Cell culture technique and Cell culture technique and its implicationits implication

341 Zoo341 Zoo

Dr. Gamal BadrDr. Gamal Badr

Associate professorAssociate professor

History of tissue cultureHistory of tissue culture 1885: Roux maintained embryonic chick cells in saline1885: Roux maintained embryonic chick cells in saline 1965:Ham introduced serum free culture medium that support 1965:Ham introduced serum free culture medium that support

living cellsliving cells 1965: Harris & Watkins successfully fused human and mouse 1965: Harris & Watkins successfully fused human and mouse

cells by viruscells by virus 1975: Kohler & Milstein produced the first Hybridomas capable 1975: Kohler & Milstein produced the first Hybridomas capable

of secreting monoclonal antibodies.of secreting monoclonal antibodies. 1978: Sato developed a serum free medium with a cocktail of 1978: Sato developed a serum free medium with a cocktail of

hormone and growth factorshormone and growth factors !982: human insulin is produced!982: human insulin is produced 1985: human growth factors were produced1985: human growth factors were produced 1986: lymphoblastoid gamaIFNlicences1986: lymphoblastoid gamaIFNlicences 1987:tissue type plasminogen activator(tPA) became 1987:tissue type plasminogen activator(tPA) became

commercially availablecommercially available 1989:Recombinant erythropoitin in trial1989:Recombinant erythropoitin in trial 1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF, 1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF,

mAbs)mAbs)

What is cell culture?What is cell culture? Cells, previously growing in a human or animal Cells, previously growing in a human or animal

modified to grow in plastic or glassmodified to grow in plastic or glass In the body = In the body = in vivoin vivo On plastic or glass = On plastic or glass = in vitroin vitro

Kept in an incubator to stay at body temperatureKept in an incubator to stay at body temperature

We use special media with nutrients so the cells We use special media with nutrients so the cells can grow and dividecan grow and divide

What can we do with cells?What can we do with cells? Test pharmaceutical drugsTest pharmaceutical drugs Watch disease mechanismsWatch disease mechanisms

Design potential treatmentsDesign potential treatments

Observe the regenerative processObserve the regenerative process How do cells and tissues repair themselves after How do cells and tissues repair themselves after

damage from illness or injury?damage from illness or injury?

Observe the developmental processObserve the developmental process

The “Do’s” and “Don’ts” of cell cultureThe “Do’s” and “Don’ts” of cell culture The Do’sThe Do’s• Use personal protective equipment (PPE)Use personal protective equipment (PPE)• Wear dedicated PPE for tissue culture facility and keep separate from PPE in Wear dedicated PPE for tissue culture facility and keep separate from PPE in

the general lab environment.the general lab environment.• Keep all work surfaces free of clutter.Keep all work surfaces free of clutter.• Correctly label reagents including flasks, medium.Correctly label reagents including flasks, medium.• Only handle one cell line at a time.Only handle one cell line at a time.• Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol).Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol).• Wherever possible maintain separate bottles of media for each cell line.Wherever possible maintain separate bottles of media for each cell line.• Examine cultures and media daily for evidence of gross bacterial or fungal Examine cultures and media daily for evidence of gross bacterial or fungal

contamination.contamination.• Quality control all media and reagents prior to use.Quality control all media and reagents prior to use.• Ensure that incubators, cabinet, centrifuges and microsocpes are cleaned.Ensure that incubators, cabinet, centrifuges and microsocpes are cleaned.• Test cells for mycoplasma on a regular basis.Test cells for mycoplasma on a regular basis.

