Lecture 7 Serum-free media, Defined media supplements

 Lecture 7 Serum-free media, Defined media supplements

• Media development
  • tissue extract, body fluid
  • Eagle MEM→calf, human, horse serum, protein hydrolysate, embryo extract
  • serum-free→facilitate downstream process & reduce risk of adventitious infectious agents
• Physiochemical
  • pH
    • normal fibroblast 7.4 to 7.7
    • transformed 7.0 to 7.4
    • phenol red at 7.4, orang at 7, yellow at 6.5, lemon yellow <6.5, pink at 7.6, purple at 7.8
  • buffer
    • open dish→CO2 cause pH high
    • overproduction of CO2 & lactic acid in transformed cell lines at high cell concentrations
    • bicarbonate HCO3 buffer
      • widely use
      • requires CO2 incubation
      • pH 7.2-7.4
      • inexpensive, non toxic
      • pH fluctuation when expose to air
    • HEPES
      • pH 6.8-8.2
      • more stable than HCO3
      • w/o CO2 incubation
      • better pH stability, less sensitive to temp
      • more expensive, potential toxicity at high concentrations
    • phosphate buffer
      • pH 6.8-7.4
      • less common
      • stable, inexpensive
      • can interfere with certain cellular process
    • maintain optimal pH, critical for cell growth, metabolism, overall health
    • purpose
      • stabilize pH
      • counteract pH changes from cellular metabolism
      • maintain optimal conditions for cell growth & function
  • CO2 HCO3
    • CO2 in gas phase dissolves in medium, establish equilibrium with HCO3→lowers pH
    • effect of elevated CO2 tension neutralized by high HCO3 concentration
    • inclusion of high concentration of Na pyruvate enable cells increase CO2→independent of exogenous CO2 & HCO3
  • O2
    • anaerobic, glycolysis for energy production, O2 still need
    • dissolved O2 may be toxic→elevation of free radicals
    • incorporation of free radical scavengers (glutathione, beta mercaptoethanol or dithiothreitol) into medium
    • most cell prefer lower O2, some organ cultures require >95% O2 in gas phase, some tumor cells prefer hypoxic
  • Osmolality
    • cell growth, metabolism, protein production
    • concentration of osmotically active particles in a solution→mOsm/kg
    • mammal 260-320
    • effects
      • low→cell swell & lysis
      • high→cell shrink, reduce growth rate
    • factors
      • base media composition
      • serum/protein supplement
      • added nutrient/GF
      • pH adjustment
      • accumulation of metabolic by product
    • hypotonic for open-plate/petri dish
  • Temperature
    • optimal depends on
      • body temp
      • anatomic variation
      • incorporation of safety factor to allow for minor errors in regulating incubator
    • +-5C
    • overheating more serious→mammalian cells can survive several days at 4C but cannot tolerate more than 39.5C for >2h
    • most maintain at 37C
    • avian cell 38.5C
    • cold blood animal 15C to 26C→maintain at room temp/cool incubator
    • influence pH due to increase CO2 solubility at low temp
    • decrease pH to 0.2 unit lower at RT than at 37C
  • Viscosity
    • cell growth
    • cell suspension is agitated
    • cells are dissociated after trypsinization
    • cell damage reduce by increasing viscosity with CMC/PVP
  • Surface tension & foaming
    • foaming arise in stirrer vessel/bioreactor when 5% CO2 bubbled
    • limit gaseous diffusion, increase contamination if foam reach neck
    • +silicone antifoam, pluronic F68→reduce surface tension, protect cells from shear stress of bubbles
• Balanced salt solution BSS
  • pH & salt concentration
  • composed of inorganic salt & may include NaCO3, glucose
  • form basis of complete media
    • modified by omitting glucose, phenol red, Ca2+, Mg2+
    • deficient in Ca2+ reduce cell aggregation & attachment
  • EMEM Hank's salt→sealed flask with a gas phase of air
  • EMEM Earle's salts→higher HCO3 with 5% CO2
  • HEPES (10-20mM)→pH 7.2-7.8
  • tricine pH 7.4-8
  • function
    • diluent for concentrates of aa & v to make complete media
    • isotonic wash/dissection medium
    • short incubation >4h with glucose present
• Complete media
  • aa
    • essential aa + cysteine, arg, glu, tyr
    • aa concentration limit max cell concentration attainable→cell survival & growth rate
    • glu→energy & carbon source, unstable as generate NH
    • glutamax→alanyl-glu dipeptide; stable & bioavailable
  • v
    • EMEM→water soluble VB exclude biotin
    • biotin, PABA, fat-soluble VADEK in complex
    • precipitation of folate reduce cell survival & growth rate
  • salt
    • Na K Mg Ca Cl SO PO HCO3
    • Earle's→high HCO3, 5% CO2 at gas phase
    • Hank's→low HCO3, air at gas phase; BSS
    • Ca2+ required by CAM→signal transduction, cell proliferation, differentiation
  • glucose
    • energy source
    • glycolysis→pyruvate to lactate/acetoacetate, enter TCA, produce CO2 + H2O
  • organic supplements
    • protein, peptide, nucleoside, TCA intermediate, pyruvate, lipids when serum concentration reduce
    • hormones & GF not specified
  • Ab
    • reduce frequency of contamination
    • encourage Ab-resistant
    • hide presence of low level cryptic contaminant
    • hide mycoplasma
    • antimetabolic
    • encourage poor aseptic technique
• Serum
  • growth factor
    • cell proliferation, adhesion factors, antitrypsin activity
    • small amount
    • PDGF
  • growth hormone
    • in conjunction with somatomedin IGF→mitogen
    • hydrocortisone promotes cell attachment and proliferation
  • CS BS
  • protein
    • major
    • increase viscosity, reduce shear stress, add to medium's buffering capacity
    • albumin→carrier of lipid, mineral, globulin
    • fibronectin→promotes cell attachment
    • a2-macroglobulin→inhibits trypsin
    • transferrin + Fe→less toxic & bioavailable
  • nutrients & metabolites→important in simple media, less in complex
  • lipid→bound to proteins (albumin)
  • minerals→bound to albumin
  • inhibitors
    • inhibit cell proliferation
    • artifact preparation/physiological negative growth regulators (TGF-B)
  • heat inactivation removes complement (-labile constituent) & reduces cytotoxic action of Ig w/o damage polypeptide growth factors
  • disadvantage
    • physiological variability
    • shelf life & consistency
    • QC
    • specificity
    • availability
    • downstream processing
    • contamination→virus
    • cost
    • growth inhibitors
    • standardization
• Selection
  • RPMI 1640, DMEM, MEM→75% sales
  • cell bank
  • FBS more expensive then FCS
  • ready made expensive than powder
  • quality of serum can be tested by plotting growth curve, perform specific functional assays, examine sterility
• Serum free media