In vitro toxicity testing methods can more cost-effectively identify potentially hazardous chemicals compared to animal testing. Common techniques include extracting chemicals from test materials and monitoring their effects on cultured cells through microscopy and assays measuring cell viability, morphology, and metabolism. While 2D cell cultures are widely used, 3D cultures better mimic in vivo conditions and may improve predictions of drug toxicity to potentially reduce clinical trial failures. A variety of quantitative assays exist to complement qualitative observations, with the MTT, neutral red, calcein, and LDH assays among the most frequently employed.

1. Submitted by:
Dr. Vijayata
(dr.viz.vet@gmail.com)

2. • In vitro toxicity testing is the scientific analysis of the effects of toxic
chemical substances on cultured bacteria or mammalian cells.
• In vitro (literally 'in glass') testing methods are employed primarily :
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to identify potentially hazardous chemicals
to confirm the lack of certain toxic properties in the early stages of
the development of potentially useful new substances such as
therapeutic drugs, agricultural chemicals and food additives.
• In vitro toxicity testing methods can be more useful and cost-effective
than toxicology studies in living animals (which are termed in vivo or
"in life" methods).
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3. • Cell culture can be used to screen for toxicity both
- by estimation of the basal functions of the cell (i.e. those processes
common to all types of cells) or
- by tests on specialized cell functions.
• For general toxicity studies, the more commonly used cell lines
include the well characterized diploid human fibroblast lines, WI38 and tumour cell lines, HeLa.
• The first and most readily observed effect following exposure of
cells to toxicants is morphological alteration in the cell layer or
cell shape in monolayer culture.
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4. • The cytotoxic concentrations of chemicals determined in vitro
have been shown to correlate well with lethal doses in
laboratory animals and man for a range of selected drugs and
chemicals (Ekwall, 1983).
• Morphological changes in cells exposed to chemicals (blebbing
- suggesting injury of the cell membrane or vacuolization)
observed by light or electron microscopy, have also been used
to demonstrate basic cytotoxicity. (Ekwall, 1983).
• These observations may provide valuable information about the
pathologic processes that occur as a consequence of exposure to
a chemical substance.
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5. End-points for the assessment of general
cellular toxicity
• Cell morphology
- Blebbing, vacuolisation, fine ultrastructural modification
• Cell viability
- Trypan blue (enters dead cells), neutral red (actively taken up
by living cells), Cr51 release
• Cell growth
- Cell count, plating efficiency, DNA or protein content, glucose
consumption, lactate production, NR-test, MTT-test
• Metabolic parameters
- O2 consumption or ATP level, pool of DNA and RNA
precursors, NADH-NAD conversion.
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Ekwall et al (1990)

6. Organ of origin
Primary cultures or isolated cells
Cell lines
Nervous system
Chick embryo ganglia;
chick embryo brain cells;
mouse and rat cerebellum cells
C 1300 (mouse);
C 6 (rat)
Lung
Human, rabbit and rat alveoloar
macrophages
P 388Dl (mouse);
A 549 (human)
Reticuloendothelial
system
Human, mouse lymphocytes and
erythrocytes;
rat and mouse peritoneal macrophages
------
Liver
Rat and chick embryo hepatocytes
Chang (human);
CC1144 (rat);
ARL (rat);
RLC-GA (rat)
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7. End-points more commonly used as markers of
toxic effects in specialized
cells
Synthesis or release of specific molecules:
– Collagen mat, heme, haemoglobin, albumin, urea, lipoprotein,
bile salts, metallothionein,glycosaminoglycans, proline and
hydroxyproline, energy-dependent choline accumulation, and
histamine release .
Synthesis, activity or release of specific enzymes:
– Glucuronidase, lactate-dehydrogenase, oubain-insensitive
ATPase, G-6-P dehydrogenase, glycogen phosphorylase,
glutamic-oxalacetic transaminase, glutamic-pyruvic
transaminase, acetylcholinesterase, and renin.
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8. Interactions of compound with cells:
– Phagocytosis, cytoplasmic inclusions, intracellular accumulation,
uptake or binding of compound to cytosol and lipoproteins,
mitogenic response.
Alterations of metabolic pathways:
– Methaemoglobin reduction, glucose-transport, 5-methyl tetra
hydropholate accumulation, hormone-stimulated
gluconeogenesis, lipid peroxidation, fat accumulation and
glucosamine and galactose incorporation.