Cell Culture ProtocolsCell Culture Protocols

The Don’tsThe Don’ts

• Do not continuously use antibiotics in culture medium.Do not continuously use antibiotics in culture medium.• Don’t allow waste to accumulate particularly within the incubators Don’t allow waste to accumulate particularly within the incubators

and culture area.and culture area.• Don’t have too many ppl in the lab at any one time.Don’t have too many ppl in the lab at any one time.• Don’t handle cells from unauthenticated sources in the main cell Don’t handle cells from unauthenticated sources in the main cell

culture suite.culture suite.• Avoid keeping cell line continually in culture without returning to Avoid keeping cell line continually in culture without returning to

frozen stock.frozen stock.• Avoid cell culture becoming fully confluent. Always sub-culture 70-Avoid cell culture becoming fully confluent. Always sub-culture 70-

80% confluency or as advised on ECACC’s cell culture data sheet.80% confluency or as advised on ECACC’s cell culture data sheet.• Don’t allow media to go out of date.Don’t allow media to go out of date.• Avoid water baths dirty.Avoid water baths dirty.• Don’t allow essential equipment to become out of ccalibration.Don’t allow essential equipment to become out of ccalibration.

Technique and instrumentTechnique and instrumentLaminar flow

Carbon dioxide incubatorCarbon dioxide incubator

MicroscopeMicroscope

Tissue culture WareTissue culture Ware

Culture Media SterilizationCulture Media Sterilization

Cell counting

Changing MediumChanging Medium

Passaging cells (subculturing cells)Passaging cells (subculturing cells) Process of diluting cell number in Process of diluting cell number in

order to keep cells actively growingorder to keep cells actively growing For adherent cells, when they cover For adherent cells, when they cover

the tissue culture dish, they need to be the tissue culture dish, they need to be passagedpassaged Otherwise, the cells will become Otherwise, the cells will become

unhealthy and stop growingunhealthy and stop growing

Other miscellaneous EquipmentOther miscellaneous Equipment

Fridge Freezer for storing mediumFridge Freezer for storing medium Liquid nitrogen Container for cryopreservation Liquid nitrogen Container for cryopreservation

of cellsof cells Incubator for warming up of the mediumIncubator for warming up of the medium

Bench centrifuges to separate out cell pellet.Bench centrifuges to separate out cell pellet.

Sterile techniqueSterile technique Procedures by which cultures are manipulated Procedures by which cultures are manipulated

without infecting the worker or contaminating without infecting the worker or contaminating the cultures or the laboratory environmentthe cultures or the laboratory environment

Important for the cell cultureImportant for the cell culture You want to be sure you are growing only the cells You want to be sure you are growing only the cells

you want to grow a single unwanted cell can ruin an you want to grow a single unwanted cell can ruin an experiment or a multimillion $ production runexperiment or a multimillion $ production run

Important for the lab workerImportant for the lab worker Some cultured cells can pose health threats to Some cultured cells can pose health threats to

workers if they are inhaled, ingested, or absorbed--workers if they are inhaled, ingested, or absorbed--sterile technique prevents exposure of the worker to sterile technique prevents exposure of the worker to cultured cellscultured cells

Sterile technique: BacteriaSterile technique: Bacteria Media is sterilized in an autoclaveMedia is sterilized in an autoclave

Very high pressure and temperature kills Very high pressure and temperature kills Minimizing contamination through awarenessMinimizing contamination through awareness

Inoculation loop, pipets, pipet tips, etc. should never touch Inoculation loop, pipets, pipet tips, etc. should never touch contaminating surfacescontaminating surfaces

Containers holding media and other cell additives should be Containers holding media and other cell additives should be kept closed until needed, then opened briefly kept closed until needed, then opened briefly

Tubes and flasks are “flamed” whenever they are openedTubes and flasks are “flamed” whenever they are opened The purpose of flaming is not to sterilize, but to warm the tube The purpose of flaming is not to sterilize, but to warm the tube

and create warm air convection currents up and away from the and create warm air convection currents up and away from the opening. This "umbrella" of warm, rising air will help to prevent opening. This "umbrella" of warm, rising air will help to prevent the entrance of dust particles carrying contaminating microbesthe entrance of dust particles carrying contaminating microbes

Sterile technique: Tissue cultureSterile technique: Tissue culture Media is usually sterilized by filtrationMedia is usually sterilized by filtration

Standard biological filters are 0.22 Standard biological filters are 0.22 m - 0.45 m - 0.45 m; these remove m; these remove most microbes by trapping them in the filtermost microbes by trapping them in the filter

This does not remove all microbes (such as mycoplasm), and will not This does not remove all microbes (such as mycoplasm), and will not remove virusesremove viruses

