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Lecture 4b  culture conditions and media
 

Lecture 4b culture conditions and media

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Industrial Microbiology Dr. Butler 2011

Industrial Microbiology Dr. Butler 2011

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  • Jan. 31, 2011 Intro - Cells are cultured in chemically complex liquid medium that is suitable for supporting growth for several generations. There are several formulations that have been developed for the growth of specific cell types. Closing – Because there are unstable components (glutamine, bicarbonate) media must be filter sterilized, not autoclaved. Storage time for media depends on the recipe. With glutamine, the half life of the media is 3 weeks at 4  C. Without glutamine or bicarbonate, the half life is indefinite.
  • Disposable filter units. 4.3, vacuum pump, 4.4 from a peristaltic pump.
  • Different formulations are used, the one that you choose depends on the cell line that you are trying to grow. You can also combine different formulas.
  • Glucose is metabolized anaerobically, converted to lactic acid.
  • Typical pattern of substrate utilization (glucose, glutamine) and formation of byproducts (lactate and ammonia).
  • In vivo , ammonia is removed by kidney, in cell cultures there is no such removal system, so the ammonia accumulates and may inhibit cell growth.
  • Do diagram on overhead: glutamine – glutamate - ketoglutarate
  • Synthesis of alanine: ammonia combines with pyruvate, alanine is not inhibitory, may be used as a carbon source if other carbon sources exhausted. Glutamate may be used, but cells require adaptation time to increase rate of membrane transport of glutamate, which is typically low.
  • The concentrations of the ingredients in cell culture medium is also an important consideration, especially with respect to the salts in solution. It is important that the concentration of the salts provide an osmotic pressure that is similar to physiological conditions. Isotonic - glucose in concentration – does not ionize - NaCl – ionization state of the particle is important – Na+ and Cl- are separte particles - moles of partciles or ions/ moles of solute – particles - ionization
  • Do numeric example on overhead - Defined media – osm can be calculated by hand or calculated
  • Colligative property – i.e. melting point, and boiling point
  • Organic buffers may be used in conjunction with bicarbonate:CO 2 system when a higher degree of pH control is required. Bicarbonate – when warm, can maintain pH, so when removed does not function properly as a buffer at lower temperatures.
  • Differs the most in every media.
  • Penicillin – disrupts cell wall synthesis, cells lyse. Streptomycin inhibits protein sythesis by preventing initiation of protein synthesis at the ribosome. Amphotericin B – binds ergosterol, a cell membrane component. Membrane function is disrupted, cell membrane is made more permeable (compounds leak out), cells die.
  • Balance of risks - Cocktail of antibiotics can mask something that when antibiotics are taken out, can be contaminated
  • pH = 7.4 Inoculate cells in the media: 1 x 10^5 cells/ml Start off = lag phase Media color = (7.4) red  purple (7.8) Some slow growth of cell, bicarbonate... Purple  red  orange  yellow (6.5) As cell starts to grow pH starts to decrease – lactic acid production – glycolysis buffer system
  • Intro - Serum is a universal supplement, effective for most cells. Helps to buffer against disturbances to the culture – pH, heavy metals, enzyme activity (i.e. trypsin)
  • Serum is still used despite technical disadvantages. These include: 1 . Expensive – accounts for 70-80% of the cost of the media formulation. 2. Chemically undefined: can have variation between batches, leading to inconsistent promotion of growth. 3. High protein content: may compromise the extraction and purification of cell-secreted proteins. Source of contamination: can stil have viral or bacterial contamination. Source of prions – mad cow disease (BSE, bovine spongiform encephalitis, Creutzfeld Jacob disease in humans. Ethical considerations , may be considered cruel to animals. Fetal calf serum is a byproduct of the meat industry. Fetus taken out of pregnant cows at the slaughterhouse, needle injected into heart, blood pumped out.
  • After first point: Want to replace serum with a set of hormones, growth factors, attachment proteins, and transport proteins that will substitute for serum in the cell culture system. After third point: The amount of each factor will be cell-specific and vary with the parameter being optimized (i.e. cell proliferation vs. cell differentiation). In general, hormone requirements of the same cell type under in vitro conditions are similar, even if the cells are derived from different species, or animals at different stages of development, or primary cultures vs. cell lines. Concluding statement: Another approach is to gradually wean cells off serum by lowering the amount of serum in the medium for each subculture process. 10% - 8% - 6% - 4%, etc. Let cells adapt and grow at lower serum amounts. This may not work for all cells.

