Transcript Defined Media and Supplements Chpt. 9

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
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Biotin
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Choline chloride
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D-Calcium pantothenate
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Folic Acid
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Niacinamide
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Para-Aminobenzoic Acid
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Pyridoxine hydrochloride
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Riboflavin 376
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Thiamine hydrochloride 337
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Vitamin B12 1355
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i-Inositol
INORGANIC SALTS
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Calcium nitrate (Ca(NO3)2 4H2O)
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Magnesium Sulfate (MgSO4) (anhyd.)
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Potassium Chloride (KCl)
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Sodium Bicarbonate (NaHCO3)
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Sodium Chloride (NaCl)
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Sodium Phosphate dibasic (Na2HPO4)
anhydrous
OTHER COMPONENTS
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D-Glucose (Dextrose) 180
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Glutathione (reduced) 307
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HEPES 238
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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
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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++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
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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
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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
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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
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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.