defined media and supplements chpt. 9. rpmi 1640 roswell park memorial institute amino acids glycine...
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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