Lecture 4b  culture conditions and media Lecture 4b culture conditions and media Presentation Transcript

  • Scaling up the production process
    • Oxygen requirements
    • Supply of oxygen to satisfy cell metabolism is one of the major problems associated with culture scale up.
    • O2 consumption rate: 0.06-0.6 mmole/hour for 10^6 cells/ml
    • For small volumes (< 1 litre) O2 diffusion form the headspace through the culture surface
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • Culture Medium
    • animal cells require chemically complex liquid media suitable for growth of several generations
    • commercially made, supplied as 1x liquid ready for use, 10x concentrated solution, or powdered form
    • dilute concentrate with presterilized distilled water
    • dissolve powdered medium in water and filter sterilize
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P56.
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 22.
  •  
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • Components of a typical culture medium:
    • Carbohydrates
    • Amino Acids
    • Salts
    • Buffering system
    • Vitamins and hormones
    • Antibiotics
    • Phenol red
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 1. Carbohydrates
    • glucose (25 mM) used most often as energy source
    • may use alternative carbohydrate sources such as fructose, galactose, or maltose (reduced lactic acid production, more stable culture pH)
    • precursor for biosynthesis ribose through the pentose phosphate pathway (for nucleic acid synthesis)
    • can also use glutamine (4 mM) for energy
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 56.
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 2. Amino acids
    • 0.1-0.2 mM added as a source of precursors for protein synthesis
    • glutamine (2-4 mM) used precursor for TCA intermediates, purines, pyrimidines, amino sugars, and asparagine
    • ammonia is released as a by-product of glutamine breakdown, can be inhibitory to growth (half-life of glutamine at 4 o C is 3 weeks)
    • occurs via cellular metabolism or thermal breakdown
  • Specific amino acid consumption in 2 cultures
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P57.
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • to reduce accumulation of ammonia:
    • -> use continuous feed of low [gln]
    • -> use gln-containing dipeptides that hydrolyze slowly in culture
    • alanine accumulates as a result of ammonia uptake/removal
    • glutamate may be used as a substitute, results in lower ammonia produced
  • Energy metabolism glucose-6P Glucose P-gluconate ribulose-5P Ribose-5P fructose-diP P-enolpyruvate pyruvate Lactate acetyl CoA citrate 2-oxoglutarate succinate oxaloacetate TCA cycle Glutamine glutamate NH 3 NH 3 CO 2 CO 2 CO 2 CO 2 Alanine transamination
  • Radioactive glucose for metabolic flux analysis CH 2 OH C C C O C HC.OH OH OH OH H H H H 1- 14 C 6- 14 C 3- 3 H
  • Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 3. Salts
    • included to make solution isotonic and to maintain balance with the intracellular content
    • Osmolarity – measure of the total number of particles dissolved in solution
    • a control parameter, can affect property of a culture
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 1 Osmolar ( 1 Osm/l) has 6.023x10 23 particles in 1 liter of water
    • ionization increases the number of osmotically active particles
    • based on total number of particles dissolved in solution
    • nature of particles is irrelevant
    • Ex. 1 mM glucose -> 1 mOsm/l
    • 1 mM NaCl -> 2 mOsm/l
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • osmolality (mOsm/kg of water) = osmolarity (in dilute solution)
    • osmolarity of standard culture medium (isotonic) ~ 250-350 mOsm/l (300 mOsm -> optimal)
    • -> hypertonic – osmolarity is too high (cells shrink)
    • -> hypotonic – osmolarity is too low (cells burst)
    • osmolarity of medium should be within 10% of optimal value
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • higher osmolarity, the lower the freezing point
