytotoxicity vary depending on nature of the study
and whether cells are killed or simply have their metabolism
altered.
Cytotoxicity
assays
Cytotoxicity assays are assays where the cell/tissue
systems are
chemicals

used as models for testing

,food

additives,

culture

the effect/toxicity of

pharmaco-therapeutic

chemotherapeutics, pesticides, oncogenic agents etc.,

drugs,

Need for cytotoxic assays

Several substances which are utilised by the population being produced
and made available in the market are tested in animal models.
Financial, ethical and limitation of animals models warrant the need for
alternative models.
Invitro toxicity testing using cell culture /tissue culture models have
been

designed

towards

the

aim

of

reducing

replacement of animal models.

Advantages of cytotoxicity assays

Cheaper than other models
The assays are easily quantifiable
The results are reproducible
More control can be exercised
Multiple designing is possible

,refinement

and

APPLICATION OF CYTOTOXIC ASSAYS

1.Anticancer drug screening MTT based or Sulforhodamine B
based
2.Predictive drug testing for tumours - Measuring the chemo
sensitivity of cells derived from patients tumour to design a
chemotherapeutic regime
3.Testing pharmaceuticals
4.Transformation and mutagenesis
Anchorage independence
Reduced density limitation of cell proliferation
Mutagenesis
Sister chromatid exchange assay
5.Testing for carcinogenicity expression analysis
6. Testing for Inflammatory responses to pharmaceuticals,
cosmetics

or

xenobioitics

-(Kits

Epiderm(MarTek),

Episkin

Fundamental requirements of invitro

cytotoxicity testing
1.An assay system should give a reproducible dose response curve
over a concentration range that includes the in vivo dose
2.There should be a linear relation ship between cell number and
assay response
3.The resultant dose response curve should relate predictively to in
vivo effect of the same compound
(Ideally the same range of concentration should give the same
effect)

ASSAY DESIGN
Exposure to study compound
Exposure time may simulate the exposure levels of invivo
The penetration time required shall be considered
Choice of assay -The sensitivity required over concentration range
Phase of the growth cycle: vary with cell cycle specific drugs and non cycle
specific drugs .

Recovery period

To allow recovery from metabolic dysfunction unrelated to cell death

when metabolic parameters are used as index
Use of controls appropriate to the toxin and mode of application
reliability can be increased by using positive control of the
compounds
Choice of cell type
Depends on type of compound to be tested
For general toxicity screening -fast growing fibroblast /epithelial
lines eg

MDCK,CHO,HeLa

Limitation transformed .dedifferentiated cells not suited for specific

cellular function

Culture methods
Primary cells and organ tissues
Spheroid cultures for penetration assay and solid tumour
modeling
Suspension cultures long term and short term assays for
drug sensitivity- systems using tumour biopsy materials
Colonogenic growth in soft agar growth of primary tumour
cells
minimises growth of anchorage dependent stromal cells
support cells with high self renewal capacity-tumour stem
cells
Monolayer culture -widely used and offers flexibility in terms

Three-dimensional cell cultures in toxicology

conventional two-dimensional cell cultures do not reproduce the tissue
architecture in vivo, and do not forecast organ-specific toxicity.
three-dimensional cultures emulate the biochemistry and mechanics of the
microenvironment in tissues more closely.

Biotechnology & Genetic Engineering Reviews Volume 26 Editor:

S.E. Harding 2010

NATURE OF ASSAYS
Choice of assay depend on the agent under study, the nature of the
response and the particular target cell
Assays are divided into 5 major classes
Viability : An immediate or short term response as alteration in membrane
permeability or perturbation of particular metabolic pathway correlated with
cell proliferation or survival.
Survival : The long term retention of self renewal capacity (5-10 generation
or more)
Metabolic : Assays usually micro titration based, of intermediate duration
that can either measure a metabolic response at the time of or shortly after
exposure (2 or 3 population doubling after exposure- preferable)
Transformation :Survival in an altered state (a state expressing genetic
mutation)
Irritancy : Difficult to model invitro Monitoring cytokine release response

GENERALIZED SCHEME REPRESENTING

AN
IN VITRO CYTOTOXICITY ASSAY PROTOCOL.

