Development of media

• Initial attempt to culture cells were performed in natural

media based on tissue extracts and body fluids
• Chemically defined media were introduced in the 1950s
Making informed choices: media,
– Eagle’s Basal Medium
serum, additives etc. – Eagle’s Minimal Essential Medium (MEM)
– Dulbecco’s modification of MEM (DMEM)
– Supplemented with serum, protein hydrolysates or
embryo extracts

Optimization of media The role of cell culture media

• Practically all cells can grow in Minimum Essential Media • Provide essential nutrients
• Optimization goes in the direction of replacing serum or – Amino acids, fatty acids, trace elements, salts, vitamins
more selective media appropriate for a particular cell type and cofactors
– RPMI 1640 for lymphoblastoid cell lines • Maintain proper chemical environment
– Ham’s F-12 with higher content of vitamin and amino – pH and osmolarity mostly through ions and bicarbs
acids • Provide energy (carbon) source
– Leibovitz L-15 for cells grown without CO2
The choice of culture medium and supplements can have a
major impact on growth, function and phenotype of cells
in vitro

Basic constituents of media Mammalian cell culture media

• Inorganic salts • Salts: K+, Na+, Cl-, Mg2+, Ca2+
• Carbohydrates – Provide osmotic balance
• Amino acids – Help regulate membrane potential of the cells
• Vitamins – Some are required as cofactors for cell attachment
• Fatty acids and lipids factors
• Proteins and peptides • Carbohydrates
– Mostly glucose and galactose but also maltose or
fructose
• Trace elements: Se2+, Zn2+, Cr2+
– Selenium is a detoxifier and removes oxygen free
radicals

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 Mammalian cell culture media Fancy stuff
• 9 essential AA • Some nonessential molecules (pyruvate, hypoxanthine
– histidine, isoleucine, leucine, lysine, methionine, thymidine, adenosine) to improve growth
phenylalanine, threonine, tryptophan, valine • Fatty acids
– Most cell require also cysteine and tyrosine • Cholesterol (usually not included in the mixture of fatty
• Vitamins acids)
– Niacin, folic acid, riboflavin, inositol, thiamine • Detergent to emulsify lipids
Essential for replication but the deficiency does not – Toxic to some cells
manifest itself until several cell doublings • Hormones and growth factors
– Vit D, C, E, A • Specific factors for growth and differentiation
Antioxidants and differentiation agents • Phenol red as pH indicator
Commonly not added because unstable!!!

Physicochemical properties of
mammalian cell culture media pH
• pH • pH 7.4-7.7
• Osmolarity • Phenol red is commonly used as an indicator
• Surface tension and foaming – Red at 7.4
– Orange at 7.0
– Yellow at 6.5
– Purple at 7.8
• Most often maintained by CO2/bicarbonate buffer
– Cells HAVE to grow in the CO2 incubator

CO2 and bicarbonate Other buffering

• Most commonly used method to stabilize pH • Culture media must be buffered under two conditions
• “natural” buffer, low cost, non-toxic – Open dishes when pH rises
• H2O + CO2 H+ +HCO3- – Overproduction of CO2 and lactic acid at high cell
• Cells grown in open vessels such as dishes need to be concentration
incubated in CO2 • HEPES in addition to bicarbonate/CO2 buffer
• CO2 level must be set to match the medium • To stabilize pH when cells are out of the incubator
• Bicarbonates are also energy source
• Serum also has considerable buffering capacity

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 It’s in the water Basic Media
• Prepared by reverse osmosis to final resistance of 16-18 MΩ Mammalian Insect
Glucose 4 g/l 2.5 g/l
Amino acids 0.01-0.15 g/l 0.1-1.5 g/l
Glutamine 1 g/l 1 g/l
HCO3 3.5 g/l 0.35 g/l
H2PO4 0.1 g/l 1 g/l
Salts 4.5 g/l NaCl 1g/l MgSO4,KCl
Vitamins More Less
pH 7.2 6.4
Osmolarity 300 mOsm 350 mOsm

How to select the appropriate medium Some special media

• Obtain info from the same source the cells are coming • DMEM is optimized for use with serum supplementation
from and high density growth
– Check the CO2 settings appropriate for this medium • Ham’s nutrient mixtures F-12 and F-10 for CHO cells and
– Change medium if CO2 is different from commonly used fibroblasts at low density
in the lab • Leibovitz l-15 growth without CO2 incubator
• Best growth is not always the fastest !!!
• Optimize media for your experiment

Serum Identified serum components

• Most widely used additive to cell culture medium • Contains release products from platelets
• Source of identified and unidentified growth factors, • Includes PDGF, PD-ECGF
metabolites, hormones etc. • Serotonin (5-HT), ADP, ATP
• Least defined and most variable component of tissue • Platelet Factor IV
culture • Insulin, transferrin, ferritin
– Variable from lot to lot • LDL’s, albumin, factors bound to albumin
– Contaminants or degradative enzymes
– Infectious agents (viruses, prions)
– Expensive
• Increases buffering capacity
• Able to bind and neutralize toxins

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 Fetal Bovine Serum (FBS) Alternatives to serum- plasma
• Most often used type of serum • Prepared by spinning out platelets, then clotting blood
• Each serum batch is produced from over 1000 fetuses to • Eliminates contributions from platelet granule factors
account for gestational stage, gender, breed and • Should contain low levels of PDGF
biochemical composition • PDGF is a growth factor for smooth muscle and fibroblasts
• Potential source of adventitious agents (mycoplasma and • Might not want this present for culturing cells not of these
viruses) origins, like endothelium
• Very sensitive to degradation, adsorption and accidental
contamination