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9. Cell surface activities:
– Adhesiveness, antibody-mediated rosette formation,
complement deposition on treated cell membrane,
chemotactic migration, GABA-mediated postsynaptic
inhibition, membrane polarization, fibre retraction or
outgrowth, and electrophysiological alteration.
Intracellular markers
– Mitochondria, lysosomes, peroxisomes
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10. Introduction
• The quality of materials used (cell lines, media and other reagents)
will affect the quality of the cultures and the subsequent scientific
data and products derived from them.
• The main areas of quality control that are of concern for tissue
culture are:
– The quality of the reagents and materials.
– The provenance and integrity of the cell lines.
– The avoidance of microbial contamination.
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11. • Cell viability and Cytotoxicity assays are used for drug screening
and cytotoxicity tests of chemicals.
• This in vitro test evaluates the potential of the materials or their
extracts to cause damage to cells in culture.(useful in evaluating
the toxicity or irritancy potential of materials and chemicals. )
• Cell cultures are suitable test systems for the determination of
cytotoxic reactions such as changes in cell cycle, inhibition of
cell division, and cell death. (caused by eluates or extracts of
products either natural or processed.)
ISO 10993-5:2009 Part 5: Tests for in vitro cytotoxicity
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12. Cytotoxicity is evaluated by qualitative and quantitative means.
Qualitative
Quantitative
A. Qualitative Cytotoxicity Tests
Three different qualitative cytotoxicity tests are commonly used :
1. Extraction method/ MEM Elution
2. Agar Diffusion or Agarose overlay assay
3. Direct contact method
In general, in these tests, toxicity is verified after a period of exposure
(typically 24–72 hours) of the cells to the extract or device.
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13. 1. MEM Elution - Test on Extracts (ISO 10993-5)
• The test material is extracted for 24 hours in Minimum Essential
Medium (MEM).
• An extract is prepared from the test material which is then placed
over the cultured cells. (L-929 mouse fibroblast cells)
• Following incubation, the cells are examined microscopically for
morphological changes, degeneration and lysis of the cells.
• Potentially cytotoxic substances are uniformly distributed
throughout the cell culture
cell line L929
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15. 2. Agar Diffusion or Agarose overlay assay - (ISO 10993-5)
• In this method, a thin layer of nutrient-supplemented agar is
placed over the cultured cells.(L-929 mouse fibroblast cells )
• The test material (or an extract of the test material dried on filter
paper/sample) is placed on top of the agar layer, and the cells are
incubated for 24 hours.
• Cytotoxic leachates diffuse into the cell layer via the agar, and a
zone of malformed, degenerative or lysed cells under and around
the test material indicates cytotoxicity.
• MRC-5 Human Embryonic Lung Cells
Material
Agar layer
Agar overlay: positive
control
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16. 3. Direct Contact (ISO 10993-5)
• In this method, a piece of test material is placed directly onto
cells growing on culture medium.(without the agar layer)
• Cell cultures are grown to a standard monolayer.
• The cells are then incubated for 24 hours at 37 C.
• During incubation, leachable chemicals in the test material can
diffuse into the culture medium and contact the cell layer.
• Subsequently, the monolayers are examined microscopically for
the presence of morphological changes, reduction in cell density
or lysis of cells around the test material.
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17. B. Quantitative cytotoxic assays:
• Evaluation of cell number and viability by measuring of
metabolic activity, membrane permeability.
MTT
assay
Neutral
Red
assay
Calcein
assay
LDH
assay
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18. 1. MTT assay:- Mitochondrial activity
[3-(4,5-dimethylthiazol-2-yl)-2,5- diphenylformazan bromide ]
• MTT assay is used often in determining cell viability (detects viable
cells).
• The MTT is a colorimetric method that measures the reduction of
yellow 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium
bromide into an insoluble purple formazan product by mitochondrial
succinate dehydrogenase.
• MTT being water soluble can penetrate through cell membrane, water
insoluble formazan is trapped inside the cell.
• Dead cells do not have active mitochondrial reductases (as the cellular
reduction is only catalyzed by living cells), MTT is not reduced and the
purple formazan is not formed.
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19. Calcein – AM
(non – fluorescent compound)
Water soluble
(Colourless substrate)
Esterases
Calcein
Nucleus
Viable cell
NADH,NADH
Dehydrogenase
Mitochondrial
Miochondria
succinate
dehydrogenase.
Formazan dye
MTT Formazan dyes
MTT
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20. • Samples are read using an ELISA plate reader at a wavelength of
570 nm.
• The amount of color produced is directly proportional to the
number of viable cells.
