Cell Tissue Culture Research medical purposes l Study

Cell & Tissue Culture Research/ medical purposes l Study cell processes e. g. l l l Cancer Development drug mechanisms, disease processes Diagnoses l e. g. Downs Syndrome, genetic disorders Skin grafts l Stem Cells l

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) l Human proteins (h. GH, Insulin) l Enzymes (streptokinase) l Complex molecules (cyclosporin) l l Steroids (Mexican Yam + bacterium or fungus) l Pill, cortisone (anti-inflammatory - $200 $0. 46 /g),

Commercial applications Food l Pruteeen (Methylophilus methylotropus) l l Animal feed grown on methane gas/ methanol Quorn fungus Fusarium graminearum) l grown on glucose l Autolysed yeast (meat/cheese flavours) l Dairy products/Brewing l Rennin in cheese making l

Commercial applications Industrial manufacture Vitamins l Plastics (biodegradable, PHBV) l Washing powders l Mining (Copper, Gold) l Enzyme production (food technology etc. ) l

Commercial applications Agriculture GM crops (Flavr savr Tomato) l Virus free strawberries l Orchid production l Monsanto Roundup resistant maize l BST for milk production l

Commercial applications Biodegradation Oil slicks l Organochlorines (PCBs, PCPs) l Sewage Treatment l Sewage contamination analyses l

General Requirements for cell culture Growth requirements l nutrient medium l l l heterotroph, photoautotroph (light), chemoautotroph surface on which to growth factors Temperature l Hyperthermophiles, mesophiles, psychrophiles p. H (buffered) l acidophiles, Prevent contamination l (aseptic technique, antibiotic, fungicide)

Bacterial Growth Media Most bacteria are heterotrophs l Require complex medium (defined) l l l Often undefined e. g l l l 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. l Blood/ milk/ acid p. H etc.

Growth conditions Oxygen requirements l Obligate aerobes l l l Facultative aerobes l l Absolutely require oxygen Large scale cultures need to aerate Can grow without oxygen, but grow better with it Obligate anaerobes l l Oxygen is toxic to them Grown in anaerobic jars (filled hydrogen & carbon dioxide)

Preventing contamination Aseptic technique Selective media (contain antibiotics or specific nutrients/ p. H) Antibiotics (selective) l Fungicides l 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 l Bacteria preparing for cell division (also low numbers) Logarithmic (exponential growth phase) l Unlimited growth rate l l Plenty food, space Wastes not yet at toxic levels Stationary (declining) phase l l Limited by food, space Toxic build up Continuous culture l Refresh part of culture

Mammalian Cell Culture More difficult than bacteria conditions more carefully regulated l Media more complex (amino acids, vitamins) l p. H indicator (phenol red – indicates CO 2) l Sera (e. g. foetal calf serum / donor horse serum - contain growth factors) l If cells divide, they usually die after a finite number of divisions. l

Mammalian Cell Culture Primary culture l l l Culture cells taken from an animal Limited life span More like reality Treat with enzymes to disrupt cells l Trypsin / collagenase Surface attachment l l l Cells are anchorage dependent Reach confluence Subculture

Mammalian cell Culture Continuous cell lines l l l Derived from tumours (He. La, PC 12) Transformed (viruses) - lost cell cycle control Immortalised Easier to grow routinely l l 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 l l Allows isolation of single cells Novel mutants

Plant cell culture Simpler requirements than animal cells. Easier to produce a whole plant from single cell l Nuclear totipotency – capable of producing all differentiated cell types because genome contains all genes (all cells are nuclear totipotent – in theory - Dt. S). 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