Tissue CultureTissue Culture(Cell Culture)(Cell Culture)

Toxicology Testing In VitroToxicology Testing In Vitro

IntroductionIntroduction

• The concept of tissue culture is not new!! (1885)

• Toxicological test LDLD5050 methods are necessary to assess the

hazard and safety of various substances such as medicine,

consumer products and industrial chemicals

• In recent years animal or replace animal use

ContentContent• Def.

• Historical usage

• Use of tissue culture

• Advantages of tissue culture

• Types of tissue culture

• Requirements of tissue culture

• Work area and Equipments

• Cytotoxicological studies

DefinitionDefinition

Tissue culture and Plant tissue culture:

Is a term used for the process of keeping

tissue alive and growing artificially in the lab

Using Liquid or Semisolid growth media

Tissue culture means :

the ability of tissues to survive and grow outside the body in an artificial environment (by using liquid,semisolid or solid growth media such as broth or agar.)

Historical UsageHistorical Usage

• Firstly, they chose the frog as a source of tissue because it was a cold-blooded animal, and

consequently incubation was not required.

• Then at 1885 they used the medullary plate of an embryonic chicken and maintained it in

warm saline solution for several days.

• In medical science medical science it is carried into warm-blooded animals, where normal and pathological

development are close to human using:

Pure strains of rodents.

Embryonated hen’s egg.

• In 1902 Leo Loeb In 1902 Leo Loeb placed fragments of the skin (tissue transplantation)(tissue transplantation) from the embryos of

guinea pigs in agar and in coagulated serum and inserted them into adult guinea pigs.

Uses of tissue cultureUses of tissue culture• Used in culturing of specific plants.

• Investigation of the normal physiology and biochemistry of the cell (development

of nerve cell).

• Study the effect of various chemicals or drugs on specific cell types

(cytotoxicological- effects)

• Study the protective or curative effect of certain substance.

• Used for growing viruses and production of anti viral vaccines.

• Cancer research

• Used medically to generate artificial tissues.(liver, articular cartilage & skin

grafting)

Advantages of Tissue CultureAdvantages of Tissue Culture

• Control of the surrounding environment

• Sample is Homogenous

• Use the specific cells

• Cheaper!

• Result in short time

• Humane!

TypesTypes1.1. Primary culturesPrimary cultures

2.2. Secondary cultures (cell line)Secondary cultures (cell line)

1. Primary cultures:1. Primary cultures:

it refers to cultures prepared directly from animals. it refers to cultures prepared directly from animals.

• Organ culture• Slice tissue culture (small pieces of tissue are

cultured in enriched media)• Histotypic (histoculture) : (high density slice

culture of one cell type)• Primary cell culture (single cells which could

be plated on a coated surface)• Reaggregated culture (single cell reaggregate

into small balls).

2. Secondary cultures:-2. Secondary cultures:-

Cells that survive the crisiscrisis and continue to growgrow are

referred to cell line.cell line.

Continuous cell line (cells can passage indefinitely

and express stable phenotype)

Transformed cell line (from tumor cells)

Clonal cell line (cells could be cloned in continuous cell

lines to obtain genetically homogenous population)

• Plating density : number of cells per volume of of tissue culture.

• Cells can grow either on suspension or adherent culture.

Critical RequirementsCritical Requirements

1.1. Appropriate tissueAppropriate tissue

2. A suitable Growth mediumsuitable Growth medium

3. Sterile condition (Aseptic)(Aseptic)

4. Specific medium (Growth regulatory)(Growth regulatory)

5.5. Frequent sub culturingFrequent sub culturing

Work area and EquipmentWork area and Equipment• Laminar flow hood

• CO2 incubator

• Inverted microscope

• Dissecting microscope

• Inverted fluorescence microscope

• Liquid nitrogen containers

• Vessels (Roller bottles, Screw cap flasks, multiwell plate)

• Hot air oven, Autoclave, Water bath, Refrigerator, Deep freezer,

Water distiller, Vacuum pump, Pipette, pH meter, syringe filters,

Magnetic stir plate…..etc

HoodHood

CoCo22 incubator incubator

Inverted MicroscopeInverted Microscope

Liquid Nitrogen ContainerLiquid Nitrogen Container

Cryo tubesCryo tubes

Roller bottlesRoller bottles

Screw cap flasksScrew cap flasks

Single use filterSingle use filter

Bottle top filterBottle top filter

Tissue culture plate (24- well)Tissue culture plate (24- well)

96 96 wellwell6 well6 well

PipettePipette

Toxicological StudiesToxicological Studies Morphomatic analysis (size, number)

Estimation of some metabolites or enzymes

Measurement of viability of the cell

a)Dye uptake (diacetyl fluorescien and fluorescien)

b)Chromium release from dead cells

Estimation of Cytokines

Detection of DNA change

Estimation of LC50

Evaluation of the curative and protective

effects of substances against toxicants