Cell & Tissue Culture
Research/ medical purposesStudy cell processes e.g.
Cancer Development drug mechanisms, disease processes
Diagnoses e.g. Downs Syndrome, genetic disorders
Skin graftsStem Cells
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
PharmaceuticalsAntibiotics (fungi)Human proteins (hGH, Insulin)Enzymes (streptokinase)Complex molecules (cyclosporin)
Steroids (Mexican Yam + bacterium or fungus) Pill, cortisone (anti-inflammatory - $200 $0.46 /g),
Commercial applications
FoodPruteeen (Methylophilus methylotropus)
Animal feed grown on methane gas/ methanolQuorn
fungus Fusarium graminearum)grown on glucose
Autolysed yeast (meat/cheese flavours)Dairy products/BrewingRennin in cheese making
Commercial applications
Industrial manufactureVitaminsPlastics (biodegradable, PHBV)Washing powders Mining (Copper, Gold)Enzyme production (food technology etc.)
Commercial applications
AgricultureGM crops (Flavr savr Tomato)Virus free strawberriesOrchid productionMonsanto Roundup resistant maizeBST for milk production
Commercial applications
BiodegradationOil slicks Organochlorines (PCBs, PCPs)Sewage TreatmentSewage contamination analyses
General Requirements for cell culture
Growth requirements nutrient medium
heterotroph, photoautotroph (light), chemoautotroph surface on which to grow growth factors
Temperature Hyperthermophiles, thermophiles, mesophiles, psychrophiles
pH (buffered) acidophiles,
Prevent contamination (aseptic technique, antibiotic, fungicide)
Bacterial Growth Media
Most bacteria are heterotrophs Require complex medium (defined)
carbon source (glucose), nitrogen source (ammonium salts), energy source (glucose), micronutrients (Fe, Co, Mn etc)
Often undefined e.g Yeast extract, peptone, casein hydrolysate
Vitamins and specific amino acids may also be included Special media contain specific requirements e.g.
Blood/ milk/ acid pH etc.
Growth conditions
Oxygen requirementsObligate aerobes
Absolutely require oxygen Large scale cultures need to aerate
Facultative aerobes Can grow without oxygen, but grow better with it
Obligate anaerobes 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
Containment (cabinets)
Sterile media (autoclave/ 0.2m filter)
Measuring Growth Rate
Haemocytometer
Flow cytometer
Plate dilution method (viable count)
Colorimetry (densitometry)
Growth curves
Slow growth phase Bacteria preparing for cell division (also low numbers)
Logarithmic (exponential growth phase) Unlimited growth rate
Plenty food, space Wastes not yet at toxic levels
Stationary (declining) phase Limited by food, space Toxic build up
Continuous culture Refresh part of culture
Mammalian Cell Culture
More difficult than bacteriaconditions more carefully regulatedMedia 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.
Mammalian Cell Culture
Primary culture Culture cells taken from an animal Limited life span More like reality
Treat with enzymes to disrupt cells Trypsin / collagenase
Surface attachment Cells are anchorage dependent Reach confluence Subculture
Mammalian cell CultureContinuous cell lines
Derived from tumours (HeLa, PC12) Transformed (viruses) - lost cell cycle control Immortalised
Easier to grow routinely 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 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 plantsProtoplasts can also be grown into whole plants – to produce hybrids or genetically modified cells.Lack of cell wall means genes are easily introduced
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