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Animal CellCulture

(SBB 4301)By

Prof Madya Dr Nakisah Mat Amin

Semester July2008/2009

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Introduction

Fundamental and applied researches. Applications of cell culture technology;

Diagnostic

Isolation and identification of agents,diseases and mechanisms.

Bioreactor Production of biological reagents/monoclonal

Abs. Stem cell research

Embryonic and adult (somatic) stem cells fortissue engineering (regenerative

medicine)/artificial organ. Drug discovery

Antiviral/anticancer/antitumor promotingactivity

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Plasticity of adult stem cells

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Embryonic Stem cells

1 2

3 4

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Risks associated

with animal cells

Viruses (including latent viruses)

Mycoplasma

Bacteria, yeast and fungi

Other pathogenic substances suchas prions

Oncogenic DNA and otheroncogenic materials

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Operator andEnvironmental Safety

Risk analysis

Classification of contaminants

Defined cell lines

Good microbial techniques/good

occupational safety and hygiene

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Classification

of Contaminants

Biosafety Type I

-Microorganisms that have never beenidentified as causative agents of disease in

man and that offer no threat to theenvironment

Biosafety Type II

- Microorganisms that may cause disease inman and might therefore offer a hazard tolaboratory workers. They are unlikely tospread into the environment. Prophylacticsare available and treatment is effective.

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Biosafety Type III

-Microorganisms that offer a severe threat tothe health of laboratory workers butcomparatively small risk to the populationat large. Prophylactics are available and

treatment is effective.

Biosafety Type IV

-Microorganisms that cause severe illness in

man and offer a serious hazard tolaboratory workers and people at large. Ingeneral, effective prophylactics are notavailable and no effective treatment isknown.

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IntroductionTo Aseptic Technique

Work in culture hood

Roll back long sleeves, no rings,

watches, etc. Hair pulled back, caps off

Wash hands with antibacterial soap,spray down with 70% EtOH(Industrial alcohol)

Wash all surface with 70% EtOH

.

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Dont cough, talk, sneeze, sing intohood

Lids on bottles when not in use

Sterile tips: change if they touchanything other than media.

Keeps tips IN hood Back surface is cleanest

Wash all surface with 70% EtOH

Ctd..

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Horizontal LaminarFlow Cabinets

Types of LaminarFlow

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Vertical LaminarFlow Cabinets

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The Class IIIBioSafety Cabinet

A. Glove ports with O-ring for attaching arm-length

gloves to cabinet

B. Sash

C. Exhaust HEPA filter

D. Supply HEPA filter

E. Double-ended autoclave or pass-through box

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A typical layout for working clean to dirty within a Class III

BSC. Clean culture (left) can be inoculated (center);contaminated pipettes can be discarded in the shallow panand other contaminated materials can be placed in thebiohazard bag (right). This arrangement is reversed for left-handed persons.

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Potential Risk to Human Recipients(WHO Study Group on Biologics)

High risk:

- Blood and bone marrow cells derived from humansor non-human primates, caprine and ovine cells,hybridomas when at least on fusion partner is of

human or non-human primate origin.Medium risk:

- Mammalian non-hematogenous cells such asfibroblasts and epithelial cells

Low risk:

- Human diploid cell lines and cells derive from aviantissues.

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Tissue Culture

A generic term for in vitroculture of organs, tissuesand cell.

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Animal Tissue Culture

Organ culture : Maintaining of whole organ orfragments of tissue.

Cell culture : culture of individual cells fromanimal tissues or established cell line in vitro

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Cell types

commonly used in cultureAnchorage dependent

Fibroblast

Epithelial cells

Muscle cells

Neuron

Suspension

Hematopoietic cells

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Primary Culture

Cells derived directly from organ ortissue or the host organisms.

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Why primary cell culture?

Not been modified in any way other thanenzymatic or physical association.

However, mixed in nature, limited lifespanand carries potential contamination.

It is possible to culture up to 50 60doublings.

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Cell Line

Cells arise from a primary culture after firstsuccessful sub-culture.

Two categories :

- Primary: Cells (culture) derived directly fromorgan or tissue of the host organisms-limitedpassage. Normally having a finite number ofpassage (less than 70 times)

- Continuous: Cells having the apparent to bepassage indefinitely.

