Cytotoxicity in animal cell culture.pptx.pdf

Cytotoxicity assays are widely used by the pharmaceutical industry to screen for cytotoxicity in compound libraries. Researchers can either look for cytotoxic compounds, if they are interested in developing a therapeutic that targets rapidly dividing cancer cells, for instance; or they can screen "hits" from initial high-throughput drug screens for unwanted cytotoxic effects before investing in their development as a pharmaceutical

Assessing cell membrane integrity is one of the most common ways to measure cell viability and cytotoxic effects. Compounds that have cytotoxic effects often compromise cell membrane integrity. Vital dyes, such as trypan blue or propidium iodide are normally excluded from the inside of healthy cells; however, if the cell membrane has been compromised, they freely cross the membrane and stain intracellular components.[1] Alternatively, membrane integrity can be assessed by monitoring the passage of substances that are normally sequestered inside cells to the outside. One molecule, lactate dehydrogenase (LDH), is commonly measured using LDH assay. LDH reduces NAD to NADH which elicits a colour change by interaction with a specific probe.[3] Protease biomarkers have been identified that allow researchers to measure relative numbers of live and dead cells within the same cell population. The live-cell protease is only active in cells that have a healthy cell membrane, and loses activity once the cell is compromised and the protease is exposed to the external environment. The dead-cell protease cannot cross the cell membrane, and can only be measured in culture media after cells have lost their membrane integrity.

Cytotoxicity can also be monitored using the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) or with 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), which yields a water-soluble product, or the MTS assay. This assay measures the reducing potential of the cell using a colorimetric reaction. Viable cells will reduce the MTS reagent to a colored formazan product. A similar redox-based assay has also been developed using the fluorescent dye, resazurin. In addition to using dyes to indicate the redox potential of cells in order to monitor their viability, researchers have developed assays that use ATP content as a marker of viability.[1] Such ATP-based assays include bioluminescent assays in which ATP is the limiting reagent for the luciferase reaction
Cytotoxicity can also be measured by the sulforhodamine B (SRB) assay, WST assay and clonogenic assay.

Suitable assays can be combined and performed sequentially on the same cells in order to reduce assay-specific false positive or false negative results. A possible combination is LDH-XTT-NR (Neutral red assay)-SRB which is also available in a kit format.

1. Cytotoxicity

2. •Carried out to check toxicity of cosmetics, food additives, drugs or
anticancer drugs
•Animal sacrifice now not feasible
•In vitro cell lines
•Toxicity: in vivo, direct cellular damage, as with a cytotoxic anticancer drug,
physiological effects, such as membrane transport in the kidney or
neurotoxicity in the brain, inflammatory effects, both at the site of
application and at other sites, and other systemic effects.
•difficult to monitor systemic and physiological effects in vitro, most assays
determine effects at the cellular level, or cytotoxicity.
•cytotoxicity vary, -cells are killed or simply have their metabolism altered.
•an anticancer agent may be required to kill cells,
•subtle analysis of specific targets such as an alteration in cell signalling or
cell interaction such as might give rise to an inflammatory or allergic
response.
Viability, Toxicity and Survival

3. In vitro limitations
Pharmacokinetics.
•The measurement of toxicity in vitro is generally a cellular event.
•difficult to recreate the complex pharmacokinetics of drug
exposure in vitro,
•using multicellular tumor spheroids for drug penetration or timed
perfusion to simulate concentration and time (C × T) effects—
•concentrate on a direct cellular response, thereby gaining
simplicity and reproducibility.
Metabolism.
•Many nontoxic substances become toxic after being metabolized
by the liver;
•substances that are toxic in vitro may be detoxified by liver
enzymes.
•must be demonstrated that potential toxins reach the cells in vitro
in the same form as they would in vivo.

4. •may require additional processing by purified liver microsomal
enzyme preparations
•coculture with activated hepatocytes or hepatoma derived cells
Hep-G2 or HepaRG
•genetic modification of the target cells with the introduction of
genes for metabolizing enzymes under the control of a regulatable
promoter
Tissue and systemic responses.
▪A toxic response in vitro may be measured by changes in cell
survival or metabolism
•in vivo may be a tissue response (e.g., an inflammatory reaction,
fibrosis, kidney failure) or a systemic response (e.g., pyrexia,
vascular dilatation).
•models of these responses constructed, utilizing organotypic
cultures reassembled from several different cell types and
maintained in the appropriate hormonal milieu.
•Assays for inflammatory responses, teratogenic disorders, and
neurological dysfunctions
• a proper understanding of cell–cell interaction and the interplay
of endocrine hormones with local paracrine and autocrine factors.