Unlike bacterial media, animal cell media cannot be autoclaved, Unlike bacterial media, animal cell media cannot be autoclaved, because this would destroy many of the growth factors and other because this would destroy many of the growth factors and other molecules needed for cell growthmolecules needed for cell growth

Working with cells in a laminar flow hoodWorking with cells in a laminar flow hood HEPA filterHEPA filter

Disinfect Disinfect 70% Ethanol is sprayed in hood, onto bottles entering hood70% Ethanol is sprayed in hood, onto bottles entering hood

Minimizing contamination through awarenessMinimizing contamination through awareness Inoculation loop, pipets, pipet tips, etc. should never touch Inoculation loop, pipets, pipet tips, etc. should never touch

contaminating surfacescontaminating surfaces Containers holding media and other cell additives should be Containers holding media and other cell additives should be

kept closed until needed, then opened brieflykept closed until needed, then opened briefly

Sterilization methodsSterilization methods AutoclaveAutoclave

Applies heat under high pressure; this increases the boiling point Applies heat under high pressure; this increases the boiling point of water to 121ºC (normal boiling point of water is 100ºC)of water to 121ºC (normal boiling point of water is 100ºC)

15-20 min. is sufficient to kill most microbes15-20 min. is sufficient to kill most microbes FiltrationFiltration

Large volumes: suction filterLarge volumes: suction filter Small volumes: syringe filterSmall volumes: syringe filter

UV radiationUV radiation Causes mutations to form in the DNA of microbes, causing Causes mutations to form in the DNA of microbes, causing

genetic damage and eventual deathgenetic damage and eventual death Used to sterilize surfaces (such as the surface of laminar flow Used to sterilize surfaces (such as the surface of laminar flow

hoods)hoods)

What is in the media?What is in the media?

Dulbecco’ Modified Eagle’s Media (DMEM)Dulbecco’ Modified Eagle’s Media (DMEM) Contains glucose, some proteins, and essential saltsContains glucose, some proteins, and essential salts Contains a pH indicator (Contains a pH indicator (phenol redphenol red)) Media looks Media looks

pink/red at pH 7.2pink/red at pH 7.2 Acidic Acidic -yellow or orange (cell growth, -yellow or orange (cell growth,

bacterial growth)bacterial growth) Basic Basic -purple (no cell growth, not enough -purple (no cell growth, not enough

COCO22))

Media preparationMedia preparation Process of combining and sterilizing ingredients of a Process of combining and sterilizing ingredients of a

particular mediumparticular medium Proper media prep is essential for cell culture systems!Proper media prep is essential for cell culture systems!

If the media is lacking any components, the cells would If the media is lacking any components, the cells would either die or be unhealthyeither die or be unhealthy

If the media is not properly sterilized, cells will be If the media is not properly sterilized, cells will be contaminated with microorganismscontaminated with microorganisms

If glassware, pipettes, etc. used to prepare media are not If glassware, pipettes, etc. used to prepare media are not properly cleaned, cell cultures can be contaminated with properly cleaned, cell cultures can be contaminated with chemical residueschemical residues

As a result, the cells would not produce the desired product As a result, the cells would not produce the desired product effectivelyeffectively

More media componentsMore media components

Antibiotics (penicillin and streptomycin)Antibiotics (penicillin and streptomycin) Prevent bacterial contaminationPrevent bacterial contamination

Salts and buffersSalts and buffers To simulate To simulate in vivoin vivo environment environment

SerumSerum Portion of blood after the cells and fibers have clottedPortion of blood after the cells and fibers have clotted From cow, horse, sheepFrom cow, horse, sheep added to media as a nutrient source for growing cellsadded to media as a nutrient source for growing cells

Lipids, proteinsLipids, proteins

Phosphate Buffered Saline Phosphate Buffered Saline Used to wash/remove excess serum that Used to wash/remove excess serum that

inhibits the function of TRED. inhibits the function of TRED. Must be warmed in the water bath before Must be warmed in the water bath before

use so cells are not shocked by cold liquid.use so cells are not shocked by cold liquid.

