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Defined Media and Supplements Chpt. 9

Defined Media and Supplements Chpt. 9. 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

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Defined Media and Supplements Chpt. 9

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  1. Defined Media and SupplementsChpt. 9

  2. 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-Inositol INORGANIC SALTS Calcium nitrate (Ca(NO3)2 4H2O) Magnesium Sulfate (MgSO4) (anhyd.) Potassium Chloride (KCl) Sodium Bicarbonate (NaHCO3) Sodium Chloride (NaCl) Sodium Phosphate dibasic (Na2HPO4) anhydrous OTHER COMPONENTS D-Glucose (Dextrose) 180 Glutathione (reduced) 307 HEPES 238 Phenol Red RPMI 1640 Roswell Park Memorial Institute

  3. 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)

  4. pH • For Most Cells pH=7.4 • Phenol Red is Used as pH Indicator • Purple at ph>7.8, ph<6.5 yellow • H2O+CO2H2CO3H++HCO3- • 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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.

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