5. Nature of the assay
1.Viability: An immediate or short-term response, such as an alteration in
membrane permeability or a perturbation of a particular metabolic
pathway correlated with cell proliferation or survival
2.Survival: The long-term retention of self-renewal capacity (5–10
generations or more)
3.Metabolic: Assays, usually microtitration based, of intermediate duration
that can either measure a metabolic response (e.g., dehydrogenase
activity; DNA, RNA, or protein synthesis) at the time of, or shortly after,
exposure.
•Making the measurement two or three population doublings after
exposure is more likely to reflect cell growth potential and may correlate
with survival.
4.Genotoxicity and Transformation: Survival in an altered state (genetic
mutation, alterations in growth control, or malignant transformation)
5.Irritancy: A response analogous to inflammation, allergy, or irritation in
vivo; in vitro by monitoring cytokine release in organotypic cultures

6. 1.Viability
•Either dye exclusion or dye retention
1.Trypan blue dye exclusion assay (naphthalene
black, erythrosin)
2.Dye uptake or retention assay (neural red or
diacetyl fluorescin and propidium iodide)
•Release of lactate dehydrogenase by leaky
cells

7. 2.SURVIVAL
•Long term testing
•Retention of regenerative capacity
•Plating efficiency

10. 2.Survival

12. •Analyze the curve for differences in sensitivity:
(a) Slope of the curve and length of the knee.
•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, although a more significant difference can be observed in the
IC90
•the IC90 is often used as a simple derivative.
(b) 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 compared by calculating the area
under the curve, but this is done for expediency and is not
mathematically valid

15. Variable parameters in survival assay
1.Concentration of agent.
• A wide range of concentrations in log increments (e.g., 1 × 10
−6
M, 1 × 10
−5
M, 1
× 10
−4
M, 1 × 10
−3
M, and control)- first attempt and a narrower range (log or
linear) for subsequent attempts.
2.Invariant agent concentrations.
•the quality of medium, water, or an insoluble plastic.
•the serum concentrations can be varied.
•As serum may have a masking effect on low-level toxicity, an effect may only be
seen in limiting serum.
3.Duration of exposure to agent.
•Exposure to ionizing radiation- matter of minutes to achieve the required dose,
•cycle dependent antimetabolic drugs may take several days to achieve a
measurable effect.
•Duration of exposure (T) and drug concentration (C) are related, although C × T is
not always a constant.
•Prolonging exposure increase sensitivity beyond that predicted by C × T, because
of cell cycle effects and cumulative damage.

16. 4.Time of exposure to agent.
•when the quality of the agent is unknown, stimulation is
expected, or only a minor effect is expected (e.g., 20%
inhibition rather than several fold)
•the agent may be incorporated during clonal growth rather
than at preincubation.
•Confirmation of anticipated toxicity—e.g., for a cytotoxic
drug—requires a conservative assay, with minimal drug
exposure as compared to that in vivo
•Confirmation of the lack of toxicity—e.g., for tap water or
a nontoxic pharmaceutical—requires a more stringent
assay, with prolonged exposure during cloning.
5.Cell density during exposure.
•HeLa cells - less sensitive to the alkylating agent mustine at
high cell densities [Freshney et al., 1975].
•To determine the effects of cell density it should be varied
in the preincubation phase, during exposure to the drug.

17. 6.Cell density during cloning.
• The number of colonies may fall at high concentrations of a toxic
agent,
•compensate for this effect by seeding more cells so that
approximately the same number of colonies form at each
concentration.
•This procedure removes the risk of a low clonal density
influencing survival and improves statistical reliability,
•Error prone-cells from higher drug concentrations are plated at a
higher cell concentration, influencing survival.
•It is preferable to plate cells on a preformed feeder layer, the
density of which (5 × 10
3
cells/cm2) greatly exceeds that of the
cloning cells.
•This step ensures that the cell density is uniform regardless of
clonal survival, which contributes little to the total cell density.
•cloning on a feeder layer can sometimes reveal a resistant fraction
of cells that is not apparent without the feeder layer

18. 7.Effect of medium constituents:
•Serum and cysteine-reduce toxicity of
formaldehyde on buccal epithelium
•Age of medium

