Download Cell & Tissue Culture - Hyndland Secondary School

Cell & Tissue Culture
Research/ medical purposes
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Study cell processes e.g.
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Cancer
Development
drug mechanisms,
disease processes
Diagnoses
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e.g. Downs Syndrome, genetic disorders
Skin grafts
 Stem Cells
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Stem Cells
Stem cells are unspecialised
Stem cells are able to divide and
differentiate
They will in the future be used to
“repair” damage in the body.
Commercial applications
Pharmaceuticals
Antibiotics (fungi)
 Human proteins (hGH, Insulin)
 Enzymes (streptokinase)
 Complex molecules (cyclosporin)
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Steroids (Mexican Yam + bacterium or fungus)
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Pill, cortisone (anti-inflammatory - $200 $0.46 /g),
Commercial applications
Food
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Pruteeen (Methylophilus methylotropus)
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Animal feed grown on methane gas/ methanol
Quorn
fungus Fusarium graminearum)
 grown on glucose
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Autolysed yeast (meat/cheese flavours)
 Dairy products/Brewing
 Rennin in cheese making
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Commercial applications
Industrial manufacture
Vitamins
 Plastics (biodegradable, PHBV)
 Washing powders
 Mining (Copper, Gold)
 Enzyme production (food technology etc.)
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Commercial applications
Agriculture
GM crops (Flavr savr Tomato)
 Virus free strawberries
 Orchid production
 Monsanto Roundup resistant maize
 BST for milk production
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Commercial applications
Biodegradation
Oil slicks
 Organochlorines (PCBs, PCPs)
 Sewage Treatment
 Sewage contamination analyses
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General Requirements for cell
culture
Growth requirements
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nutrient medium
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heterotroph, photoautotroph (light), chemoautotroph
surface on which to grow
growth factors
Temperature
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Hyperthermophiles, thermophiles, mesophiles,
psychrophiles
pH (buffered)
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acidophiles,
Prevent contamination
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(aseptic technique, antibiotic, fungicide)
Bacterial Growth Media
Most bacteria are heterotrophs
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Require complex medium (defined)
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Often undefined e.g
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carbon source (glucose),
nitrogen source (ammonium salts),
energy source (glucose),
micronutrients (Fe, Co, Mn etc)
Yeast extract, peptone, casein hydrolysate
Vitamins and specific amino acids may also be included
Special media contain specific requirements e.g.
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Blood/ milk/ acid pH etc.
Growth conditions
Oxygen requirements
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Obligate aerobes
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Facultative aerobes
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Absolutely require oxygen
Large scale cultures need to aerate
Can grow without oxygen, but grow better with it
Obligate anaerobes
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Oxygen is toxic to them
Grown in anaerobic jars (filled hydrogen & carbon
dioxide)
Preventing contamination
Aseptic technique
Selective media (contain antibiotics or
specific nutrients/ pH)
Antibiotics (selective)
 Fungicides
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Containment (cabinets)
Sterile media (autoclave/ 0.2m filter)
Measuring Growth Rate
Haemocytometer
Flow cytometer
Plate dilution method (viable count)
Colorimetry (densitometry)
Growth curves
Slow growth phase
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Bacteria preparing for cell division (also low numbers)
Logarithmic (exponential growth phase)
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Unlimited growth rate
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Plenty food, space
Wastes not yet at toxic levels
Stationary (declining) phase
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Limited by food, space
Toxic build up
Continuous culture
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Refresh part of culture
Mammalian Cell Culture
More difficult than bacteria
conditions more carefully regulated
 Media more complex (amino acids, vitamins)
 pH indicator (phenol red – indicates CO2)
 Sera (e.g. foetal calf serum / donor horse
serum - contain growth factors)
 If cells divide, they usually die after a finite
number of divisions.
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Mammalian Cell Culture
Primary culture
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Culture cells taken from an animal
Limited life span
More like reality
Treat with enzymes to disrupt cells
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Trypsin / collagenase
Surface attachment
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Cells are anchorage dependent
Reach confluence
Subculture
Mammalian cell Culture
Continuous cell lines
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Derived from tumours (HeLa, PC12)
Transformed (viruses) - lost cell cycle control
Immortalised
Easier to grow routinely
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Continue to divide provided correct conditions
maintained
Subculturing required when confluent (covering the entire
plate
Risk of cell line changing (mutating)
Can lose anchorage dependence
Cloning
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Allows isolation of single cells
Novel mutants
Plant cell culture
Simpler requirements than animal cells.
Easier to produce a whole plant from single cell
 Nuclear totipotency – capable of producing all
differentiated cell types because genome contains
all genes (all cells are nuclear totipotent – in
theory - DtS).
Explants (cells or pieces of tissue) grown in
appropriate media (light required – photoautotroph)
Growth regulators (plant hormones) induce
differentiation to produce whole plants
Protoplasts can also be grown into whole plants – to
produce hybrids or genetically modified cells.
Lack of cell wall means genes are easily introduced
Method