Introduction to tissue culture cell line

Dr Ghada Abou El-Ella

Definition of Tissue Culture

Tissue culture is the branch of biology in which tissues or cells of higher animals and plants are grown artificially in a controlled environment.

Such studies were undertaken in the hope that the behavior of various body components could be studied and their potentialities more readily analyzed under the simpler and more readilymanipulated conditions possible in the test tube.

Definition of Tissue CultureTissue culture is the term used for Tissue culture is the term used for ““the process of growing cells artificially in the laboratory””

Tissue culture involves both plant and animal Tissue culture involves both plant and animal cells cells

Tissue culture produces clones, in which all Tissue culture produces clones, in which all product cells have the same product cells have the same genotypegenotype (unless (unless affected by mutation during culture)affected by mutation during culture)

History of tissue cultureThe 1st culture of animal tissue was done in 1907 by The 1st culture of animal tissue was done in 1907 by Ross Harrison where he cultured nerve tissue of frog in Ross Harrison where he cultured nerve tissue of frog in blood clot also cultured at room Temp cold blooded.blood clot also cultured at room Temp cold blooded.

Tissue culture had its origins at the beginning of the 20th Tissue culture had its origins at the beginning of the 20th century with the work of century with the work of GottleibGottleib HaberlandtHaberlandt (plants) (plants) and Alexis Carrel (animals) and Alexis Carrel (animals)

The first commercial use of plant The first commercial use of plant clonalclonal propagationpropagation on on artificial media was in the germination and growth of artificial media was in the germination and growth of orchid plants, in the 1920orchid plants, in the 1920’’s s

History of tissue cultureIn the 1950In the 1950’’s and 60s and 60’’s there was a great deal of s there was a great deal of research, but it was only after the development of a research, but it was only after the development of a reliable artificial mediumreliable artificial medium

A more recent advance is the use of plant and A more recent advance is the use of plant and animal tissue culture along with genetic animal tissue culture along with genetic modification using viral and bacterial vectors and modification using viral and bacterial vectors and gene guns to create genetically engineered gene guns to create genetically engineered organisms organisms

Basic requirements for plant and animal tissue culture

Both plant and animal tissue culture have several critical requirements:Appropriate tissue (some tissues culture better than others) A suitable growth medium containing energy sources and inorganic salts to supply cell growth needs. This can be liquid or semisolid

Basic requirements for plant and animal tissue culture

Aseptic (sterile) conditions, as microorganisms grow much more quickly than plant and animal tissue and can over run a culture Growth regulators as auxins & cytokinins in plants or it will be provided in serum as in animalsFrequent sub culturing to ensure adequate nutrition and to avoid the build up of waste metabolites

Animal tissue /cell culture Animal tissue /cell culture differences from plant tissue culturedifferences from plant tissue cultureAnimal cell lines have limited numbers of cell cycles Animal cell lines have limited numbers of cell cycles before they begin to degradebefore they begin to degradeAnimal cells need frequent sub culturing to remain Animal cells need frequent sub culturing to remain viable viable Animal Tissue culture media need in addition to Animal Tissue culture media need in addition to inorganic salts, energy sources, amino acids, vitamins, inorganic salts, energy sources, amino acids, vitamins, etc., they require the addition of serum (bovine serum is etc., they require the addition of serum (bovine serum is very common, but others are used) very common, but others are used) Animal tissue cultures can pose biohazard concerns, and Animal tissue cultures can pose biohazard concerns, and cultures require special inactivation with hypochlorite cultures require special inactivation with hypochlorite (e.g. (e.g. JanolaJanola, , ChloroxChlorox, etc.) and then incineration , etc.) and then incineration

Uses of Animal Tissue Culture

Growing viruses - these require living host cells Making monoclonal antibodies, used for diagnosis and research Studying basic cell processes Genetic modification & analysis ‘Knockout’ technology - inactivating certain genes and tracing their effects Providing DNA for the Human Genome Project (and other species’ genome projects)

Advantages of Tissue Culture

Need fewer animalsNeed fewer animalsCan look at direct effect of a substance on a cell type Can look at direct effect of a substance on a cell type without interaction with any other substance.without interaction with any other substance.Homogeneity of cell typeHomogeneity of cell typeEconomyEconomy

aa-- where we used fewer reagentswhere we used fewer reagentsbb-- less costly than maintaining animalsless costly than maintaining animals

N.BN.B(A)(A) Cancer cells divide forever, i.e are immortal.Cancer cells divide forever, i.e are immortal.(B)(B) Cancer cells don't show contact inhibition.Cancer cells don't show contact inhibition.