VIABILITY ASSAYS

Relies on membrane integrity and inference is immediate

Measured by assessment of
Dye exclusion -Uptake of a dye (Trypan Blue, Erythrosine or
Naphthalene black)

to which the cell is normally impermeable-

Cell suspension mixed with dye and examine by microscopy

Dye uptake and release of a dye( diacetyl fluorescein or neutral
red)

normally taken up and retained by viable cells- Cell

suspension mixed with propidium iodide and diacetyl fluorescein

and examined under fluorescent microscope
In

Neutral red uptake assays end point is measured with an

ELISA reader at 570nm

Isotope release method Reduced
and oxidized to

51

Cr3 is taken up by viable cells

51

Cr2+ which cannot pass through live cell

membrane
Limitation
Does not predict ultimate survival
Overestimates viability

SURVIVAL ASSAY
A long term test to demonstrate survival implying retention of
regenerative capacity
It is measured by means of plating
efficiency which measures
For high grade or
acute
cytotoxin,
proliferating capacity for several cell generation.
treat flasks for 1 Trypsinize
Trypsinize the
the
72 h depending on
monolayer,
Colonogenic assay protocol
resuspend cells, and
nature
of
cytotoxin and cell
cycle duration

Treat
the
cells
with
experimental agent at a
range of concentrations for
24 h.
Cells
are
trypsinized,
counted, and diluted as for
monolayer dilution seeded at
a low cell density, and
incubated for 13 weeks. The
colonies are fixed and stained
when they reach a reasonable
size for counting by eye but
before they overlap.

count
count.

Dilute
Dilute serially
serially to
to between
between 10
10

and
For
low
grade
and 200
200 cells/ml
cells/ml (based
(based on
on
cell
cell count
count of
of controls)
controls)
cytotoxin
or
presumed
chronic exposure
treat
cloning
cultures for 1 3
Incubate for from 1 - 3
weeks,
weeks (depending on
depending on the
growth rate)
nature
of
the
cytotoxin
. concentration of
Increasing
cytotoxin

Relative Plating efficiency at each drug concentration is calculated as

a fraction of the control (surviving fraction) .
A Plot of surviving fraction on a log scale against

the

concentration on a linear or log scale is done , and the concentration

of compound promoting 50% inhibition of colony formation(IC50) or
90% (IC90) is calculated , and analysed

for

difference in

sensitivity
A shallower slope and/or longer knee means reduced sensitivity;
A steeper slope and/or shorter knee means increased sensitivity.
Both the length of knee and the slope influence the IC50 and the
IC90,
The IC90 is often used as a simple derivative.
Resistant fraction. The fraction of resistant cells is indicated by
a flattening of the lower end of the curve.
(c) Total resistance is indicated by the lack of any gradient on
the curve.
(d) Area under the curve: Complex survival curves may be

Interpretation

of

Survival

Survival Curve. Semilog plot of the Curves. Semilog plot of cell survival
surviving fraction of
forming

from

test

cells (colonies against

the

concentration

of

cells/colonies cytotoxin. The slope increases with

forming from control cells) against increasing sensitivity and decreases

the

concentration

of

cytotoxin. with

reduced

sensitivity

until

it

Typically, the curve has a knee, becomes totally flat for complete
and the IC90 lies in the linear range resistance. Partial resistance can be
of the curve. The

expressed as the resistant fraction

IC50, falling on the knee, is a less shown by the curve flattening out at
stable value.

the lower end.

Variable parameters in survival assay

Concentration of agent- various dilutions
Invariate agent concentrations certain condition cannot be altered
Duration of exposure to agent- effects are rapid /slow
Time of exposure to agent- according to the requirement/agent
Cell density during exposure - HeLa cells less sensitive to mustine
at high cell densities
Cell density during cloning- High level of toxicity seeding of more
cells.
Colony size- cytostatic agents may reduce size of colonies
Solvents-The solvents in which the agents are prepared shall
influence the effects on cell
Limitation of Plating efficiency tests
Labour intensive
Time consuming to set up and analyse large samples
Duration of each experiment would be 2-4 weeks
some cell lines have poor plating efficiency esp freshly isolated
normal cells)

