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Using Cultured Cells
• Cells can be grown as individuals– Bacteria and yeast– Animal cells– Plant cells
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Tuesday (last year)
00.20.40.60.8
11.21.41.61.8
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Series1
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Growth Curve Data
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Primary vs. Secondary Metabolites
• Growing cells focus on “primary metabolites”– Required for living and
making macromolecules– Only kind in eukaryotic
• Cells after log growth phase make secondary metabolites– Often useful to us– Micro-organisms mainly
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Cell Culture Requirements
• For all cells– Nutrients– Dissolved oxygen– Waste removal (or dilution)– Appropriate pH and temperature
• For some cells– Surface for attachment– Growth stimulating signals
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Nutritional Requirements
• Basics are common to all cells– Energy source (carbon)– Nitrogen source– Salts (Na+, K+, Ca++)
• Details vary with specific cells– Bacteria more self-sufficient– Mammalian cells more sensitive, helpless
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Bacterial Media
• Carbon source– Molasses– Malted grain– Cellulose– Plant oils and fats– Wood pulp waste
• Nitrogen source– Corn steep liquor– Yeast extracts– Peptones – Soybean meal
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Other Ingredients
• Water
• Trace minerals
• Inhibitors (if needed)
• Dissolved oxygen
• Antifoam agents
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Oxygen
• Must be dissolved, but water has low capacity
• Continually supplied from above
• Continually supplied from below
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Growth Conditions
• Proper temperature• Proper pH• Low levels of waste
metabolites• Growth conditions are
maintained by a fermentor
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pH
• Cell growth affected by pH– Biphasic curve (rate vs. pH)– Low at low and high– Best in middle
• pH is altered by cell growth– Metabolic wastes include acids, ammonia– Control by buffering, medium replacement or
adding alkaline or acid
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Temperature
• Cell growth affected by temperature– Biphasic curve (rate vs. temperature)– Low at low and high temperature– Best in middle (range defined by cell type)
• Cell growth produces heat• Regulate temperature of environment
– Incubator– Cooling/warming coil– Water jacket
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Jacketed Tank (concept)
• Resembles a thermos bottle
• Container surrounded by hollow space
• Difference: inlet and outlet for heat exchange solution
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Other Tank Types
• Rely on air for mixing• Bubble tank
– Sparged air acts to mix– As air rises, mixing occurs
• Air-lift tank– Divided tank– Shown at left
• Both tend to have foam
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Mammalian Medium
• More complicated recipes– Cells less capable than microbes– Need more “processed” ingredients
• May need lipids and amino acids• May need vitamins and hormones• May require serum supplementation
• Animal serum provides many “factors”– Growth factors– Attachment factors
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Simple Mammalian Medium
INORGANIC SALTS: Calcium Chloride 1.80 Ferric Nitrate 0.000248 Potassium Chloride 5.30 Magnesium 0.813 Sodium Chloride 110.34 Sodium Bicarbonate 44.10 Sodium Phosphate 0.906 OTHER: D-Glucose 5.55 Phenol red 0.0346 Sodium Pyruvate 1.00
VITAMINS: D-Ca pantothenate 0.0083 Choline Chloride 0.0285 Folic Acid 0.00906 i-Inositol 0.04 Niacinamide 0.0328
AMINO ACIDS: L-Arginine-HCl 0.398 L-Cystine 0.200 L-Alanyl-Glutamine 4.03 Glycine 0.399 L-Histidine HCl-H2O 0.20 L-Isoleucine 0.802 L-Leucine 0.802 L-Lysine-HCl 0.798 L-Methionine 0.201 L-Phenylalanine 0.400 L-Serine 0.400 L-Threonine 0.078 L-Tryptophan 0.078 L-Tyrosine 0.398 L-Valine 0.803
+ 10% Fetal Bovine Serum
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Serum Supplements
• Provide important nutrients
• Add growth factors
• Recreate “animal” environment
• Potential source of infectious agents
• Expensive
• Lot-to-lot variation
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Attachment Surface
• Mammalian cells– Most “normal” cells require– Exceptions are blood cells
• Glass or treated plastic– Surface charge groups– Proteins in serum help with attachment
• Bacteria and yeast grow in suspension• Commonly used insect cells go either way
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Surface Growing vs. Suspension
Tank withsuspendedcells at aconcentrationof 106/ml
64 plates with 106 cells/plate
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Cell Density
• Cells grow best in specific density range– Range is wider for single cell organisms– Narrower for dissociated cells (tissue or organ)
• Mammalian cells secrete growth factors– “Conditioned medium” stimulates growth– Density too low and these signals too low
• Use gradual increase in culture volume– Small initial volume, large final volume– Density of cells ~ same throughout process
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