Transcript Defined Media and Supplements

Defined Media and
Supplements
Chapter 9
Reasons for development of media
Cells cultured in natural media
 Cells cultured in chemically defined media
based on analyses of body fluids and nutritional
biochemistry
 Eagle’s basal Medium, Eagle’s Minimal Essential
medium (MEM), Dulbecco’s modification of
Eagle’s medium (DMEM) etc
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Physicochemical properties
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Most cell lines grow well at pH 7.4
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Transformed cell lines @ pH 7.0-7.4
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Phenol red is used as an indicator
Role of Co2, Bicarbonate ions and pH
Requires both - optimum cell growth
 Table 9.1 shows the amount of Co2, HCO3- and
HEPES
 Inclusion of pyruvate in medium enables cells to
increase their endogenous production of CO2 making
them independent of exogenous CO2 as well as
HCO3
Co2, Bicarbonate and pH
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Co2 in gas phase dissolves in medium
Establishes equilibrium with HCO3- ions and lowers
pH.
H2O + CO2
H2CO3
H+ + HCO3-
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Balanced by bicarbonate concentration from a base.
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Reduction of Oxygen Toxicity
In vivo – oxygen required for respiration
 In vitro – glutathione
 Cell cultures – require low oxygen tensions
 Organ cultures – late-stage embryos, newborns or
adults require 95% O2
 Selenium tolerance is provided
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Reduction of Viscosity
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Cell damage can be reduced by –
Carboxymethylcellulose (CMC) or
Polyvinylpyrrolidone (PVP)
Reduction of surface tension and
foaming
Foaming can lead to Protein Denaturation
 Contamination
 Limit gaseous diffusion
 Arises in suspension cultures in stirrer vessels or
bioreactors
 Silicone antifoam or Pluronic F68 – 0.01-0.1% prevents foaming
 Prevents foaming by reducing surface tension and
may protect cells against shear stress from bubbles
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What is Balanced Salt Solutions?
BSS is composed of inorganic salts, may include
sodium bicarbonate and glucose
 Forms basis of complete media
 BSS recipes are modified
 PBS without Ca2+ and Mg2+ = known as PBS
solution A
 D-PBSA
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Usage of BSS
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Depends on CO2 tension
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Tissue disaggregation or monolayer dispersal
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Suspension or adherent cell culture
What is Complete Media?
media – all constituents (glutamine)
and supplements (serum, growth factors or
hormones)
 Complete
9.4.1 Amino acids
Essential amino acids – cysteine, arginine,
glutamine and tyrosine
 Individual requirements vary with cell type
 Responsible for cell growth and survival
 Other nonessential amino acids are added
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9.4.2 Vitamins
Eagle MEM - Water soluble vitamins – B group,
choline, folic acid, inositol and nicotinamide
 M199 - Fat soluble vitamins (A,D, E and K)
 LHC-9 has Vitamin A
 MCDB 110 has Vitamin E
 Individual requirements vary with cell type
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9.4.3 Salts
Na+, K+, Mg 2+, Ca2+, Cl-, SO4, PO4 and HCO3- maintain osmolality of medium
 Ca2+ - signal transduction process, whether
proliferate or differentiate
 Na+, K+ and Cl- regulate membrane potential
 SO4, PO4- and HCO3- act as nutritional precursors
for macromolecules and regulate intracellular charge
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9.4.4 Glucose
Source of energy
 Metabolism of glucose – by Glycolysis to form
Pyruvate
 Pyruvate - converted to lactate or acetoacetate –
enters into citric acid cycle
 More energy derived from glutamine than glucose
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9.4.5 Organic Supplements
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Proteins, Peptides, Nucleosides, Citric acid cycle
intermediates, Pyruvate and Lipids
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Help in cloning and maintaining certain specialized
cells (in presence or absence of serum)
9.4.7 Antibiotics
Disadvantages: Encourage development of
antibiotic-resistant organisms
 Hide cryptic contaminants
 Hide mycoplasma infections
 Antimetabolic effects that can cross-react with
mammalian cells
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9.5 Serum
Commonly used : bovine calf, fetal bovine, adult
horse and human serum
 Calf (CS) and fetal bovine (FBS) – used for cell
lines and cloning
 Human serum – used for human cell lines
 Horse serum is consistent from batch to batch
- Less polyamines
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9.5.1 Protein
Albumin – important carrier of lipids, minerals and
globulins
 Fibronectin – promote cell attachment
 Fetutin – enhance cell attachment
 Transferrin – binds iron
 Increases viscosity of medium, reducing shear stress
during pipetting and stirring and adds to medium’s
buffering capacity
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9.5.2 Growth Factors
Platelet-derived growth factor (PDGF)
 Fibroblast growth factor (FGFs)
 Epidermal growth factor (EGF)
 Insulin-like growth factors IGF-I and IGF-II
 Help in Mitogenic activity and stability
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9.5.3 Hormones
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Hydrocortisone – present in fetal bovine serum – can
promote cell attachment and proliferation or cell
differentiation
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Insulin – uptake of glucose and amino acids – binds
to IGF receptors
Selection of medium and serum
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RPMI 1640, DMEM and MEM – 75 % sales
DMEM/F12 – 4%
DMEM has twice the amino acid concentration of MEM,
four times the vitamin concentrations and twice the HCO3and CO2 concentrations to achieve better buffering
MEM has additional amino acids, vitamins, nucleosides
and lipoic acids
Testing serum
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Plating efficiency: check growth of cells in Cloning,
count them
Stain and count colonies
Plating efficiency (survival) and colony size (cell
proliferation)
Tested at a range 2-20%
Testing serum
Growth curve: lag period, doubling time and
saturation density
- Lag period – culture has to adapt to serum
- Saturation density – more cells will grow in a given
amount of serum
 Preservation of cell culture characteristics
 Sterility
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