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Cell Culture Environment
How to grow a plant ?
Water
Soil
Air (CO2 and O2 )
Nutrition
Water : Culture medium
Soil : substrate for cell growth
Air (CO2 and O2 ) : Air in incubation chamber
Nutrition : growth supplement
Culture Medium
Functions of culture medium:
1. Maintain the pH
2. Maintain the osmolarity
3. Provide nutrient
4. Provide energy
Basic components of cell culture environment
The in vitro environment must meet the
fundamental physiological requirements of the cell
1. Culture medium
2 . Physiological factors
pH 7.27.5
osmolarity 280320 mOsmol/kg
CO2
25% in air
temperature 35oC-37oC
3. Stationary versus dynamic media
supply enough
replenish at proper time
vol. 0.20.3ml/cm2
A complete culture media:
1. A basal medium specifies for all cellular
requirements
2. A set of components that specify other
cellular requirements and permit
growth of cells in the basal medium
Basic Energy source for cell growth
Carbohydrate
sugar
starch
Basal Medium
Types of basal medium
1.Eagles medium and derivative
e.g. BME, EMEM, AMEM, GMEM, JMEM
2. Media designed at Roswell Park
Memorial Institute ( RPMI)
e.g.RPMI 1629, RPMI 1630, RPMI1640
3. Basal medium designed for use after
serum supplement
e.g. Fischer’s Lieboutz, Trowell, Will
4.Basal medium designed for serum-free formulation
e.g. CMRL 1060
Ham’s F10 and derivatives
TC199 and derivatives
NCTC
Waymouth
5. For insects culture
e.g. Grace’s medium
Schneider’s medium
Mitsuhashi and maramerosch medium
IPL-41 medium
Chiu and black medium
D-22 medium
What does cells need to grow better?
Basic components of the culture medium
1.Buffer system
ß - glycerophosphate
organic components
e.g. HEPES, Tes, Bes
2. Energy Source
glucose, maltose, sucrose, fructose, galactose,
mannose
3. Amino Acid
essential amino acid
4. Vitamins
precursor for the cofactors
e.g. biotin, choline, folic acid, nicotinic acid…
5 . Hormones and growth factor
e.g. insulin, hydrocortisone, NGF, EGF
6. Proteins and Polypeptide supplement with:
fetuin, -globulin, fibronectin, albumin, transferrin….
7. Fatty acid and Lipid
8. Accessory
e.g. Zn,Cu,Se….
10. Antibiotics
factors to be considered:
 absence of cytoxisity
 broad anti-microbial spectrum
 accepted cost
 minimum tendency to induce
 formation of resistant micro- organism
e.g. penicillin, streptomycin gentamycin.
Buffer System
Bicarbonate buffer
Recommended CO2 concentration and gas phase to use with common
basal media
basal medium
NaHCO3
concentration
Eagle’s MEM(Hank's salt)
grace’s (Hank salt )
IPL-41 (Hank salt )
TC100 (Hank salt )
Schneider’s (Hank salt )
IMDM
TC199
DMEM/Ham’sF12
RPMI1640
Ham’sF12
DMEM
4
4
4
4
4
36
26
29
24
14
44
% of CO2
gas phase
atmospheric
atmospheric
atmospheric
atmospheric
5
5
5
5
5
10
Choice of basal medium
from literature
e.g. BME: for HeLa, L-cells, BHK,
primary culture of human rodent and avian
fibroblast
e.g. RPMI: for human haemopoietic cells
e.g. Iscove’s modified Dulbecco’s medium( IMDM):
for haemopoietic origin, growth and
differentiation of human and murine primary
bone marrow culture
e.g. sf9,sf21: for Drosophilla and insect culture
Preparation of basal medium
Factors to be considered:
 Avoid contamination
 Use pure water
 Use analytical chemicals
 Glassware used must be cleaned
Equipment for preparation of media:
 High purity chemicals and biologicals
 Good analytical balance
 Hot plate with magnetic stirrer
 Volumetric flasks of various volume
 pH meter
 Osmometer
 Autoclave and membrane filtrate
 medium kept at 4oC
 storage:
storage should not be over 3 months
Serum
Why use serum ?
Advantages of using serum
1. Serum represent a cocktail of most of the factors
2. Required for cell proliferation and maintenance.
3. An universal growth supplement which is effective
with most cells.
