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Cell And Tissue Culture
Mammalian Cells
Advanced Higher Biology
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What is it?
� Tissue culture is the term used for ³the process
of growing cells artificially in the laboratory´
(OSMS.otago.ac.nz/main/bursary)
� Tissue culture involves both plant and animal
cells
� Tissue culture produces clones, in which all
product cells have the same genotype (unlessaffected by mutation during culture)
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What¶s the Background?
� Tissue culture had
its origins at the
beginning of the20th century with
the work of Gottleib
Haberlandt (plants)
and Alexis Carrel(animals)
Haberlandt
Carrel
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Introduction
� Cell culture is the process by which prokaryotic,
eukaryotic or plant cells are grown under
controlled conditions. But in practice it refers to
the culturing of cells derived from animal cells.
� Cell culture was first successfully undertaken by
Ross Harrison in 1907
� Roux in 1885 for the first time maintainedembryonic chick cells in a cell culture
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The Background, III
� A more recent advance is the use of plant
and animal tissue culture along with
genetic modification using viral andbacterial vectors and gene guns to create
genetically engineered organisms
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Why is cell culture used for?
Areas where cell culture technology iscurrently playing a major role.
� Model systems for Studying basic cell biology, interactions between disease
causing agents and cells, effects of drugs on cells, process andtriggering of aging & nutritional studies
� Toxicity testingStudy the effects of new drugs
� Cancer researchStudy the function of various chemicals, virus &
radiation to convert normal cultured cells to cancerous cells
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Contd«.
� Virology
Cultivation of virus for vaccine production,
also used to study there infectious cycle.
� Genetic Engineering
Production of commercial proteins, large
scale production of viruses for use in vaccine
production e.g. polio, rabies, chicken pox,
hepatitis B & measles
� Gene therapy
Cells having a functional gene can be
replaced to cells which are having non-functional
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What is needed?Tissue culture, both plant and animal has
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
� Aseptic (sterile) conditions, as microorganisms
grow much more quickly than plant and animaltissue and can over run a culture
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What is Needed, II
� Growth regulators - in plants, both
auxins & cytokinins. In animals, this is
not as well defined and the growthsubstances are provided in serum from
the cell types of interest
� Frequent subculturing to ensureadequate nutrition and to avoid the build
up of waste metabolites
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Major development¶s in cell culture
technology� First development was the use of
antibiotics which inhibits the growth of
contaminants.
� Second was the use of trypsin to remove
adherent cells to subculture further from
the culture vessel
� Third was the use of chemically defined
culture medium.
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What do you need to do it?
� Source of cell material
-freshly prepared
-stock of cell line-bacterial culture
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Suitable container
� Simple flask
� Sophisticated fermenter with computer-
controlled monitoring
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Growth medium
� Glucose
� Water
� Amino acids� Salts
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Opportunity for Gas Exchange
� Oxygen
� Carbon dioxide
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Animal serum
� Foetal Bovine Serum
� Essential for animal cell proliferation
� 5% - 10% of growth media
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Indicator
� Waste products causes change in pH
� Use indicator like phenol red
� Changes from red to yellow
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Control of Temperature and pH
� 37.5 OC
� pH 7.5
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Method for Measuring Cell Growth
� Counting cell numbers in culture
(haemocytometer)
� Measure optical density in
spectrophotometer
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Sterilisation
� Antibiotics
� Sterilisation
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Cells are either«.
� Anchorage ± dependant
� Anchorage - independant
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Anchorage ± independant cells
� Cells associated with body fluid
-blood cells
� Grown in suspension
� Will eventually need subculturing
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Anchorage ± dependant cells
� Most animal derived cells
� Adhere to bottom of a flask and form a monolayer
� Eventually cover entire surface of substratum(confluence)
� Proliferation then stops
� Need to subculture cells at this point (remove to freshmedium)
� Proliferation can begin again
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2 main categories of animal cell
cultures«.
� Primary culture
� Continuous cell line
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Primary Cultures
� Taken from fresh tissue
� Limited life span in culture
� Treated by pr oteolytic enzyme (Trypsin)
� Separate into single cells-epithelial cells
-fibroblasts
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Primary culture
� Cells when surgically or enzymatically removed froman organism and placed in suitable cultureenvironment will attach and grow are called as primaryculture
� Primary cells have a finite life span� Primary culture contains a very heterogeneous
population of cells
� Sub culturing of primary cells leads to the generationof cell lines
� Cell lines have limited life span, they passage severaltimes before they become senescent
� Cells such as macrophages and neurons do not dividein vitro so can be used as primary cultures
� Lineage of cells originating from the primary culture iscalled a cell strain
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Continuous Cell Line
� Derived from humans
� Been transfor med
-lose sensitivity to factors associated with growth control
� Produce immor talised cell lines
� Cell lines are neoplastic
� Often lose their anchorage-dependence
-associated with an altered xsome pattern
� More easily cultured
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Continuous cell lines
� Most cell lines grow for a limited number of generationsafter which they ceases
� Cell lines which either occur spontaneously or induced
virally or chemically transformed into Continous cell lines
� Characteristics of continous cell lines
-smaller, more rounded, less adherent with a higher
nucleus /cytoplasm ratio
-Fast growth and have aneuploid chromosome number
-reduced serum and anchorage dependence and grow
more in suspension conditions-ability to grow upto higher cell density
-different in phenotypes from donar tissue
-stop expressing tissue specific genes
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Cell
line
Species
of origin
Tissue of
origin
Cell
morphology
Growth in
suspension?
