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 L-Valine VITAMINS Biotin Choline chloride D-Calcium pantothenate Folic Acid Niacinamide Para-Aminobenzoic Acid Pyridoxine hydrochloride Riboflavin 376 Thiamine hydrochloride 337 Vitamin B 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 H 2 O+CO 2  H 2 CO 3  H + +HCO 3 - To Compensate NaHCO 3 is Added (4mM) Why Not Use HEPES to Control pH? Lack of Dissolved CO 2 and HCO 3 - Limits Cell Growth Sodium Pyruvate Yields Endogenous CO 2 NaHCO 3 in Equilibrium With Dissolved CO 2 The Higher the CO 2, The More HCO 3 - 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 CO 2 ? Why Not Use HEPES? –Turns out HCO 3 - AND CO 2 Help Growth

Oxygen Cultured Cells Rely Mainly on Glycolysis Organ Cultures Require High (95%) Dispersed Cultures Require Low Oxygen Tension Atmospheric O 2 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 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 -196 °C

Balanced Salt Solutions Solution Composed of Inorganic Salts –Ex. KCl, CaCl 2, NaCl Earle’s vs Hanks –Earle’s good for CO 2 –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) –HCO 3 - and CO 2 (10%) To Improve Buffering If No Info Available, Do Simple Growth Expt.