TAMILNADU VETERINARY AND ANIMAL SCIENCES UNIVERSITY

MADRAS VETERINARY COLLEGE

ANIMAL BIOTECHNOLOGY

COURSE ABT 604 ANIMAL CELL CULTURE: PRINCIPLES AND APPLICATIONS

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

Toxicity is a complex event in vivo where there may bedirect cellular damage (cytotoxic anticancer drug)Physiological effects (membrane transport in kidney, neurotoxicity ) Inflammatory effects Systemic effectsCytotoxicity assays Cytotoxicity assays are assays where the cell/tissue culture systems are used as models for testing the effect/toxicity of chemicals ,food additives, pharmaco-therapeutic drugs, chemotherapeutics, pesticides, oncogenic agents etc.,Definition of cytotoxicity vary depending on nature of the study and whether cells are killed or simply have their metabolism altered.CYTOTOXICITY ASSAYS

Need for cytotoxic assaysSeveral 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 ,refinement and 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

ASSAY DESIGNExposure to study compoundExposure time may simulate the exposure levels of in vivo The penetration time required shall be consideredChoice of assay -The sensitivity required over concentration rangePhase of the growth cycle Vary with cell cycle specific drugs and non cycle specific drugs Recovery periodTo 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 compoundsChoice of cell typeDepends 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 functionASSAY DESIGN Contd..

Three-dimensional cell cultures in toxicology contd.,

Three-dimensional cell cultures in toxicology contd.,

Three-dimensional cell cultures in toxicology contd.,

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.

Assays Based on Cell Proliferation assessing effect of agents on cell counts- Can be adopted for small samples in early stage of testing

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, 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

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

APPLICATION OF CYTOTOXIC ASSAYSAnticancer drug screening MTT based or Sulforhodamine B basedPredictive drug testing for tumours - Measuring the chemo sensitivity of cells derived from patients tumour to design a chemotherapeutic regimeTesting pharmaceuticalsTransformation and mutagenesisAnchorage independenceReduced density limitation of cell proliferationMutagenesis Sister chromatid exchange assayTesting for carcinogenicity expression analysis Testing for Inflammatory responses to pharmaceuticals, cosmetics or xenobioitics -(Kits Epiderm(MarTek), Episkin (Saduc),Skin Ethic)Drug interaction studies

Culture Episkin is an invitroreconstructed human epidermis from normal human keratinocytes cultured on a collagen matrix at the air-liquid interface. This model is histologically similar to the in vivo human epidermis. The human keratinocytes come from mammary samples obtained from healthy consenting donors during plastic surgery. HIV 1 & 2,B and Chepatitis tests are carried out on the donor bloodsas well as verification of the bacteriological & fungal sterility of the cells and absence of mycoplasma. Episkin is an invitroreconstructed human epidermis from normal human keratinocytes cultured on a collagen matrix at the air-liquid interface. This model is histologically similar to the in vivo human epidermis.The human keratinocytes come from mammary samples obtained from healthy consenting donors during plastic surgery. Epidermis in vivoAfter 3 days of immersed culture conditions, the epidermis is airlifted during 10 days allowing differentiation and formation of a horny layer.

EpiDermTM Tissue ModelMatTek's patented EpiDermTM System consists of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis. These "ready-to-use" tissues, also known generically as reconstructed human epidermis (RhE), are cultured on specially prepared cell culture inserts using serum free medium, attain levels of differentiation on the cutting edge of in vitro skin technology. Ultrastructurally, the EpiDermTM Skin Model closely parallels human skin, thus providing a useful in vitro means to assess dermal irritancy and toxicology.

Formalin fixed, paraffin embedded, H&E stained

LIMITATIONS OF IINVITRO CYTOTOXIC ASSAY Pharmacokinetics : Difficult to recreate the complex invivo milieu 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 techniquesMetabolism :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 simulate invitro - Need for model to measure tissue responses like inflammation fibrosis, etc and systemic responses like pyrexia vascular dilatation required-organotypic culture my help overcome this

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

4.. http://www.promega.in/resources/product-guides-and-selectors/protocols-and-applications-guide/

Thank You