• The MTT can be used to evaluate the cytotoxicity of:
‐
‐
‐
‐
Toxic compounds
Toxins and environmental pollutants
Potential anti-cancer drugs
Antibodies to examine growth inhibiting potential
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21. 2. Calcein assay :-
• The acetomethoxy derivate of calcein (calcein AM) is used in
testing of cell viability as it can be transported through the
cellular membrane into live cells.
• After transport into the cells, intracellular esterases remove the
acetomethoxy group, the molecule gets trapped inside and
gives out strong green fluorescence.
• As dead cells lack active esterases, only live cells are labeled
and counted by flow cytometry.
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22. Calcein – AM
(non – fluorescent compound)
Water soluble
(Colourless substrate)
Esterase
Calcein
Nucleus
Viable cell
NADH,NADH
Dehydrogenase
Miochondria
Formazan dye
MTT Formazan dyes
MTT
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23. 3. Neutral Red (NR) assay:- Lysosomal activity
- Membrane permiability
• The neutral red (NR) assay is a cell survival chemo sensitivity assay.
• This assay is based on the incorporation of NR into the lysosomes of
viable cells after being incubation with test agents.
• NR (3-amino-7-dimethyl-2-methylphenazine hydrochloride) is a
weak cationic dye that readily penetrates cell membranes by nonionic diffusion, accumulating intracelluarly in lysosomes, where it
binds with anionic sites in the lysosmal matrix.
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24. • Therefore, it is possible to distinguish between viable, damaged
or dead cells as viable cells take up the NR dye, damaged or
dead cells do not.
• When a reduced cytotoxic effect was determined by neutral red
(NR-test), which shows the activity of lysosomal enzymes,
microscopically multiplication or enlargement of lysosomes
was observed.
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25. 4. Lactate dehydrogenase (LDH) leakage assay : (membrane
integrity)
• Lactate dehydrogenase (LDH), which is a soluble cytosolic
enzyme present in most eukaryotic cells, releases into culture
medium upon cell death due to damage of plasma membrane.
• The increase of the LDH activity in culture supernatant is
proportional to the number of lysed cells.
• LDH activity, therefore, can be used as an indicator of cell
membrane integrity and serves as a general means to assess
cytotoxicity resulting from chemical compounds or
environmental toxic factors.
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26. • LDH Assay measures LDH activity present in the culture
medium using a coupled two-step reaction.
• In the first step, LDH catalyzes the reduction of NAD+ to NADH
and H+ by oxidation of lactate to pyruvate.
• In the second step of the reaction, diaphorase (NADPH
dehydrogenase) uses the newly-formed NADH and H+ to
catalyze the reduction of a tetrazolium salt to highly-colored
formazan which absorbs strongly at 490-520 nm.
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27. Calcein – AM
(non – fluorescent compound)
Water soluble tetrazolium
(Colourless substrate)
Esterase
Calcein
Nucleus
Viable cell
NADH,NADH
Dehydrogenase
Miochondria
Formazan dye
MTT Formazan dyes
MTT
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28. • Currently, pharmaceutical firms spend a large amount of
money on the compound efficacy and cytotoxicity test.
• There is still a 78% failure rate for all drugs, which may be
devastating to developing companies.
• Effective compounds in vitro may be non-effective in vivo for
many reasons, including differences between in vitro and in
vivo target biology, interrelated biochemical mechanism,
metabolism, poor penetration into solid tissues, etc.
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29. • Currently, almost all cell-based assays or biosensors are
developed in 2-D culture systems, although conventional 2-D
cultures usually suffer from contact inhibition and a loss of
native cell morphology and functionality.
• In comparison with 2-D cultures, 3-D cell models create a
more realistic representation of real human tissues, which is
critical to many important cell functions, including
morphogenesis, cell metabolism, gene expression,
differentiation and cell-cell interactions.
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30. • 3D cell culture models can provide a platform for high throughput
and systematic experimentation, reducing the need for animals,
and permitting a more straightforward understanding of cause and
effect in drug safety and efficacy studies.
• Fibrous materials are used, such as non-woven polyethylene
terephthalate (PET), to maintain tissue native morphology,
because they can provide high
specific surface areas,
mechanical
properties and void volumes.
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31. •
In studying cytotoxicity and drug testing, maintaining cells in
their native functional state in a proper 3-D environment
would improve predictions and have the potential to reduce
clinical trial failures.
• Therefore, although designing 3-D models is much more
complicated than designing the 2-D counterparts, cell- and
tissue-based assays with a 3-D model are superior and are the
assays of choice for HTS (High-throughput screening) of drug
cytotoxicity.
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