Defined Media and SupplementsChpt. 9

RPMI 1640 Roswell Park Memorial Institute AMINO ACIDS• Glycine • L-Arginine• L-Asparagine• L-Aspartic acid• L-Cystine 2HCl• L-Glutamic Acid• L-Glutamine • L-Histidine • L-Hydroxyproline• L-Isoleucine • L-Leucine• L-Lysine hydrochloride• L-Methionine• L-Phenylalanine• L-Proline• L-Serine• L-Threonine• L-Tryptophan• L-Tyrosine disodium salt dihydrate• L-Valine

VITAMINS• Biotin• Choline chloride• D-Calcium pantothenate• Folic Acid• Niacinamide• Para-Aminobenzoic Acid• Pyridoxine hydrochloride• Riboflavin 376• Thiamine hydrochloride 337• Vitamin B12 1355• i-InositolINORGANIC SALTS• Calcium nitrate (Ca(NO3)2 4H2O)• Magnesium Sulfate (MgSO4) (anhyd.)• Potassium Chloride (KCl) • Sodium Bicarbonate (NaHCO3)• Sodium Chloride (NaCl)• Sodium Phosphate dibasic (Na2HPO4)

anhydrousOTHER COMPONENTS• D-Glucose (Dextrose) 180• Glutathione (reduced) 307• HEPES 238• Phenol Red

Development of Media

• Initial Media Were Body Fluids, Lymph, Serum, Chick Embryo Extract

• Need For Larger Amounts Gave Rise to Chemically Defined Media

• Eagle’s Basal Medium (1955)• Followed by MEM (Minimum Essential Medium)• Horse Serum, Calf Serum or Human Serum

Supplementation• Common Media: MEM, DMEM, RPMI 1640

(hematopoietic cells)• Serum-free is Preferred (minimize infectious agents)

pH

• For Most Cells pH=7.4• Phenol Red is Used as pH Indicator• Purple at ph>7.8, ph<6.5 yellow• H2O+CO2H2CO3H++HCO3

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• To Compensate NaHCO3 is Added (4mM)• Why Not Use HEPES to Control pH?• Lack of Dissolved CO2 and HCO3

- Limits Cell Growth• Sodium Pyruvate Yields Endogenous CO2

• NaHCO3 in Equilibrium With Dissolved CO2

• The Higher the CO2, The More HCO3- Needed

Buffering

• Bicarbonate Buffer Most Common– Cheap– Nutritional benefit– Non-toxic– Not so good buffering capacity

• HEPES Buffer Stronger– Expensive– Toxic At High Concentrations

• Why Use CO2? Why Not Use HEPES?– Turns out HCO3

- AND CO2 Help Growth

Oxygen

• Cultured Cells Rely Mainly on Glycolysis

• Organ Cultures Require High (95%)

• Dispersed Cultures Require Low Oxygen Tension

• Atmospheric O2 Is More Than Adequate

-Mercaptoethanol Used To Neutralize Oxyen Radicals

Osmolality

• Measured by Depression of Freezing Point

• Put Cells in Distilled Water, They Burst

• Put Cells in Salty Water, They Shrink

• Human plasma=290 mosmol/kg

• 260-320 mosmol/kg Acceptable

• Osmolality Is Primarily From Inorganic Salts, Amino Acids and Vitamins

Temperature

• Typically 37°C

• Cells Tolerate Lower Temp (35 °C)

• Do Not Tolerate High Temp (39.5 °C), Few Hours and They Die

• Cells Can Survive For Years @ -196 °C

Balanced Salt Solutions

• Solution Composed of Inorganic Salts– Ex. KCl, CaCl2, NaCl

• Earle’s vs Hanks– Earle’s good for CO2

– Hanks good for sealed flasks and atmospheric air as gas phase

• HEPES Buffer Can Be Added, Expensive Though • If HEPES Added, NaCl Omitted to Maintain Osmolality• BSS Used For Tissue Dissaggregation• BSS Rely on Phosphate for Buffering, Relatively Weak

Buffering Capacity

Serum

• Several Types of Serum– FCS– Horse Serum– Human Serum (has to be tested for viruses)– FBS

• Anti-trypsin Activity• Contains Growth Factors

– Promote Cell Proliferation– Ex. IGF-I, Insulin, FGF, EGF, IL-1, IL-6– Albumins Major Component Of Serum

Glucose

• Included In Most Media

• Energy Source

• Metabolized into pyruvate

• May! Enter CAC (citric acid cycle)

• This Explains Need For Glutamine/glutamate For Carbon and Energy Needs

Antibiotics

• Antibiotics Should Be Avoided– Mask cryptic contaminations (mycoplasma)– Can generate antibiotic resistant bacteria– Good aseptic technique should be sufficient

• Several Antibiotics/Anti-mycotics– Ampicilin, Gentamycin, Amphotericin B,

Tetracyclin

• Primary Cultures-Use Antibiotics

Conditioned Medium

• Feeder Cells Are Used • Useful in Hard To Grow Cells

– Ex. Myoblasts grow well in conditioned medium

– Collagen was found to be released by feeder cells

• Growth Factors Released By Feeder Cells– IGF-I

– PDGF

– FIBRONECTIN

How Do You Select The Correct Medium

• Check Literature

• 75% of Media Sales is RPMI 1640, DMED AND MEM

• DMEM is Basically MEM With Higher– Amino Acid (2x)– Vitamins (4x)

– HCO3- and CO2 (10%) To Improve Buffering

• If No Info Available, Do Simple Growth Expt.