Trypsin EDTA Trypsin EDTA An enzyme used to detach the cells from a culture An enzyme used to detach the cells from a culture

dish.dish. EDTA binds calcium ions in the media that would EDTA binds calcium ions in the media that would

normally inhibit trypsin.normally inhibit trypsin.

BleachBleach

Used to destroy any remaining cells in Used to destroy any remaining cells in dishes and tubes before they are tossed in dishes and tubes before they are tossed in the trash can.the trash can.

Add enough to change media to clear, Add enough to change media to clear, wait 5 minutes, wait 5 minutes, rinse solution down sinkrinse solution down sink throw away the dish/flask/plate in the trash can.throw away the dish/flask/plate in the trash can.

Potential sources of contamination in Potential sources of contamination in cell culturecell culture

EquipmentEquipment Glassware, instruments, incubators.Glassware, instruments, incubators.

SolutionsSolutions Media or reagents added into mediaMedia or reagents added into media

Room airRoom air Work surfacesWork surfaces OperatorsOperators

Hands, hair, clothing, breath, etc.Hands, hair, clothing, breath, etc. Incoming cellsIncoming cells

Types of contamination in animal cell Types of contamination in animal cell culture systemsculture systems

BiologicalBiological BacteriaBacteria FungiFungi Cross-contamination by other cell culturesCross-contamination by other cell cultures

ChemicalChemical Residues left from detergents or disinfectants on Residues left from detergents or disinfectants on

glassware, pipettes, instruments, etc.glassware, pipettes, instruments, etc. Metal ions, other impurities in waterMetal ions, other impurities in water Endotoxin: highly bioreactive part of the cell wall of Endotoxin: highly bioreactive part of the cell wall of

some types of bacteria (endotoxin molecules are shed some types of bacteria (endotoxin molecules are shed from bacteria and are left behind even after bacteria die)from bacteria and are left behind even after bacteria die)

Characteristics of microbial Characteristics of microbial contamination in cell culturescontamination in cell cultures

pHpH Sudden change in pH is often a strong Sudden change in pH is often a strong

indicator of contaminationindicator of contamination TurbidityTurbidity

Media looks cloudyMedia looks cloudy Microscopic evaluationMicroscopic evaluation

Can see individual microorganisms, often Can see individual microorganisms, often because their motion can be seen easily under because their motion can be seen easily under the microscopethe microscope

Further detection of contamination in cell Further detection of contamination in cell culturescultures

MycoplasmaMycoplasma Smallest free-living prokaryotesSmallest free-living prokaryotes Not killed easily by many antibioticsNot killed easily by many antibiotics Contamination can’t be seen by microscopic Contamination can’t be seen by microscopic

evaluationevaluation Mycoplasma testing should be done Mycoplasma testing should be done

routinely (several tests are available)routinely (several tests are available) Long-term effects of mycoplasma contamination include Long-term effects of mycoplasma contamination include

reduced growth rate, changes in cell shape and reduced growth rate, changes in cell shape and metabolism, and chromosome abnormalitiesmetabolism, and chromosome abnormalities

EndotoxinEndotoxin LAL test: an extract from the blood of LAL test: an extract from the blood of

horseshoe crabs is used to test for endotoxin horseshoe crabs is used to test for endotoxin (horseshoe (horseshoe crab blood contains a protein that binds endotoxin & can be crab blood contains a protein that binds endotoxin & can be detected)detected)

Minimizing contaminationMinimizing contamination Contamination is a fact of life when dealing Contamination is a fact of life when dealing

with cell cultureswith cell cultures Very difficult to prevent entirely, but good lab Very difficult to prevent entirely, but good lab

practices can keep contamination incidents to a practices can keep contamination incidents to a minimumminimum

Proper cleaning and sterilization of glassware, Proper cleaning and sterilization of glassware, pipettes, and other lab instruments. pipettes, and other lab instruments.