Assays Based on Cell Proliferation assessing effect of agents

on cell counts
- Can be adopted for small samples in early stage of testing
Metabolic Cytotoxicity Assays- measuring survival as alteration of
metabolism, retention of metabolic or proliferative ability of the cell
population as a whole
total amount of protein, DNA or
metabolic activity such as
Respiration and glycolysis using manometry
reduction of tetrazolium salt (3-(5,5 dimethyl thiazole-2-yl)-2,5diphenyltetrazolium bromide-MTT)to formazan or
synthesis of protein or DNA
Ability to incorporate labelled precursors such as [ 3H] thymidine

Limitation : cannot discriminate between reduction in

activity and reduction in number of cells

Micro titration Assays

Large number of samples can be handled with few cells per sample
End point determined by cell count, isotope incorporation or cell viability measured
as MTT reduction

MTT-BASED CYTOTOXICITY ASSAY - [Plumb et al., 1989].

Set up microtitration plate and incubate for

about two population doublings

When cells are in exponential growth, add

drug or toxin

Remove drug and allow cells to recover in

growth medium

After two or three more population doublings,

remove
medium and replace with MTT or XTT.
Read on plate reader after 3-4 h. Use
absorbance to plot
inhibition curve and calculate IC50

Analysis of MTT Assay:

Plot a graph of the absorbance (y-axis) against the concentration of drug (x-axis)
Calculate the IC50 as the drug concentration that is required to reduce the absorbance
to half that of the control.
The mean absorbance reading from the wells in columns 2 and 11 is used as a control.
The absorbance values in columns 2 and 11 should be the same.
Occasionally, they are not, however, and this is taken to indicate uneven plating of cells
across the plate.
The absolute value of the absorbance should be plotted so that control values may be
compared, but the data can then be converted to a percentage-inhibition curve, to
normalize a series of curves.

Micro titration assays can be automated, saves time and are comparable
to Colonogenic assays
Limitation-cannot distinguish between the differential responses between
cells within a population and degree of response in each cell

Some commercial assays for Cell Viability, and Cytotoxicity Assays

Characteristic

Incubation
Parameter
measured

Cell Titer-Glo
Luminescent Cell
Viability Assay

CellTiter-Blue
Cell Viability
Assay

10 minutes

30 minutes

14 hours

14 hours

ATP

live- and deadcell protease

activity

resazurin
reduction

MTS reduction

Sensitivity:
96-well/384well

50 cells/15 cells
(also 1536-well
format)

Sample Type

suspension or
adherent cells

Detection

CellTiter 96

Cyto Tox 96
Non-Radioactive
Cytotoxicity
Assay

luminescent

several hundred
cells or cell
equivalents

AQueous One
Solution Cell
Proliferation
Assay

390 cells/50 cells 800 cells/200 cells

(also in 1536well format)

suspension or
adherent cells

fluorometric

suspension or
adherent cells

suspension or
adherent cells

fluorometric
or
colorimetric
colorimetric
Promega Corporation

INVITRO LIMITATIONS
Pharmacokinetics :
Difficult to recreate the complex of drug exposure in vitro
Significant difference in exposure times concentration of drug rate of
change of
concentration, metabolism tissue penetration, clearance and
excretion
overcome by use of spheroids/timed perfusion techniques
Metabolism :

The detoxification mechanism of animal model is difficult in cell

culture system
(requires processing of the agents, coculture, genetic
modification of target cells)
Tissue and systemic response :
Complex tissue and even systemic reaction are very difficult to

REFERENCE
1. Culture of Animal Cells: A Manual of Basic technique, Fifth Edition,
by R. Ian Freshney Copyright 2005 John Wiley & Sons, Inc. pp 359373
2. Methods in Biotechnology. Vol 8.animal Cell Biotechnology
Edited by N.Jenkins Humana press Inc. Totowa NJ
3. Biotechnology & Genetic Engineering Reviews Volume 26 Editor:
S.E. Harding 2010
4.. http://www.promega.in/resources/product-guides-andselectors/protocols-and-applications-guide/

Thank