19. 8.Colony size.
•Some agents are cytostatic (i.e., they inhibit cell proliferation) but
not cytotoxic, and during continuous exposure they may reduce
the size of colonies without reducing the number of colonies.
•the size of the colonies should be determined by densitometry
[McKeehan et al., 1977], automatic colony counting, or visually
counting the number of cells per colony.
•For colony counting, the threshold number of cells per colony (e.g.,
50 as in Protocol 21.9) is purely arbitrary, and it is assumed that
most of the colonies are greatly in excess of this number.
•Colonies should be grown until they are quite large (>1 × 10
3
cells),
• when the growth of larger colonies tends to slow down and
smaller, but still viable, colonies tend to catch up with these larger
colonies.
•For colony sizing, stain the cultures earlier, before the growth rate
of larger colonies has slowed down, and score all of the colonies.

21. 9.Solvents.
•Ethanol, propylene glycol, and dimethyl sulfoxide have been used for this
purpose, but may themselves be toxic to cells.
•Hence, the minimum concentration of solvent should be used to obtain a
solution.
•The agent may be made up at a high concentration in, for example, 100%
ethanol, then diluted gradually with BSS and finally diluted into medium.
•The final concentration of solvent should be <0.5%, and a solvent control
must be included (i.e., a control with the same final concentration of
solvent but without the agent being tested).
•Precaution while using solvents in plastic or rubber ware.
•glass with undiluted solvents
• plastic only when the solvent concentration is <10%.
•plating efficiency - only when the cloning efficiency is high enough for
colonies that form to be representative of the whole cell population.
•Ideally, this means that controls should plate at 100% efficiency.
•In practice, however, this is seldom possible, and control plating
efficiencies of 20% or less are often accepted.

22. Assays based on cell proliferation
•Cell counts after a few days in culture
• in the early stages of testing, a complete growth curve is required
•because cell counts at a single point in time can be ambiguous.
•Growth curve analyses, using cell counting, are feasible only with
relatively small numbers of samples,
•In cases for which there are many samples, a single point in
time—e.g., the number of cells three to five days after
exposure—can be used.
•The time should be selected as within the log phase, and
preferably mid-log phase, of control cells.
•backed up with a complete growth curve over the whole growth
cycle or by an alternative assay, such as a survival curve by
clonogenic assay or MTT

23. Metabolic cytotoxicity assay
•Plating efficiency tests are labor intensive and time consuming
• some cell lines have poor plating efficiencies,
• None of these tests measures survival directly
•the net increase in the number of cells (i.e., the growth yield),
•the increase in the total amount of protein or DNA, or
•Continued metabolic activity, such as the reduction of a
tetrazolium salt to formazan or the synthesis protein or DNA, is
determined.
•Survival in these cases is defined as the retention of metabolic or
proliferative ability by the cell population as a whole some time
after removal of the toxic influence.
• such assays cannot discriminate between a reduction in metabolic
or proliferative activity per cell and a reduced number of cells, and
therefore any novel or exceptional observation should be
confirmed by clonogenic survival assay.

24. Microtritration assays
•plates are economical to use, lend themselves to automated handling, and can be of
good optical quality.
•The most popular is the 96-well microtitration plate, each well having 28–32 mm2 of
growth area, 0.1 or 0.2 mL medium, and up to 1 × 105 cells.
•Large numbers of samples may be handled simultaneously, but with relatively few
cells per sample.
•With this method, the whole population is exposed to the agent, and viability is
determined subsequently, usually by measuring a metabolic parameter such as the
ATP or NADH/NADPH concentration.
•The end point of a microtitration assay is usually an estimate of the number of cells.
•Although this result can be achieved directly by cell counts or by indirect methods,
such as isotope incorporation, cell viability as measured by MTT reduction
[Mosmann, 1983] is now widely chosen as the optimal end point [Cole, 1986; Alley
et al., 1988].
•MTT is a yellow water-soluble tetrazolium dye that is reduced by live, but not dead,
cells to a purple formazan product that is insoluble in aqueous solutions.
•However, a number of factors can influence the reduction of MTT [Vistica et al.,
1991].