Practicing sterile technique when working with Practicing sterile technique when working with cell culturescell cultures

Types of cell cultureTypes of cell culture

Primary Cell culturePrimary Cell culture

Continuous Cell linesContinuous Cell lines

Adherent vs Suspension cells for tissue Adherent vs Suspension cells for tissue cultureculture

Adherent cellsAdherent cells: cells grow in a : cells grow in a single layer (called a monolayer) single layer (called a monolayer) attached to the tissue culture dishattached to the tissue culture dish Cell growth is limited by available Cell growth is limited by available

surface area on which cells can growsurface area on which cells can grow To passage adherent cells, the cells To passage adherent cells, the cells

must be released from the dish (done must be released from the dish (done either enzymatically, chemically, or either enzymatically, chemically, or mechanically)mechanically)

Suspension cellsSuspension cells : cells are : cells are suspended in liquid as single cells or suspended in liquid as single cells or as free-floating clumps of a few cellsas free-floating clumps of a few cells To passage suspension cell cultures, a To passage suspension cell cultures, a

proportion of the cells in culture are proportion of the cells in culture are diluted into a larger volume of mediumdiluted into a larger volume of medium

Secondary or continuous Secondary or continuous Cell Lines Cell Lines

Attached Cell Lines    

Name Species and tissue of origin Morphology

MRC-5  (Prod. No. 84101801) Human lung Fibroblast

HELA  (Prod. No. 93021013) Human cervix Epithelial

VERO  (Prod. No. 84113001) African Green Monkey Kidney Epithelial

NIH 3T3  (Prod. No. 93061524) Mouse embryo Fibroblast

L929  (Prod. No. 85011425) Mouse connective tissue Fibroblast

CHO  (Prod. No. 85050302) Chinese Hamster Ovary Fibroblast

BHK-21  (Prod. No. 85011433) Syrian Hamster Kidney Fibroblast

HEK 293  (Prod. No. 85120602) Human Kidney Epithelial

HEPG2  (Prod. No. 85011430) Human Liver Epithelial

BAE-1  (Prod. No. 88031149) Bovine aorta Endothelial

SUSPENSION Cell linesSUSPENSION Cell linesSuspension Cell Lines    

Name Species and tissue of origin Morphology

NSO  (Prod. No. 85110503) Mouse myelomaLymphoblastoid-

like

U937  (Prod. No. 85011440) Human Hystiocytic

Lymphoma Lymphoblastoid

Namalwa  (Prod. No. 87060801) Human Lymphoma Lymphoblastoid

HL60  (Prod. No. 98070106) Human LeukaemiaLymphoblastoid-

like

WEHI 231  (Prod. No. 85022107) Mouse B-cell Lymphoma Lymphoblastoid

YAC 1  (Prod. No. 86022801) Mouse Lymphoma Lymphoblastoid

U 266B1  (Prod. No. 85051003) Human Myeloma Lymphoblastoid

SH-SY5Y  (Prod. No. 94030304) Human neuroblastoma Neuroblast

Storing and maintaining Continuous Storing and maintaining Continuous Cell LinesCell Lines

Advantages of Cell CultureAdvantages of Cell Culture

Cell types kept Constant and homogenous.Cell types kept Constant and homogenous. There is direct access to the cells so effect of There is direct access to the cells so effect of

toxicity of drug and chemicals studied without toxicity of drug and chemicals studied without being lost to other tissues and excreted.being lost to other tissues and excreted.

Replace/ reduce the number of animals. Legal, Replace/ reduce the number of animals. Legal, moral, ethical issues?(animal rights Group).moral, ethical issues?(animal rights Group).

Control of the environment (pH, osmotic Control of the environment (pH, osmotic pressure, temperature, oxygen and Carbon pressure, temperature, oxygen and Carbon dioxide tension.) dioxide tension.)

Disadvantages of Cell CultureDisadvantages of Cell Culture

Contamination can be Chemical (culture Contamination can be Chemical (culture medium) Or Biological (adding antibiotics).medium) Or Biological (adding antibiotics).

Finding a Happy Environment.Finding a Happy Environment.