3T3 Mouse Connective Fibroblast No
CHO
ChineseHamster
Ovary Epithelial Yes
BHK21 Syrian
Hamster
Kidney Fibroblast Yes
HeLa Human Cervical
Carcinoma
Epithelial Yes
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Why sub culturing.?
� Once the available substrate surface is coveredby cells (a confluent culture) growth slows &ceases.
� Cells to be kept in healthy & in growing statehave to be sub-cultured or passaged
� It¶s the passage of cells when they reach to 80-90% confluency in flask/dishes/plates
� Enzyme such as trypsin, dipase, collagenase incombination with EDTA breaks the cellular gluethat attached the cells to the surface
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Adherent cells
� Cells which are anchorage dependent
� Cells are washed with PBS (free of ca & mg ) solution.
� Add enough trypsin/EDTA to cover the monolayer
� Incubate the plate at 37 C for 1-2 mts
� Tap the vessel from the sides to dislodge the cells� Add complete medium to dissociate and dislodge the
cells
� with the help of pipette which are remained to be
adherent
� Add complete medium depends on the subculture
� requirement either to 75 cm or 175 cm flask
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Suspension cells
� Easier to passage as no need to detach them
� As the suspension cells reach to confluency
� Asceptically remove 1/3rd of medium
� Replaced with the same amount of pre-warmed medium
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Freezing cells for storage
� Remove the growth medium, wash the cells by PBS
and remove the PBS by aspiration
� Dislodge the cells by trypsin-versene
� Dilute the cells with growth medium� Transfer the cell suspension to a 15 ml conical tube,
centrifuge at 200g for 5 mts at RT and remove the
growth medium by aspiration
� Resuspend the cells in 1-2ml of freezing medium
� Transfer the cells to cryovials, incubate the cryovials
at -80 C overnight
� Next day transfer the cryovials to Liquid nitrogen
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Working with cryopreserved cells
� Vial from liquid nitrogen is placed into 37 C water
bath, agitate vial continuously until medium is thawed
� Centrifuge the vial for 10 mts at 1000 rpm at RT, wipe
top of vial with 70% ethanol and discard thesupernatant
� Resuspend the cell pellet in 1 ml of complete medium
with 20% FBS and transfer to properly labeled culture
plate containing the appropriate amount of medium
� Check the cultures after 24 hrs to ensure that they
are attached to the plate
� Change medium as the colour changes, use 20%
FBS until the cells are established
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Cell viability
� Cell viability is determined by staining the cellswith trypan blue
� As trypan blue dye is permeable to non-viable
cells or death cells whereas it is impermeable to
this dye
� Stain the cells with trypan dye and load to
haemocytometer and calculate % of viable cells
- % of viable cells= Nu. of unstained cells x 100
total nu. of cells
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Basic aseptic conditions
� If working on the bench use a Bunsen flame to heat theair surrounding the Bunsen
� Swab all bottle tops & necks with 70% ethanol
� Flame all bottle necks & pipette by passing very quicklythrough the hottest part of the flame
� Avoiding placing caps & pipettes down on the bench;practice holding bottle tops with the little finger
� Work either left to right or vice versa, so that all material
goes to one side, once finished� Clean up spills immediately & always leave the work
place neat & tidy
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Safety aspect in cell culture
� Possibly keep cultures free of antibiotics in order to
be able to recognize the contamination
� Never use the same media bottle for different cell
lines. If caps are dropped or bottles touched
unconditionally touched, replace them with new ones
� Necks of glass bottles prefer heat at least for 60 secs
at a temperature of 200 C
� Switch on the laminar flow cabinet 20 mts prior tostart working
� Cell cultures which are frequently used should be
subcultered & stored as duplicate strains
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Other key facts««.?
� Use actively growing cells that are in their log phase of
growth, which are 80-90% viable
� Keep exposure to trypsin at a minimum
� Handle the cells gently. Do not centrifuge cells at highspeed or roughly re-suspend the cells
� Feeding & sub culturing the cells at more frequent
intervals then used with serum containing conditions
may be necessary
� A lower concentration of 104cells/ml to initiate
subculture of rapidly growing cells & a higher
concentration of 105cells/mlfor slowing growing cells
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Thanks
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