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Defined Media and Supplements Chpt. 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 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 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++HCO3To 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.