2. Reasons for development of media
• Initially cells were cultured in natural media
(chick embryo extract, serum, lymph etc.)
• Cells cultured in chemically defined media
based on analyses of body fluids and
nutritional biochemistry
• Eagle’s basal Medium, Eagle’s Minimal
Essential medium (MEM), Dulbecco’s
modification of Eagle’s medium (DMEM)
etc
4. What is Balanced Salt Solutions?
• BSS is composed of inorganic salts, may include
sodium bicarbonate and glucose
• Forms basis of complete media
• BSS recipes are modified
• PBS without Ca2+ and Mg2+ = known as PBS
solution A
• D-PBSA
• Hank’s Salt- for sealed flask
• Earl’s Salt- for 5%CO2
5. What is Complete Media?
• Complete media – all constituents (glutamine)
and supplements (serum, growth factors or
hormones)
• Complex media – often supplemented with
extra metabolites (nucleosides, TCA cycle
intermediates and lipids)
• Nutrients are low in F12 media and high in
DMEM.
6. Amino acids
• Essential amino acids – cysteine, arginine, glutamine and
tyrosine
• Individual requirements vary with cell type
• Responsible for cell growth and survival
• Other nonessential amino acids are added
• Glutamine used as a energy source/ carbon source by oxidation
to glutamate by glutaminase and enter into TCA cycle by
transamination to 2-oxoglutarate.
• Deamination of glutamine – produce ammonia. So, use
dipeptide (glutamyl alanine or glutamyl glycine): minimize the
ammonia production and more stable in medium.
7. Vitamins
• Eagle MEM - Water soluble vitamins – B
group, choline, folic acid, inositol and
nicotinamide, excluding biotin.
• M199 - Fat soluble vitamins (A,D, E and K)
• LHC-9 has Vitamin A
• MCDB 110 has Vitamin E
• Individual requirements vary with cell type
8. Salts
• Na+, K+, Mg 2+, Ca2+, Cl-, SO4, PO4 and
HCO3- - maintain osmolality of medium
• Ca2+ - signal transduction process, whether
proliferate or differentiate
• Na+, K+ and Cl- regulate membrane potential
• SO4, PO4- and HCO3- act as nutritional
precursors for macromolecules and regulate
intracellular charge
9. Glucose
• Source of energy
• Metabolism of glucose – by Glycolysis to
form Pyruvate
• Pyruvate - converted to lactate or
acetoacetate – enters into citric acid cycle
• More energy derived from glutamine than
glucose
10. Organic Supplements
• Proteins, Peptides, Nucleosides, Citric acid
cycle intermediates, Pyruvate and Lipids
• Help in cloning and maintaining certain
specialized cells (in presence or absence of
serum)
11. Hormones and growth factor
• Not specified in most regular media.
• Frequently added to serum free media.
12. Antibiotics
• Disadvantages: Encourage development of
antibiotic-resistant organisms
• Hide cryptic contaminants
• Hide mycoplasma infections
• Antimetabolic effects that can cross-react with
mammalian cells.
• Encourage poor aseptic techniques.
14. DMEM Media
Steps involved in the Preparation and Sterilization of DMEM media
1. Suspend 13.5gms in 900ml tissue culture grade water with constant, gentle stirring until the
powder is completely dissolved. Do not heat the water.
2. Add 3.7gms of sodium bicarbonate powder (TC230) or 49.3 ml of 7.5% sodium bicarbonate
solution (TCL013) for 1 litre of medium and stir until dissolved.
3. Adjust the pH to 0.2-0.3 pH units below the desired pH using 1N HCl or 1N NaOH since the pH
tends to rise duringfiltration.
4. Make up the final volume to 1000ml with tissue culture grade water.
5. Sterilize the medium immediately by filtering through a sterile membrane filter with a porosity
of 0.22 micron or less, using positive pressure rather than vacuum to minimize the loss of
carbon dioxide.
6. Aseptically add sterile supplements as required and dispense the desired amount of sterile
medium into sterile containers.