25. •MTT:
Variations in MTT assay
•A similar assay has also been used to determine cellular radiosensitivity [Carmichael et al.,
1987b].
•growth factor stimulation.
•essential to ensure that the treatment itself does not affect the ability of the cell to reduce
the dye.
✔Duration of exposure.
•Fast acting compounds, the exposure period and recovery may be shortened.
•The cells must remain in exponential growth throughout and the cell concentration at the
end should still be within the linear range of the MTT spectrophotometric assay.
•If cells are moving into the stationary phase or the absorbance is nonlinear when plotted
against cell concentration, shorten the assay and proceed directly to addition of MTT
✔ Duration of exposure is related to the number of cell cycles that the cells have gone through
during exposure and recovery.
•Not only will the cell density increase more rapidly during exposure, but in addition the
response to cycle-dependent drugs will be quicker.
•Cell cycle time will influence the choice between a short-form and long-form assay.
•When first trying an assay, it may be desirable to sample on each day of drug exposure and
recovery.
•If a stable IC50 is reached earlier, then the assay may be shortened.

•SRB

29. •Endpoint :
•SRB
•Labeling with [3H]thymidine (DNAsynthesis), [3H]leucine or [35S]methionine
[Freshney & Morgan, 1978] (protein synthesis), or other isotopes can be
substituted forMTTreduction.
•Quantitation is achieved by microtitration plate scintillation counting.
•Two types of scintillation counting are available:
(1)The cellular contents may be aspirated onto filters by trypsinization and onto
glass fiber filters by suction transfer, and
•the filters may be dried and counted in scintillant, or
(2) The whole plate may be counted on a specially adapted scintillation counter
(PerkinElmer).
•the design of the assay, e.g., duration of drug exposure and recovery, phase of the
growth cycle (cell density, growth rate, etc.), that is more important.
•In a short assay with no or minimal recovery period, the end point must measure
only viable cells (e.g., MTT),
•in a longer assay the end point measures the difference between wells that have
increased and those that have not, or have even decreased.
•In a monolayer assay, at least, nonviable cells will have been lost, and the increase
or decrease relative to control wells is measured;
•by MTT, sulforhodamine, or isotope incorporation into DNA or protein becomes
less important.

30. Applications of cytotoxicity assays
1.Anticancer Drug Screening
•Drug screening for the identification of new anticancer drugs
tedious and often inefficient method of discovering new
active compounds.
•The trend is now more toward monitoring effects on specific
molecular targets.
•the MTT incubation step may be omitted and the end point
determined by measuring the amount of total protein with
sulforhodamine B [Boyd, 1989].
•Although this method is quicker and easier than the MTT
assay,
•nonviable, and certainly nonreplicating, cells will still stain,
•assay should be confirmed when activity is detected, using a
more reliable indicator such as clonogenicity or MTT
reduction.

31. 2.Predictive Drug Testing for Tumors
•measurement of the chemosensitivity of cells derived from a patient’s tumor might
be used in designing a chemotherapeutic regime for the patient [Freshney, 1978].
•the development of reliable and reproducible culture techniques for neoplastic
cells from the most common tumors (e.g., breast, lung, colon), such that cultures
of pure tumor cells capable of cell proliferation over several cell cycles may be
prepared routinely.
•As many of the cells within a tumor have a limited life span, the main targets for
chemotherapy are the clonogenic populations with infinite repopulation capacity
[Al-Hajj et al., 2004; Jones et al., 2004].
•Advances in stem cell recognition may make isolation of tumor stem cells feasible.
•the soft agar clonogenic assay [Hamburger & Salmon, 1977] might isolate
transformed stem cells for assay,
•isolated clones did not seem to have long-term regenerative capacity.
•Routine isolation of tumor cell populations with long-term repopulation efficiency
has yet to be achieved,
•to improve targeting and specificity of anticancer drugs, making predictive testing
more meaningful.
•Assays might then be performed in a high proportion of cases, hopefully within 2
weeks of receipt of the biopsy

32. •The major problem, however, is one of logistics.
•The number of patients with tumors for which the correct
target cells
(1)will grow in vitro sufficiently to be tested,
(2)can be expected to respond, and
(3) will produce a response that can be followed up, is
extremely small.
•Hence, it has proved difficult to use any in vitro test as a
predictor of response or even to verify the reliability of the
assay.
• The correlation of insensitivity in vitro with nonresponders
is high, but few clinicians would withhold chemotherapy
because of an in vitro test,
•particularly when the agent in question would probably
not be used alone.

33. 3.Testing Pharmaceuticals
•A number of pharmaceutical companies maintain
a program of in vitro testing on the assumption
that it might prove more economical and ethically
acceptable than animal testing.
•Legislation enforcing the use of animal tests is
difficult to introduce as the complexity of the wide
range of effects seen in vivo is still very difficult to
model in vitro.
•large-scale comparative surveys to determine
whether any of the many existing tests may be
acceptable [Knight & Breheny, 2002;Vanparys,
2002].