Lecture 5 Flashcards
list the basic components of cell culture media and describe their major function/role
inorganic salts carbohydrates amino acids vitamins fatty acids and lipids proteins and peptides serum trace elements (Fe, Cu, etc) pH indicator
describe the two most common buffering systems used in cell culture. how they work, advantages, common uses
pH drops over time as cells proliferate and metabolic activity produces acids (eg lactic acid). two systems:
- “Natural” buffering system: gaseous CO2 balances with the CO2/H2O content of the culture medium. high CO2 lowers pH and high HCO3- raises pH. this is done with 5-10% CO2 in the incubator air, low cost and non toxic
- “Chemical” buffering: uses zwitterion called HEPES with a superior buffering capacity (pH 7.2-7.4 physiological range), but is expensive and can be toxic. does not require controlled gaseous atmosphere
describe considerations regarding oxygen availability in cell culture
tissue O2 is around only 2-9% with hypoxic (cancer) cells being <2%. cells in culture are anaerobic and use glycolysis. there is no hemoglobin so some O2 is needed in atmosphere, but not too much as O2 forms toxic radicals. compromise between respiratory requirements and toxicity is necessary.
describe the special consideration given to glutamine
Glutamine is important for cell culture. it supports cell growth, protein turnover, glucose utilization, and is an energy and carbon source. BUT Gln is unstable and degrades releasing toxic ammonia! must change media frequently to remove this problem. Options are to supplement media with Gln prior to use or use Glutamax, a stable dipeptide as Gln source
describe the important role fetal bovine serum has in cell culture
Serum is a complex mix of proteins (albumins), growth factors, growth inhibitors, minerals, hormones. one of the most important components in media, quality and type affects cell growth. always screen batches of serum! can heat inactivate serum to reduce contamination risk.
describe osmolarity considerations in cell culture
the tolerated range is 260-320 mOs/kg with human being around 290. can be adjusted with NaCl. big dishes have more evaporation so need to start with a higher osmolarity
what are different “balance salt solutions”
Eagle’s BSS, Dulbecco’s PBS, Hank’s BSS
vary in salt composition (Ca2+ and Mg2+) and glucose to provide water and ions while maintaining osmotic pressure and pH
what are different “balance salt solutions”
Eagle’s BSS, Dulbecco’s PBS, Hank’s BSS
vary in salt composition (Ca2+ and Mg2+) and glucose to provide water and ions while maintaining osmotic pressure and pH
what is the role of antibiotics in media?
reduces contaminations. useful in the early days but unnecessary now! it encourages antibiotic resistant strains to develop and can hide low level contaminations/mycoplasma infections and encourage poor aseptic technique
role of inorganic salts
retain osmotic balance, regulate mom potential, required for cell attachment, act as cofactors
role of carbohydrates
energy source, higher conc supports more cell types
role of amino acids
essential aa must be added to culture (all in vivo essentials + Cys, Arg, Gln, Tyr), controls max cell density, can supplement non-essential aa’s for growth and prolonged viability
role of vitamins
varies with medium, necessary for growth/proliferation. in serum
role of proteins and peptides
important in serum free media. common ones are Albumin, Transferrin, Fibronectin. Increase viscosity and reduce shear stress
role of fatty acids and lipids
important in serum free media. normally in serum bound to albumin. includes cholesterol and steroids essential for specialized cells
role of trace elements
eg zinc, copper, selenium
Enzymatic cofactors
selenium is a detoxifier and removes oxygen free radicals
role of hormones
insulin promotes glucose uptake into cells, promotes division. growth factors (usually in serum)
explain considerations when considering serum free media
more sensitive: pH, temp, osmolality, mechanical forces, and enzyme treatment
don’t use antibiotics: without serum to bind some antibiotic it can be toxic
higher seeding density during adaptation: cells die as they transfer to new media, seed higher density to overcome
optimize nutrient composition: must add hormones, growth factors, attachment factors, transport proteins, and trace elements
provide other serum functions: protect against proteases, protect agains shear, bind/inactivate cytotoxins
advantages vs disadvantages of serum
advantages: contains growth factor and hormones with stimulate cell growth and function, helps attachment, spreading factor, buffering agent, binding protein, minimizes mechanical damages by viscosity increase
disadvantages: lack of uniformity in composition, testing needs to be done on each batch, may contain growth inhibitors, increase contamination risk, may interfere with purification and isolation of cell culture products
disadvantages of serum free media
serum free media requires various cell type specific media formulations (expensive) and adaptation of each cell line. during adaptation, selective pressure may lead to change of cell properties. There is also a higher degree of reagent purity, slower cell proliferation, and info about composition is not always available
describe serum substitutes
various products have been developed commercially to replace serum. still vary in batches and are often composition proprietary. must individually verify which serum substitute or serum free media works best for your cell line