4. Serum buffers the cell culture system against a variety
of perturbation
Types of serum
1. FBS ( Fetal Bovine serum) from abattoir
2. Horse serum, calf serum from donor )
should pass virus test)
Constituents of serum
1.Growth factors ; 5—30 kDa polypeptide
2.Albumin
a. a carrier protein
b. carry thyroxin and metal
c. pH buffering
3. Transferrin-ion transport protein
4. Anti-protease: prevent cell from proteolytic damage
1-trypsin
b2-macroglobulin
Nutritional and protective factors which may be supplied by
serum
1.Specific growth factors-EGF,PDGF,IGF,FGF, IL-1,IL-6
2.Trace elements-Iron, Zinc, Selenium
( Co,Cu,I,Mn,Mo,Cr,Ni,V,As,Si,F,Sn)
3. Lipids-- cholesterol, linoleic acid, steroids
4. Polyamines--putrescine, ornithin, spermidine
5. Attachment factors-- fibronectin, fetuin, Laminin
6. Mechanical protection– albumin
7. Buffering capacity– albumin
8. Neutralization of toxic factors– albumin
9. Transport of metals
transferrin/Fe+3,
ceruloplasmin/Cu+2
10.Protease inhibitors– antitrypsin,b1macroglobulin
Potential problems with the use of serum
1. Lack of reproducibility
 Serum batches varies considerably
 The presence of specific antibodies may
also affect the results
 Serum may vary during the process of collection
 Sterity
2. Risk of contamination
 To prevent viral infection:
b-propiolactone
-irradiation
heat inactivation( 56oC, 1hr)
3. Availability and cost
4. Influence of downstream process
 difficulties in purifying proteins
 serum protein 4—8 mg/ml,
 recombinant protein---- 0.1ug/ml
 difficulties in purifying monoclone antibody
Sourcing and selection of serum
 sourcing:
dealing with reputable supplier selection:
test the capability for growth on cheaper serum type
test different batches of serum for growth performance
test of cell growth from very low seeding
test of cloning efficiency
Serum storage and use
kept frozen, -20oC
thaw rapidly, mix gently
kept at 4oC once thawed
Replacement of serum in medium
 Replace by Controlled Process Serum Replacement
( CPSR)
CPSR: derived from bovine plasma,
lower endotoxin
lower protein
 Replace by supplement or fortified serum
fortified serum: supplement with mitogen, growth factor,
hormones, proteins, protein stabilizer, trace elements
Serum Free medium
A properly designed serum free medium
 is reproducible
 is not reliant on economics of the world cattle market
 simplifies down stream purification
 has no unknown factors e.g. viruses, growth inhibitors
The most common requirement of serum free medium
e.g. polypeptide hormone, insulin, iron transport protein,
transferrin
Supplement of serum free medium
e.g. steroid growth factors, trace elements reducing
agents, diamines, vitamins albumin complexes with
unsaturated fatty acid
Design of serum free media
Reduce serum gradually
Adding other components
Growth assay study in the presence of added
supplements for basal medium
Add components singly or in combination to a basal
medium in a step wise manner
Mechanical stabilizers and adhesion factors
 For suspension culture:
 To prevent shear damage: damage by air bubble, stirrer,
shaker…
improved by increase viscosity of the medium by adding:
carboxy methyl cellulose ( viscosity modifier) and
prolyvinylpyrrolidone( PVP), pluronic-F68
principles: formation of an interfacial
structure of adsorbed molecule on cell
surface)
Considerations :
 Energy source of basal medium
 Carrier of lipids
 Proper environment for metabolism
 Growth function of cells ( good buffering capacity)
 Good maintenance of electrolyte balance
 Fe+2---Zn+2----Cu+2 trace elements: Na+,K+,
Ca+2,Mg+2,Cl-,HPO4-2…..
 For anchorage dependent cells
 substrate treat with adhesive glycoprotein,
fibronectin, laminin, chondrotin, serum spreading
factors
Difficulties that may encountered with serum free medium
a. Design of a dedicated medium for each cell type
b. Culture condition become more critical
c. Serum free medium has a reduced capacity to inactivate
or absorb toxic materials
Selection of components
e.g. transport protein,stabilizing proteins growth
regulators, growth factors, attachment proteins,
crude extracts, essential nutrient
Practical hints on solubilizing specific components
e.g. riboflavin, folic acid, tyrosine, cystein
dissolve in NaOH
e.g. fatty acid, lipids, fat soluble vitamins
in alcohol solutions
e.g. pluoronic F-68
soluble in cold water
e.g. hypoxanthine
heat to dissolve