Sakeena Asmi MSc Biotechnology

1. CELL
CULTURE
MEDIA
SAKEENA ASMI
MAHATMA GANDHI
UNIVERSITY

2. INTRODUCTION
• The influence of cell culture technology on human society has been
immeasurable.
• Progress in biology in recent years has depended heavily on cell
culture technology.
• In addition, cell culture based practical technologies have been
developed in various areas, including the assessment of the
efficacy and toxicity of new drugs, manufacture of vaccines, and
biopharmaceuticals and assisted reproductive technology.
• Hence, cell culture technology is regarded as a foundation for
further development and popularization.
• In this context, the cell culture medium is the most important
factor in cell culture technology.
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3. • A medium supports cell survival and proliferation, as well as
cellular functions, hence the quality of the medium directly affects
the research results, the biopharmaceutical production rate and
treatment outcomes of assisted reproductive technology.
• It is essential therefore, for investigators who are working with cell
culture to select an appropriate medium that is suitable for their
aims.
• In some cases researchers should modify a medium themselves.
• In addition, when facing problems, researchers have to know the
properties of the medium in order to identify the cause of any
problem with their experiments.
Conti…
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HISTORY
• In 1882, Sydney Ringer developed Ringer’s solution, a balanced
salt solution of a composition that is close to that of bodily fluids,
and successfully kept frog hearts beating after dissection and
removal from the body.
• This is said to be the first instance of in vitro cultivation of animal
tissue. Balanced salt solutions were developed one after another in
the wake of Ringer’s report, including Locke’s solution, Tyrode’s
solution, the Krebs–Ringer bicarbonate solution, Gey’s solution,
Earle’s solution and Hanks’ solution.
• The composition of these balanced salt solutions is simple and
includes only inorganic salts, sometimes with glucose added as a
nutrient.
• Nonetheless, their pH, osmotic pressure, and inorganic salt
concentrations were calibrated to physiological conditions -

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and these solutions can be used successfully to keep tissues and
cells outside the body alive for short periods, generally up to a few
days.
• In 1907, Ross G. Harrison successfully monitored an apparent
outgrowth of nerve fibers of a frog for several weeks in lymph
fluid that had been freshly drawn from the lymph sacs of an adult
frog.
• This experiment is considered to be the beginning of animal cell
cultivation.
Conti…

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TYPES OF CELL
CULTURE MEDIA
Mainly 2 types of culture media are used in animal tissue culture :
1) NATURAL MEDIA
2) ARTIFICIAL / SYNTHETIC MEDIA
The type of medium depends on the type of cells to be cultured and
its objectives.

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NATURAL MEDIA
DEFINITION :
• Consisting of natural biological substances such as plasma, serum
and embryo extract.
• Natural media consists solely of naturally occurring biological
fluids.
• Natural media are very useful and convenient for a wide range of
animal cell culture.

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NATURAL
MEDIA
MEDIA TYPE EXAMPLES
COAGULANT OR
CLOTS
TISSUE EXTRACTS
BIOLOGICAL FLUIDS
Plasma separated from
heparinized blood, serum
and fibrinogen
Extracts of chicken embryos,
liver and spleen and bone
marrow extract.
Plasma, serum, lymph,
amniotic fluid and pleural
fluids
Conti…

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SYNTHETIC MEDIA / ARTIFICIAL
MEDIADEFINITION :
• Composed of a basal medium and supplements, such as serum,
growth factor and hormones.
• It is prepared by adding nutrients (both organic and inorganic),
vitamins, salts, O2 and CO2 gas phases, serum proteins,
carbohydrates, cofactors etc.
• Different artificial media have been devised to serve one or more
of the following purposes:
• 1) immediate survival (a balanced salt solution, with specific pH
and osmotic pressure)
• 2) prolonged survival (a balanced salt solution supplemented with
various formulation of organic compounds and/or serum)
• 3) indefinite growth
• 4) specialized functions.

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SYNTHETIC
MEDIA
MEDIA TYPE EXAMPLES
SERUM CONTAINING
MEDIA
SERUM FREE MEDIA
XENO FREE MEDIA
Human, bovine, equine or
other serum is used as a
supplement
Crude protein fractions such
as bovine serum albumin or
α or β globulin are
supplements
Human source components such
as human serum albumin are
used as supplements but animal
components are not allowed as
supplements
PROTEIN FREE
MEDIA
CHEMICALLY
DEFINED MEDIA
Undefined components such as
peptide fractions are used as
supplements
Highly purified components such
as recombinant proteins are
appropriate supplements
Conti…

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USE OF NATURAL MEDIA
• Alexis Carrel was a French surgeon and biologist who received the
Nobel Prize in Physiology and Medicine in 1912 for his research
on the vascular suture and transplantation of blood vessels and
organs.
• He contributed greatly to tissue culture technology by devising a
prototype of the cell culture flask that is used widely today and by
establishing the aseptic manipulation technique.
• The first success of animal cell culture by Harrison inspired Carrel
to send Montrose T. Burrows to work under Harrison’s supervision
in 1909.
• There, Burrows found that lymph is unsuitable for the cultivation
of cells from warm-blooded animals and used plasma instead.
• Thereafter, blood plasma had become a major culture medium for a
variety of animal cells.

