Please enable JavaScript.
Coggle requires JavaScript to display documents.
cell culture (gas phase (temp (Anatomical region, Experiment setting, •…
cell culture
gas phase
temp
Anatomical region
Experiment setting
• better lower than over as cells die at 40ºC
• temperature will affect pH as CO2 dissolve better at lower temperature
Optimal temperature depends: Body temperature
gas
CO2 (5%) – prevents pH related toxicity
O2 - fulfils respiratory requirements +on O2 dependent pathways +Too much O2 leads to the introduction of free radicals
buffer and pH
hc03 buffer
low cost, non-toxic and also provides other nutritional value to the cells
CO2 is released thus causing the media to become more acidic
most cell culture media employs the bicarbonate buffering system
•Increased CO2 will increase the H+ and hence decrease the pH
•To regulate, sodium bicarbonate (NaHCO3) is added into media •Presence of HCO3- push the equation to the left until equilibrium is reached at pH 7.4 (neutral)
HEPES
• superior buffering capacity in the pH range 7.2-7.6
• expensive
• can be toxic to some cell types at higher concentrations
• do not require a controlled gaseous atmosphere
• when used with exogenous CO2, HEPES must be of 2x the bicarbonate concentration for adequate buffering
liquid phase
BSS
• Simple mixtures of salts with glucose
• Provide water and certain bulk ions for cell metabolism
• Maintain intra and extracellular osmotic balance
• Buffering system • Used for cell isolation
Choice dependent on:
i) CO2 tension ii) Divalent cations
COMPLEX
amino acids
synthesis of nucleotides and proteins •Reduce cell capacity to synthesize amino acids •Lack of correct precursor or co-factor in vitro •Cells divide too fast
vitamins
co-factors and Natural anti-oxidants , B group vitamins are necessary (B2,1,7) , toxic if used in excess VA,D,E,K, Serum is an important source.
carbohydrates
• Main source of energy and to generate CO2 •containing higher concentration of sugars are able to support the growth
Other Elements in Culture Media
inorganic salts
• Na+, K+
osmotic balance •
Mg2+ and Ca2+
ECM for cell attachment and cofactor •
Iron
for respiratory pigments
• PO42-
Energy carrier
• HCO3-
Buffering effect
serum
• Yellowish, clear liquid left over after fibrin and cells
are removed from blood • Contain 200+ ingredients • 2%-10% (v/v) serum •
HIS
choice
Batch of serum, certificate of
analysis (COA), Country of origin AND Sterility level.
SFM
DIS ADV
• Composition is not well defined • Batch to batch variation • Short shelf life • Risk of contamination • High cost • Presence of unknown inhibitors • Protein content complicates purification
water
• Must be very high quality and free from impurities
• Types
i. Inorganic contaminants - Heavy metals,
iron, calcium, chlorine
ii. Organic contaminants - Detergents or byproducts
of animal/plant decay
iii. Bacterial products - Endotoxins,
lipopolysacharides
L-Glutamine
Osmolality