COURSE ABT 604 ANIMAL CELL CULTURE: PRINCIPLES AND APPLICATIONS

CYTOTOXICITY ASSAYS

Term paper submitted byP.S.VinodI year M.V.Sc., I semesterAnimal Biotechnology

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

Fundamental requirements of invitro cytotoxicity testing

An assay system should give a reproducible dose response curve over a concentration range that includes the in vivo doseThere should be a linear relation ship between cell number and assay responseThe 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 DESIGNExposure to study compoundExposure time may simulate the exposure levels of invivo The penetration time required shall be consideredChoice of assay -The sensitivity required over concentration range

Recovery periodTo allow recovery from metabolic dysfunction unrelated to cell death when metabolic parameters are used as indexUse of controls appropriate to the toxin and mode of application reliability can be increased by using positive control of the compoundsPhase of the growth cycle: vary with cell cycle specific drugs and non cycle specific drugs .

Three-dimensional cell cultures in toxicologyBiotechnology & Genetic Engineering Reviews Volume 26 Editor: S.E. Harding 2010conventional 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.

GENERALIZED SCHEME REPRESENTING AN IN VITRO CYTOTOXICITY ASSAY PROTOCOL.

SURVIVAL ASSAYA long term test to demonstrate survival implying retention of regenerative capacityIt is measured by means of plating efficiency which measures proliferating capacity for several cell generation.Colonogenic assay protocolFor high grade or acute cytotoxin, treat flasks for 1 - 72 h depending on nature of cytotoxin and cell cycle durationFor low grade cytotoxin or presumed chronic exposure treat cloning cultures for 1 3 weeks, depending on the nature of the cytotoxin.Dilute serially to between 10 and 200 cells/ml (based on cell count of controls)Incubate for from 1 - 3 weeks (depending on growth rate)Trypsinize the monolayer,resuspend cells, and count.Increasing concentration of cytotoxin

Survival Curve. Semilog plot of the surviving fraction of cells (colonies forming from test cells/colonies forming from control cells) against the concentration of cytotoxin. Typically, the curve has a knee, and the IC90 lies in the linear range of the curve. TheIC50, falling on the knee, is a less stable value.Interpretation of Survival Curves. Semilog plot of cell survival against the concentration of cytotoxin. The slope increases with increasing sensitivity and decreases with reduced sensitivity until it becomes totally flat for complete resistance. Partial resistance can be expressed as the resistant fraction shown by the curve flattening out at the lower end.

Limitation of Plating efficiency testsLabour intensiveTime consuming to set up and analyse large samplesDuration of each experiment would be 2-4 weeks some cell lines have poor plating efficiency esp freshly isolated normal cells)

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

Set up microtitration plate and incubate for about two population doublingsWhen cells are in exponential growth, add drug or toxin

Remove drug and allow cells to recover in growth mediumAfter two or three more population doublings, removemedium and replace with MTT or XTT.Read on plate reader after 3-4 h. Use absorbance to plotinhibition curve and calculate IC50

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

Promega Corporation

Some commercial assays for Cell Viability, and Cytotoxicity AssaysCharacteristic Cell Titer-Glo Luminescent Cell Viability Assay Cyto Tox 96 Non-Radioactive Cytotoxicity AssayCellTiter-Blue Cell Viability Assay CellTiter 96 AQueous One Solution Cell Proliferation Assay Incubation 10 minutes 30 minutes14 hours 14 hours Parameter measured ATP live- and dead-cell protease activityresazurin reduction MTS reduction Sensitivity: 96-well/384-well 50 cells/15 cells (also 1536-well format)several hundred cells or cell equivalents (also in 1536-well format)390 cells/50 cells 800 cells/200 cells Sample Type suspension or adherent cells suspension or adherent cellssuspension or adherent cells suspension or adherent cells Detection luminescent fluorometricfluorometric or colorimetric colorimetric

REFERENCE1. Culture of Animal Cells: A Manual of Basic technique, Fifth Edition, by R. Ian Freshney Copyright 2005 John Wiley & Sons, Inc. pp 359-3732. 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

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