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Application of animal cell culture for in vitro testing of drugs, testing of toxicity of environmental pollutants in cell culture Submitted by: Dr. Vijayata Mail – dr.viz.vet@gma

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Page 1: Drug Toxicity

Application of animal cell culture for in vitro testing of drugs, testing

of toxicity of environmental pollutants in cell culture

Submitted by:

Dr. Vijayata

Mail – [email protected]

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In vitro testing of drugs

• 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 :

�̵ 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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In vitro methods for the assessment of general cellular toxicity

• 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, WI-

38 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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• 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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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. Ekwall et al (1990)5

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Specialized cells commonly used in toxicology

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)

Reticuloendothelialsystem

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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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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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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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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Testing of toxicity of environmental pollutants in cell culture

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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Cytotoxicity testing

• 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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Cytotoxicity is evaluated by qualitative and quantitative means.

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.

QualitativeQuantitative

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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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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

Agar overlay: positive control

Material

Agar layer

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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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B. Quantitative cytotoxic assays:• Evaluation of cell number and viability by measuring of

metabolic activity, membrane permeability.

MTT assayNeutral Red assay

Calcein assayLDH assay

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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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Nucleus

Calcein – AM(non – fluorescent compound)

Esterases

Calcein

NADH,NADH Dehydrogenase

Formazan dye

Water soluble(Colourless substrate)

Miochondria

MTT Formazan dyes

Viable cell

MTT

Mitochondrial succinate dehydrogenase.

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• 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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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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Nucleus

Calcein – AM(non – fluorescent compound)

Esterase

Calcein

NADH,NADH Dehydrogenase

Formazan dye

Water soluble(Colourless substrate)

Miochondria

MTT Formazan dyes

Viable cell

MTT

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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 non-

ionic diffusion, accumulating intracelluarly in lysosomes, where it

binds with anionic sites in the lysosmal matrix.

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• 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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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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• 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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Nucleus

Calcein – AM(non – fluorescent compound)

Esterase

Calcein

NADH,NADH Dehydrogenase

Formazan dye

Water soluble tetrazolium(Colourless substrate)

Miochondria

MTT Formazan dyes

Viable cell

MTT

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2-D and 3-D Cell-based Assays in Drug Screening

• 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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• 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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• 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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• 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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Thank

you