• Other type of serum – newborn calf serum or horse serum

Serum free media are more defined

Optimization of nutrient composition and contain
• Serum free media • Transferrin/ferrous citrate
• Co-culture on the feeder layer • Lipid concentrate
• Yeast extract
• Insulin
• Bovine Serum Albumin
• Pluronic

• Completely mammalian origin free (MOF)

Breast carcinoma on feeder layer of human
fetal intestinal cells
Carcinoma cells display tubular, more in vivo
like morphology

To add or not to add…

antibiotics Last but not the least - temperature
• Reduce the frequency of contamination • The optimal temperature is dependent on body temp. of
the animal from which the cells were obtained
• Encourage the development of antibiotic resistant strains – Mammals 37oC
• Hide the low level cryptic contaminants – Birds 38.5oC
• Have antimetabolic effect – Fish 22oC
• Encourage poor aseptic technique • Anatomical variation of temperature (such as skin and
testis)

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 What is cryopreservation?
• Storage below –130oC
• A state of suspended animation
– Ultra low temperatures
– Stops cell division & metabolic processes
Cryopreservation
– Very long-term (indefinite?) storage
• Viability decreases over time

Need for cryopreservation Equipment for cryopreservation

• Maintain reserves without constant care • Liquid nitrogen
– Save time and reagents – Liquid phase (-196oC) or vapor phase (-156oC)
– Replacement of contaminated cultures • Fancy freezing chambers to regulate the freezing rate
• Reduce alterations or loss of culture characteristics – Controlled rate freezing unit
– Genotypic drift due to genetic instability – Styrofoam containers work too
– Senescence • Water bath for resuscitation
– Transformation
– Phenotypic instability due to selection and
dedifferentiation
• Need for distribution to other users

Optimal Freezing Freezing cells

• Highest survival rate • The most critical phase
• Is Accomplished By • Freeze only well growing, contamination free cells
– Cooling slowly to allow for removal of free water from – Culture medium changed 24 hours prior to freezing
the cytosol – Visually examine
– Avoid crystal formation – Check for contamination
– Use hydrophylic cryoprotectant • Cells have to be in suspension (if needed trypsinize them)
– Storing below -130oC • Remove old media and resuspend in “freezing” media that
– Thawing rapidly to minimize crystal growth contain cryoprotective agent
– Media with cryoprotective agent has to be cold (see
controlled rate of freezing)

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 Cryoprotective agents Controlled rate of freezing
• To protect against ice damage (help in dehydration) and • Optimal freezing rate is 1-3o/hour (do not forget about
alter the form of ice crystals cryopreservative agents)
• Most often used – DMSO (dimethylsulfoxide) and glycerol – Compromise between minimizing crystal formation and
but also methanol, isopropanol, ethylene glycol, methyl allowing for water migration
acetamide • Water moves out of the cell if the temperature slowly goes
• Cryoprotective agents are used in 5 –10 % concentration from room temp to – 80oC
in media with serum • No crystals form below –130oC
– Increase in serum concentration increases survival and • Cells are frozen in tubes inside of styrofoam box
sometimes 95% serum (no medium just cryoprotective • Use cold “freezing” media to initiate slow cooling + cells
agent + serum) is used with difficult cells do not like cryoprotective agents in warm temperatures

Storage Storage
• Usually in liquid nitrogen(-196oC) to avoid changes in • Remember to LABEL your cells. You might not be around
ice crystals that occur above -130oC when they are taken out from storage
• Always keep the temperature below –130oC • Make sure your labeling system is suitable for liquid
• After cooling on ice transfer to –20oC, -70 oC and finally nitrogen
liquid nitrogen • Keep good records of the locations and characteristics of
• If you have a controlled freezing unit cells can be put in your cultures
–70oC after initial cooling on ice • Remember liquid nitrogen is not sterile and behave
• Transfers have to be done quickly and efficiently to accordingly
avoid even partial rises in temperature • Be prepared for emergencies

The living rate Thawing Stored Ampoules

• Various companies offer to freeze your body after death • Thawing should be rapid
for potential reviving later. – Water bath @ 37°C
• Current prices: – Reason: minimize crystal formation
– Whole body- $1500/yr • Wash vial with alcohol (liquid nitro is not sterile)
– Head only- $250/yr • Dilution should be done slowly
– Brain only- $200/yr – DMSO will cause severe osmotic damage if done fast
– "Perpetual storage"- $120,000 • Change media to remove DMSO within 24 hours
– Suspension growing cells have to be centrifuged

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 Freezing cells Protocol
• Cells should be in excellent condition, change media a day
before if needed
• Remove old media
• Trypsinize cells (if they are in suspension skip this step)
• Inactivate trypsin
• Centrifuge them and remove media with trypsin
• Add cold media with cryoprotectant
• Put immediately on ice
• Transfer to -20oC after 1-2 hours
• Transfer to -70oC next day
• Transfer to liquid nitrogen next day for storage

Recovery
• Thawing
– Usually rapid thawing to avoid damage from ice crystal
growth
– Immerse in water bath and transfer to prewarmed
media
– Avoid excessive alkalinity – dish should be placed in the
incubator to allow the saturation with CO2
• Recovery
– Thawed cells must be washed of cryoprotectants within
24 hours and nursed back to normal growth
• Dilute cells slowly