Primary culture How to get cells from the animal

A) A) ExplantExplant method:method:-Cut block of tissue from animal-Let cells migrate out, remove tissue

B) B) DisaggregationDisaggregation method:method:--Cut small tissues from animalCut small tissues from animal--Incubate in Incubate in trypsintrypsin--Agitate to disaggregate tissue to cell suspensionAgitate to disaggregate tissue to cell suspension--Plate in culture mediumPlate in culture medium

C) C) Can count cells in Can count cells in hemacytomerhemacytomer before platingbefore plating--Count # cells in 0.l Count # cells in 0.l µµl , multiply by 10,000 to get cells / ml of mediuml , multiply by 10,000 to get cells / ml of medium

viable cells / total cells plated = plating efficiencyviable cells / total cells plated = plating efficiency

TYPES OF CELLS GROWN IN CULTURE

Tissue culture is often a generic term that refers to both organ culture and cell culture.Cell cultures are derived from either primary tissue explants or cell suspensions. Primary cell cultures typically will have a finite life span in culture .Continuous cell lines are, by definition, abnormal and are often transformed cell lines.

II. WORK AREA AND EQUIPMENTA. Laminar flow hoods.

There are two types of laminar flow hoods, vertical and horizontal.The vertical hood( biology safety cabinet): is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment.Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures.

A. Laminar flow hoods

Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion. NOTE: these are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work.

A. Laminar flow hoods

The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light. Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10-20 minutes before being used.Wipe down all surfaces with ethanol before and after each use. Keep the hood as free of clutter as possible because this will interfere with the laminar flow air pattern.

B. CO2 IncubatorsThe cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained.Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times.

C. Microscopes

Inverted phase contrast microscopes are used Inverted phase contrast microscopes are used for visualizing the cells. Microscopes should for visualizing the cells. Microscopes should be kept covered and the lights turned down be kept covered and the lights turned down when not in use. Before using the microscope when not in use. Before using the microscope or whenever an objective is changed, check or whenever an objective is changed, check that the phase rings are aligned.that the phase rings are aligned.

D. VesselsCells require a nontoxic, biologically inert, and Cells require a nontoxic, biologically inert, and optically transparent surface that will allow cells optically transparent surface that will allow cells to attach and allow movement for growth. to attach and allow movement for growth. The most convenient vessels are speciallyThe most convenient vessels are specially--treated treated polystyrene plastic that are supplied sterile and are polystyrene plastic that are supplied sterile and are disposable. disposable. Suspension cells are shaken, stirred, or grown in Suspension cells are shaken, stirred, or grown in vessels identical to those used for anchoragevessels identical to those used for anchorage--dependent cells. dependent cells.

III. Media and growth requirementsIII. Media and growth requirementsPhysiological parameters

TemperatureTemperature -- 3737ººC for cells from C for cells from homeothermhomeothermpHpH -- 7.27.2--7.5 7.5 humidityhumidity is requiredis requiredgas phasegas phase -- bicarbonate conc. and CO2 tension in bicarbonate conc. and CO2 tension in equilibriumequilibriumvisible lightvisible light -- can have an adverse effect on cells; can have an adverse effect on cells; light induced production of toxic compounds can light induced production of toxic compounds can occur in some media; cells should be cultured in the occur in some media; cells should be cultured in the dark and exposed to room light as little as possible;dark and exposed to room light as little as possible;

Medium requirementsMedium requirements

Bulk ionsBulk ions -- Na, K, Ca, Mg, Na, K, Ca, Mg, ClCl, P, , P, BicarbBicarb or CO2or CO2Trace elements Trace elements -- iron, zinc, seleniumiron, zinc, seleniumsugars sugars -- glucose is the most commonglucose is the most common

amino acidsamino acids -- 13 essential13 essentialvitaminsvitamins -- B, etc.B, etc.cholinecholine, , inositolinositol..

Growth and morphology

Visually inspect cells frequently. Cell culture is sometimes more an art than a science. Get to know what makes your cells happy!!! Frequent feeding is important for maintaining the pH balance of the medium and for eliminating waste products. Cells do not typically like to be too confluent so they should be subcultured when they are in a semi-confluent state. In general, mammalian cells should be handled gently. They should not be vortexed, vigorously pipetted or centrifuged at greater than 1500 g.

Cell feeding

prewarmed media and have cells out of the incubator for as little time as possible. a. Suspension cultures: Feeding and subculturingsuspension cultures are done simultaneously. About every 2-3 days, dilute the cells into fresh media. b. Adherent cells. About every 2-3 days, pour off old media from culture flasks and replace with fresh media.