    • water has a freezing point of 0 o C, a saline solution with an osmolality of 1 Osm/kg has a freezing point of -1.858 o C
    • can use osmometer to determing freezing point of a solution relative to water
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 4. Buffering system
    • Bicarbonate is typically included to act as a buffer system in conjuction with a CO 2 atmosphere (5-10%)(enriched air), maintains pH range of 6.9-7.4
    • Advantage: cheap
    • Disadvantage: cultures may become alkaline very quickly upon removal from incubator
    • alternate buffers may be used
    • -> HEPES (pKa = 7.3 at 37  C)
    • -> MES (pKa = 6.5 at 37  C)
    • -> CHES (pKa = 9.5 at 37  C)
    • 10-20 mM HEPES is optimal, cultures can be grown without an enriched CO 2 atmosphere, expensive
  •  
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 5. Vitamins and hormones
    • present in micromolar amounts, used as metabolic cofactors
    • varies from one medium formulation to another
    • different cell lines have different requirements
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 6. Antibiotics
    • often included in media for short-term cultures in order to reduce the risk of bacterial or fungal contamination
    • often used in combinations as an antibiotic cocktail
    • -> Penicillin G (100 u/mL) inhibits Gram-positive bacteria by inhibiting cell wall synthesis
    • -> Streptomycin (50 mg/L) inhibits Gram-positive and Gram-negative bacteria by inhibiting protein synthesis
    • -> Amphotericin B (25 mg/l) as an antifungal agent by inhibiting ergosterol synthesis
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • not used for routine subculture or stock culture
    • low levels of bacteria or fungus may be masked
    • may allow for selective retention of antibiotic resistant contaminants
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media
    • 7. Phenol red
    • pH indicator of the medium
    • sensitive to slight pH changes around the growth optimum for cells
    • at lower pH, the phenol red turns orange (pH 7.0) or yellow (pH 6.5)
    • overnight change from red to yellow indicates bacterial contamination
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum
    • Serum
    • added as a supplement ( 10% v/v )
    • supernatant of clotted blood , contains undefined materials essential for cell growth, esp. for anchorage dependent cells
    • -> pH 6.85 – 7.05
    • -> osmolarity – 250-295 mOsmol/L
    • -> protein content – 60-80 mg/mL
    • -> albumin content - 30-50 mg/mL
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum
    • Serum provides:
    • Growth factors
    • Lipids
    • Attachment factors
    • Metal transporting proteins (i.e. transferrin)
    • Protease inhibitors (neutralizes trypsin)
    • High protein content protects against toxins, shear damage by stirring in fermenters
  • Lecture 4 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum
    • cow or horse serum most commonly used
    • fetal calf serum (FCS) most effective due to high content of embryonic growth factors
    • -> newborn calf serum (under 10 days)
    • -> donor calf serum (under 8 months)
    • serum is tested for performance and cytotoxicity effects on plating efficiency and cell growth
    • filtered and tested for bacterial, fungal, and viral contamination
  • Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum
    • Disadvantages of serum:
    • Expensive
    • Chemically undefined
    • High protein content
    • Source of contamination
    • Ethical considerations?
  • Lecture 4.5 Animal Cell Biotechnology Cell Culture Conditions and Media - Serum
    • want to use serum-free media
    • supplement media with insulin, transferrin, ethanolamine, and selenite
    • specific for different cell types
  • Component characteristics of media formulations Serum based media Serum-free media Protein free media Animal-component free media Chemically-defined media
    • Media formulated goal:
    • Chemically defined
    • Protein-free
    • Animal component-free