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Cell Strain

Cells derived from a primary culture

or a cell line by selection or cloningof cells having specific properties ormarkers.

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Cell Cloning

Technique in getting pure cell line/strain

Adopt from traditional microbiological approach tothe problem of culture

Technique used in selecting a hybridoma cloneproducing monoclonal antibody

Several approaches:

- Limiting dilution-multiwell technique

- Semi solid agar

- Cloning ring technique-anchorage dependent cells.

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Initiation of Primary Culture(Single cells)

The commonest way to initiate cellcultures

Disaggregate the tissue into single cellsby

Mechanically

Enzymatically

Chelating agents

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Mechanical disaggregation

Mainly used to prepare;

Primary explants cell culture (original methoddeveloped at turn of the century).

Skin biopsies.

Cell culture from soft tissues. Methods to disaggregate tissues.

vigorous pipetting.

pressing tissue into a mesh.

wash cells through sieve.

Faster than enzymes but less yield Causes mechanical damage to cells.

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Enzymatic disaggregation

Variety of enzymes.

Trypsin and pronase.

Collagenase and dispase.

Etc Crude or purified preparations.

Alone or in various combinations.

Usually in combination with EDTA.

EDTA alone can also be used to detach cells

and seems to be gentler on the cells thantrypsin

EDTA/EGTA/Citrate

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Trypsin

Papain

Elastase

Hyaluronidase

Collagenase Type II

Collagenase Type I

Collagenase Type IV

Collagenase Type III DNase

List of enzymescommonly used

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Initiation of Primary Culture

(Solid culture)

Chop the tissue aseptically into pieces (1mm2)

Culture the pieces in growth medium.

The tissues adhere to the substratum and cellsmigrate out from the cut ends

Epithelial cells emerge first and fibroblasts

gradually come to dominate the culture.

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Chicken embryo cell culture

Easier to prepare than mouse embryo.

Isolation, identification and propagation of viruses ofanimal and human of origins.

Preparation of chicken embryo from ; Whole embryo : fibroblast-based (mesenchymal).

Specific organs : - liver, kidney, heart, etc.

Prepare cell culture that will confluent/monolayerwithin 24 hours.

Recommended % of serum.

Can be maintained ~ 50 generations prior to crisis. Requires immortalization or transformation to

become a continuous cell line.

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Human Biopsy Material

Need authorization and consent.

Reliability of courier agent for transportation.

Nature/condition of the biopsy material isimportant.

Handle in class II biohazard cabinet.

Application;

Diagnostic: - identification andcharacterization of surface markers,genetic make-up, etc.

Cancer treatment : sensitivity tests againstchemotherapy and immunotherapy.

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Growth And Maintenance

Of Cells In CultureInoculation

A culture can be initiated by inoculating the cells into sterilegrowth medium. Cells should be inoculated at a density of 104 -

105

cells/mL. Cell growth around 106 cells/mL or to 105/cm2 about 3 4 days

Cell growth stops because

- Nutrient limitation

- Accumulation of a toxic metabolite

- Lack of growth surface (for anchorage-dependent cells)

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Sub-culture/sub-passage

Inoculating some of the cells into fresh medium.

Cell will lose their viability if they are left for too long before sub-culture.

Cells grown in suspension, sub-culture involves dilution of the

high density culture with fresh medium. Dilution from 1:2 to 1:10(v/v) would be suitable.

Anchorage-dependent cell involves the detachment of cell fromthe growth surface.The cells are detached from their anchor bythe process of trypsinization. The proteolytic enzyme, trypsin is

used to break down the proteins that bind the cells to the culturesurface.

Cultures are given a passage number which indicates thenumber of sub-cultures performed since when the cells wereobtained or isolated.

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The phases of culture

The lag phase

-This is an early phase in which there is no apparent increasein cell concentration.

-This phase associated with the cellular synthesis of growth

factors which may be required to reach a criticalconcentration before growth takes place.

-The length is dependent upon the culture mediumformulation as well as the initial concentration and state ofthe cells.

-Tends to be longer at low inoculation densities or if theviability of the inoculated cells is low.

