animalcelllines-140615061128-phpapp02.ppt

8/10/2019 animalcelllines-140615061128-phpapp02.ppt

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CELL CULTURING

Girija Maganti

M.Pharm

(pharmacology)


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What is Cell Culture?

Cell culture refers to the removal of cellsfrom an animal or plant and their subsequentgrowth in a favorable artificial environment.


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Mouse, mammals,EmbryoEmbryonated Eggs

because stage of differentiation)

organ

explant

Grow in media

-monolayer-suspension cells

Cell culture

Finely cut

Finely cuttissue or explant

Enzymic digestion


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STAGES OF CULTURE

Isolated tissue

(disaggregation)

Primary cell culture(limited lifespan after certain

proliferations undergo senescence)

Finite cell cultures

continous cell lines(immortalized cell line acquires ability toproliferate indefinitely by transformation)


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Growth Curve


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ISOLATION OF TISSUES

Sterilize the site with 70% alcohol.

Remove tissue aseptically.

Transfer to the laboratory in transport medium

If delay in transporting to lab, keep at 4C forup to 72hour.


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Enzymatic disaggregation

Warm trypsin, 37C for 30 mins, cell damaged if too long

exposure.

Cold pre exposure, soak at 4C overnight and 37C for less 30

mins. Advantage: higher yield of viable cells, preserve

more cell types Other enzyme

-collagenase benefit for connective tissues and muscle

(fibrous tissue)

- pronase, dipase, DNase, hyaluronidase

Mechanical disaggregation(prevent proteolytic

damage)

Scrapping or spillage

Sieving

Syringes

DISAGGREGATION


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MEDIA AND SERUM

Common basal medium includeEagle minimal effectivemedium,Dulbeccos Modified

eagle medium,RPMI 1640,Ham

f10. The above medium includes

aminoacids,glucose,salts,vitamins and nutrients.

L-glutamate,serum,antibiotics,fungicidesare added for complete

medium.


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Serum may or may not be used Most frequently used serum-fetal calf serum

Less expensive sera such as horse or calf seracan be used.

Serum supports growth of cell.

Antibiotics and fungicides prevent microbial

contamination. Some cell types like primary cell requrie

additional supliments(collagen,fibronectin,epidermal growth factor)to attach

culture vessel and proliferate. Sterility of the medium is tested by incubatingsmall aliquot at 37c for 48 hours,ifany microialgrowth the medium should be discarded.


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CULTURE VESSELS


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MODES OF PROPAGATION

Depending on their origin, animal cells groweither as adherent monolayers or insuspension.

Adherent cells are anchorage dependent and

propagate as monolayer attached to the cellculture vessel.

Eg:cells derived from tissues

Suspension cells:proliferate without being

attached to a substratum.Eg:haemopoietic cell,cells derived from

tumours


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PRIMARY CULTURE

Capable of only limited number of cell divisions

cells that are placed in culture directly from thetissue of origin.

these are called primary cultures until the firstsubculture

Epithelial cell


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TYPES OF PRIMARY CELL CULTURE

Mouse embryos

Chick embryos

Human biopsy materials

Transplantable animal tumour

Chick embryo organ rudiments ( brain,heart, lungs, liver, kidney, spinal cord, skin,)


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SUBCULTURING

Subculturing or "splitting cells," is required toperiodically provide fresh nutrients and growingspace for continuously growing cell lines.

The frequency of subculture and the split ratio,

or density of cells plated depend on thecharacteristics of each cell line being carried.

Sub culturing -

Adherent Cells Suspension culture.


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CONTINOUS CELL LINES

After the first subculture, primary culture maybe called secondary cultures, and thereafter, ifcontinued passage is possible, a continous cellline are formed.

An established or immortalised cell line has

acquired the ability to proliferate indefinitelyeither through random mutation or deliberatemodification, such as artificial expression of the

telomerase gene.


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EXAMPLES OF ESTABLISHED CELL

LINES May be derived from Normal or Tumor cells.

Cell line Organism Origin Tissue

HeLa Human Cervical cancer

293-T Human Kidney (embryonic)

A-549 Human Lung carcinoma

ALCMurine Bone marrow

CHO Hamster Ovary

HB54 Hybridoma Hybridoma

FM3 Human Metastatic lymph node


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Mouse kidney cells

Secondary Hamsterkidney cellsPrimary cell cultures

split several times:


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INCUBATION CONDITIONS

Cell cultures should incubated in incubatorswith tightly regulated temperatures(eg:water

jacketed incubator)and co2 con.

Most cell lines grows at 37c and 5%co2 withsaturating humidity.


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STERILIZATION OF MEDIUM

The various media constituents and other reagentsused in cell cultures must be carefully sterilizedeither by autoclaving or by filtration. Heat stableconstituents tike water, salts, supplements likepeptone or tryptase etc. are autoclaved at 121C for

20 min.

But heat labile constituents like serum, trypsin,proteins, growth factors etc. must be sterilized byfiltration through a 0.2 mm porosity membranefilter. Each filtrate should be tested for sterility toavoid failure due to contamination.

.


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CRYOPRESERVATION Freeze preservation of animal cells

is now routine in all cell line banks.

A cryoprotective agent like DMSOor glycerol (at-130cis generally

added to minimize injury to cellsduring freezing and thawing.

Frozen ampoules are generally

stored in liquid nitrogenrefrigerators which are ratherconvenient and quite safe.


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Contamination

Contamination with other cell lines

(cross contamination)

Yeast

FungiViruses

Bacteria

mycoplasma


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CHARACTERISTICFEATURE O0FMICROIALCONTAMINATION

BACTERIA YEAST FUNGI

CHANGE IN PH PH DROP PH CHANGE WITHHEAVYINFECTIONS

PH CHANGESSOME TIMES

CLOUDY MEDIUM SHIMMERING INSPACES BETWEENCELLS;RODS ORCOCCI MAY BEOBSERVED

ROUND OR OVOIDPARTICLES THATBUDD OFFSMALLERPARTICLES

THINFILAMENTOUSMYCELIA;SOMETIMES CLUMPS OFSPORES

C t i ti


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Cross contamination


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APPLICATIONSModel Systems : Cell cultures provide a good model

system for studying 1) basic cell biologyandbiochemistry,2) the interactionsbetween disease-causing agents and cells, 3) the effects of drugs oncells, 4) the process and triggers for aging, and 5)nutritional studies

Drug screening and development Toxicity testing

Cancer research

Virology

Largescale production of monoclonalantibodies and vaccines

Genetic counselling

Gene therapy


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animalcelllines-140615061128-phpapp02.ppt

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