De-differentiationDe-differentiation

Origin of CellsOrigin of Cells

Major differences from Major differences from in-vivoin-vivo

Implication for Cell CultureImplication for Cell Culture Model system (many specialized functions are restored)Model system (many specialized functions are restored) Toxicity Test (viability of cells)Toxicity Test (viability of cells) Cancer researchCancer research VirologyVirology Cell Based manufacturingCell Based manufacturing Genetic councellingGenetic councelling Genetic engineeringGenetic engineering Drug screening & developmentDrug screening & development Gene TherapyGene Therapy

Production of monoclonal antibodiesProduction of monoclonal antibodies

Tissue EngineeringTissue Engineering

Carbon nanotube Carbon nanotube scaffoldingscaffolding

Name of Name of scaffoldigscaffoldig

Made up ofMade up of

NanofiberNanofiber Like carbonLike carbon

TextileTextile PolyglycolidePolyglycolide

Gas FoamGas Foam Foam like Foam like structure due structure due to CO2 gasto CO2 gas

Growing cells in 3D Forming Growing cells in 3D Forming Tissue in BioreactorTissue in Bioreactor

Application of Monoclonnal Application of Monoclonnal AntibodiesAntibodies

IMMUNOLOCALIZATIONIMMUNOLOCALIZATION IMMUNOBlottingIMMUNOBlotting Cancer TreatmentCancer Treatment

ImmunolocalizationImmunolocalization

ImmunoblottingImmunoblotting

Cancer TreatmentCancer Treatment

Stem cell researchStem cell research

Embryonic Cell LinesEmbryonic Cell Lines

Adult Cell LineAdult Cell Line

Homeiopoitic Stem CellHomeiopoitic Stem Cell

Different types of stem cellsDifferent types of stem cells

Early embryonic stem cellsEarly embryonic stem cells TotipotentTotipotent: can become any kind of cell in the body: can become any kind of cell in the body

Blastocyst embryonic stem cellsBlastocyst embryonic stem cells PluripotentPluripotent: can become virtually any kind of cell : can become virtually any kind of cell

in the bodyin the body Umbilical cord stem cells: Umbilical cord stem cells:

MultipotentMultipotent: can differentiate into only a limited : can differentiate into only a limited number of cell typesnumber of cell types

Adult stem cells:Adult stem cells: MultipotentMultipotent: can differentiate into only a limited : can differentiate into only a limited

number of cell typesnumber of cell types

Future of stem cell-mediated therapyFuture of stem cell-mediated therapy {stem cells to replace damaged {stem cells to replace damaged organsorgans}}

Stem cells are induced to Stem cells are induced to differentiate into a cell type that is differentiate into a cell type that is damaged or missing in the patientdamaged or missing in the patient

Cells are grown on tissue-Cells are grown on tissue-promoting matrix or scaffoldingpromoting matrix or scaffolding

Healthy tissue is transplanted into Healthy tissue is transplanted into patient--unlike other organ patient--unlike other organ transplants, tissue is an immune transplants, tissue is an immune match to patient, so no match to patient, so no immunosuppression drugs would immunosuppression drugs would be necessarybe necessary

Medical applications of embryonic stem Medical applications of embryonic stem cell researchcell research

DiabetesDiabetes Caused by body’s Caused by body’s

destruction of insulin-destruction of insulin-producing cells in the producing cells in the pancreas.pancreas.

Researchers in Spain Researchers in Spain (2001) used mouse (2001) used mouse embryonic stem cells that embryonic stem cells that they differentiated into they differentiated into glucose-responsive, glucose-responsive, insulin-producing cells; insulin-producing cells; the cells reversed diabetes the cells reversed diabetes symptoms when injected symptoms when injected into the spleens of mice.into the spleens of mice.

Animal cloningAnimal cloning

Therapeutic cloning

Remove nucleusfrom egg cell

Add somatic cellfrom adult donor

Grow in culture to produce anearly embryo (blastocyst)

Implant blastocyst insurrogate mother

Remove embryonic stemcells from blastocyst andgrow in culture

Induce stem cells toform specialized cells(therapeutic cloning)

Clone of donor is born(reproductive cloning)

Donorcell

Nucleus fromdonor cell

Reproductive cloning

The successful "cloning" of mammals has resulted in a flurry of research, Dolly July ‘96