-Transformed cells have a lower requirement for growthfactors, always show no lag phase

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Growth phase

-Involves an exponential increase in cell number which can berepresented by the following equation:

N= N0.2

or log 10N0 + .log102

N = final cell concentration, N0 = initial cell concentration, and=number of generations of cell growth

-During the exponential phase, cells go through the cell cycle :G1-S-G2-M

- The doubling time during cell growth can be calculated fromthe equation:

D = T/X ; D= doubling time, T= total elapsed time, andX= number of generations

Thus, for a culture in which an initial cell density of 105 cells/mLreaches 106 cells/mL in 3 days the average doubling time is

0.904 days or 22h

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The stationary phase

Occurs when there is no further increase in cell concentration.

Cell growth is limited by one of these conditions:Nutrients may have been depleted to a level that cannot

support further cell growth

The accumulation of metabolic by products may have reached alevel which is inhibitory to cell growth

The cells may have formed a complete cover over the growthsurface. Growth may stop when a single monolayer of cellscovers the available substratum (called confluence).

The cell may be metabolically active even though growth is not

occurring. For example, high productivity of secreted proteinsmay occur during this phase.

Death rate = growth rate

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The decline phase

-Occurs as a result of cell death

-Cell viability is lowest during the decline phase of culture-shown by a large difference between total and viable cell count.

-Cell concentration decreases as the cells lyse and theirintracellular metabolites are released into the growth medium.

There are two possible mechanisms of cell death in culture-apoptosis or necrosis

-Apoptosis is a normal physiological mechanism of cell death.Apoptosis is a cell suicide mechanism observed to occur inculture or in-vivo under normal physiological conditions.

Apoptosis may be initiated by the depletion of nutrients.

..ctd/..

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- ctd..

-The alternative mechanism of cell death is necrosis, whichfollows cellular injury. This does not involve the cellfragmentation characteristic of apoptosis, and the loss of

cell viability in culture is relatively slow.

-Necrosis is a passive process that normally occurs when

cell are subjected to sudden severe stress. This ischaracterised by a breakdown of the plasma membrane

leading to cell swelling and eventually cell rupture.

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Cell growth inculture

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Most of the cancer cells are removed by surgery. The remainingcancer cells begin to proliferate rapidly and cancer chemotherapy

is started. Many tumor cells are killed by the chemotherapy, buteventually some cancer cells that are resistant to thechemotherapy drug begin to grow rapidly. The chemotherapy is nolonger useful and is discontinued.

Tumorgrowth

curve

http://en.wikipedia.org/wiki/Image:Cancergrowth.PNG

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Mediarequirements

Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2.

Trace elements - iron, zinc, selenium.

Sugars - glucose is the most common.

Amino acids - 13 essential.

Vitamins - B, etc : choline, inositol. Serum - contains a large number of growth

promoting activities such as buffering toxicnutrients by binding them, neutralizes trypsin andother proteases, has undefined effects on the

interaction between cells and substrate, andcontains peptide hormones or hormone-like growthfactors that promote healthy growth.

Antibiotics - although not required for cell growth,antibiotics are often used to control the growth ofbacterial and fungal contaminants.

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Media requirements growth factors

Animal Serum : Mixture of growth factors, hormones, etc.

Special preparations : Epidermal Growth Factor (EGF), Fibroblast

Growth Factor (FGF), Endothelial Cell GrowthFactor (ECGF), Keratinocyte Growth Factor,Hepatocyte Growth Factor (HGF), Insulin-likeGrowth Factor (IGF-1, IGF-II), Nerve GrowthFactor (NGF), Platelet-derived Growth Factor

(PDGF) and variety of cytokines. transforming growth factor (TGF)

tumor necrosis factor (TNF)

interleukins

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Incubator

CO2 incubator is generally used to maintain cultures at anoptimal temperature and pH for cell growth.

Water jacket provides a constant temperature environmentwith minimal danger of local heating effects in the inner

chamber of the incubator.

Carbon dioxide is supplied from a gas cylinder into the innerchamber of the incubator

The purpose of this is to maintain the optimal pH of the

cultures by the bicarbonateCO2 buffering systems.

