Lecture 5

Question 1

list the basic components of cell culture media and describe their major function/role

Answer 1

inorganic salts
carbohydrates 
amino acids
vitamins 
fatty acids and lipids
proteins and peptides 
serum
trace elements (Fe, Cu, etc)
pH indicator

Question 2

describe the two most common buffering systems used in cell culture. how they work, advantages, common uses

Answer 2

pH drops over time as cells proliferate and metabolic activity produces acids (eg lactic acid). two systems:

1. “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
2. “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

Question 3

describe considerations regarding oxygen availability in cell culture

Answer 3

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.

Question 4

describe the special consideration given to glutamine

Answer 4

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

Question 5

describe the important role fetal bovine serum has in cell culture

Answer 5

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.

Question 6

describe osmolarity considerations in cell culture

Answer 6

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

Question 7

what are different “balance salt solutions”

Answer 7

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

Question 8

what are different “balance salt solutions”

Answer 8

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

Question 9

what is the role of antibiotics in media?

Answer 9

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

Question 10

role of inorganic salts

Answer 10

retain osmotic balance, regulate mom potential, required for cell attachment, act as cofactors

Question 11

role of carbohydrates

Answer 11

energy source, higher conc supports more cell types

Question 12

role of amino acids

Answer 12

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

Question 13

role of vitamins

Answer 13

varies with medium, necessary for growth/proliferation. in serum

Question 14

role of proteins and peptides

Answer 14

important in serum free media. common ones are Albumin, Transferrin, Fibronectin. Increase viscosity and reduce shear stress

Question 15

role of fatty acids and lipids

Answer 15

important in serum free media. normally in serum bound to albumin. includes cholesterol and steroids essential for specialized cells

Question 16

role of trace elements

Answer 16

eg zinc, copper, selenium
Enzymatic cofactors
selenium is a detoxifier and removes oxygen free radicals

Question 17

role of hormones

Answer 17

insulin promotes glucose uptake into cells, promotes division. growth factors (usually in serum)

Question 18

explain considerations when considering serum free media

Answer 18

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

Question 19

advantages vs disadvantages of serum

Answer 19

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

Question 20

disadvantages of serum free media

Answer 20

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

Question 21

describe serum substitutes

Answer 21

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