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CO2 incubator

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A facility for maintaining cell stocks is required unless all

cultures are established from primary tissue

Cells are stored in cryogenic plastic vials which are lowered

into liquid nitrogen for long-term storage.

The liquid nitrogen reservoir needs to be replenished atregular intervals. It is highly recommended that a liquidnitrogen level indicator with alarms is used to lower the riskof damaging important cell stocks by inadvertently allowing

the liquid nitrogen level to drop too low.

Liquid nitrogen storage

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The liquid nitrogen storage units areused for the cryopreservation of cells

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Viability measurement

Primarily based on the integrity of the plasmamembrane;

Trypan blue and naphthalene.

Other dyes acridine orange and propidiumiodide.

Based on cell function ;

de novoprotein synthesis .

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Separation of cells

Nonviable from viable cells ;

Media change

Centrifugation.

Based on surface markers ; Sorting by flow cytometry.

Affinity separation by nonmagnetic andmagnetic techniques.

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Terminology of cell death

Necrosis and Apoptosis

Two mechanism of cell death may briefly be defined:

Necrosis- (accidental cell death) is the pathological

process occurs when cells are exposed to a seriousphysical or chemical insult.

Apoptosis - (normal or programmed cell death) is thephysiological process by which unwanted or useless cellsare eliminated during development and other normal

biological processes.

Diff i l f d i ifi

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Differential features and significanceof necrosis and apoptosis

Necrosis Apoptosis

Loss of membraneintegrity

Membrane blebbing, but no loss ofintegrity

Aggregation of chromatin at thenuclear membrane

Begins with swelling ofcytoplasm andmitochondria

Begins with shrinking of cytoplasmand condensation of nucleus

Ends with total cell lysis Ends with fragmentation of cell into

smaller bodies

No vesicle formation,complete lysis

Formation of membrane boundvesicles (apoptotic bodies)

Disintegration (swelling)

of organelles

Mitochondria become leaky due to

pore formation involving proteins ofthe bcl-2 family

Morphological features:

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Picture of Necrosis and Apoptosis

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Necrosis Apoptosis

Affects groups of contiguouscells

Affects individuals cells

Evoked by non-physiologicaldisturbances (complementattack, lytic viruses,hypothermia, hypoxia,ischemica, metabolicpoisons)

Induced by physiologicalstimuli (lack of grownfactors, changes inhormonal environment)

Phagocytosis bymacrophages

Phagocytosis by adjacent ormacrophages

Significant inflammatoryresponse

No inflammatory response

Physiological significance:

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Necrosis Apoptosis

Loss of regulation of ionhomeostasis

Tightly regulated process involvingactivation and enzymatic steps

No energy requirement(passive process, also occursat 4oC)

Energy (ATP)-dependent (activeprocess, does not occur at 4oC)

Random digestion of DNA(smear of DNA after agarosegel electrophoresis)

Non-random mono-andoligonucleosomal length fragmentationof DNA (ladder pattern after agarose gelelectrophoresis)

Postlytic DNA fragmentation (=

late event of death)

Prelytic DNA fragmentation

Release of various factors (cytochoromeC, AIF) into cytoplasm by mitochondria

Activation of caspase cascade

Alteration in membrane asymmetry (i.e.,translocation of phosphatidyl-serine fromthe cytoplasmic to the extracellular side of

the membrane)

Biochemical features:

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C TreatedM

DNA smearing-apoptosis

Treatedcells

Controlcells

AOPIstaining

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Cytotoxicity

Cell-killing property of a chemical compound(such as food, cosmetics or pharmaceutical)

or a mediator cell (cytotoxic T cell). Incontrast to necrosis and apoptosis, the termcytotoxicity does not indicates a specificcellular death mechanism.

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Summary

Plan your work methods and materials.

Prepare and storage reagents and otherrequirements separately.

Embryonic tissue is preferable than adult tissue.

Fully equipped dedicated tissue culture room. Work fast and maintain sterility.

Remove fat, necrotic, specific tissues, etc.

Enzymatic disaggregration trypsin or collagenase incrude preparation.

Plate cells at high concentration. Close observation first 24 to 72 hours.

Appropriate condition - rich medium, temperatureand environment.

Continuous cell line -immortalization & transformation.