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• He successfully cultivated chicken embryonic cells by using
chicken blood plasma, which is readily available, and later
successfully cultivated mammalian cells as well.
• In 1912, Carrel demonstrated that the long-term cultivation of
the cells that have been obtained from the connective tissues of
chick fetuses is possible (for several months) with a periodic
exchange of the medium.
• In 1913, he discovered that adding embryonic extract to blood
plasma can dramatically increase cellular proliferation and
extend the culture period of fibroblasts from the chick embryo
heart.
Conti…

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• Meanwhile, because the composition of the lymph, plasma, and
embryonic extract was unknown, it became a new scientific
inquiry regarding which of their components affected the
survival and growth of animal tissues and cells.
• This situation led to a period when researchers attempted to
identify the growth-promoting substances within these
ingredients of natural origin and to replace them with ingredients
of definite composition.
Conti…

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ARTIFICIAL MEDIA
(mainly composed of a BASAL media)
• Each basal medium has been designed in each case on the basis of
the cell type, the origin (animal species), and the purpose of the
culturing.
• In fact, the medium composition can differ greatly depending on
such background factors. Whether supplementation with natural
products is allowed is another important presupposition for the
choice of a basal medium.
• For example, MEM (developed by Eagle) was designed under the
assumption of serum supplementation and accordingly includes
only the minimum necessary components (inorganic salts, sugar,
essential amino acids, and water-soluble vitamins).
• In contrast, Medium 199 and Ham’s F-12, intended for serum-free
culture, contain various other components.

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TYPES AND CHARACTERISTICS OF
BASAL MEDIUM

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Conti…

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1) SERUM CONTATINING MEDIUM
• Serum contains growth factors, which promote cell proliferation,
and adhesion factors and antitrypsin activity, which promote cell
attachment. Serum is also a source of minerals, lipids, and
hormones, many of which may be bound to protein.
• The sera used most in tissue culture are bovine calf, fetal bovine,
adult horse, and human serum. Calf (CS) and fetal bovine (FBS)
serum are the most widely used, the latter particularly for more
demanding cell lines and for cloning.
• Human serum is sometimes used in conjunction with some human
cell lines, but it needs to be screened for viruses, such as HIV and
hepatitis B.
• Serum also adds buffering capacity to the medium and binds or
neutralizes toxic components.

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• Horse serum is preferred to calf serum by some workers, as it can
be obtained from a closed donor herd and is often more consistent
from batch to batch.
• Horse serum may also be less likely to metabolize polyamines,
due to lower levels of polyamine oxidase; polyamines are
mitogenic for some cells.
Conti…
FETAL BOVINE
SERUM (FBS)

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COMPONENTS OF SERUM
1. Protein:
• proteins are a major component of serum, the functions of many
proteins in vitro remain obscure; it may be that relatively few
proteins are required other than as carriers for minerals, fatty acids,
and hormones.
• Those proteins for which requirements have been found are
albumin, which may be important as a carrier of lipids, minerals,
and globulins.
• fibronectin (cold-insoluble globulin), which promotes cell
attachment, although probably not as effectively as cell-derived
fibronectin; and α2-macroglobulin, which inhibits trypsin.
• Fetuin in fetal serum enhances cell attachment and transferrin
binds iron, making it less toxic and bioavailable. Other proteins, as
yet uncharacterized, may be essential for cell attachment and
growth.

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2. Growth factor:
• PDGF stimulates growth in fibroblasts and glia, but other
platelet-derived factors, such as TGF-β, may inhibit growth or
promote differentiation in epithelial cells.
• Fibroblast growth factors (FGFs), epidermal growth factor
(EGF), endothelial cell growth factors such as vascular
endothelial growth factor (VEGF) and angiogenin and insulin-
like growth factors IGF-I and IGF-II which have been isolated
from whole tissue or released into the medium by cells in
culture, have varying degrees of specificity and are probably
present in serum in small amounts.
3. Nutrients and Metabolites:
• Serum may also contain amino acids, glucose, oxo(keto) acids,
nucleosides, and a number of other nutrients and intermediary
metabolites.
Conti…

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• These may be important in simple media but less so in complex
media, particularly those with higher amino acid concentrations
and other defined supplements
4. Lipids, Minerals and Inhibitors:
• Linoleic acid, oleic acid, ethanolamine, and phosphoethanolamine
are present in serum in small amounts, usually bound to proteins
such as albumin.
Conti…

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ADVANTAGES DISADVANTAGES
• Serum contains various
growth factors and
hormones which stimulates
cell growth and functions.
• Helps in attachment of the
cells
• Acts as a spreading factor
• Acts as a buffering agent
which helps in maintaining
the pH of the culture media.
• Functions as a binding
protein
• Lack of uniformity in the
composition of serum.
• Testing needs to be done to
maintain the quality of each
batch before using.
• May contain some of the
growth inhibiting factors
• Increase the risk of
contamination.
• Presence of serum in media
may interfere with the
purification and isolation of
cell culture products

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BASIC COMPONENTS OF A COMPLETE
MEDIA
• Components vary among cell lines, and these differences are
partly responsible for the extensive number of medium
formulations
• Each component performs a specific function:
Buffering systems:
Regulating pH is critical for optimum culture conditions and is
generally achieved by one of the two buffering systems:

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Natural buffering system :
• In a natural buffering system, gaseous CO2 balances with the
CO3/HCO3 content of the culture medium.
• Cultures with a natural buffering system need to be maintained in
an air atmosphere with 5-10% CO2, usually maintained by an CO2
incubator
• Natural buffering system is low cost and non-toxic.

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HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
• Chemical buffering using a zwitterion,
HEPES, has a superior buffering capacity in
the pH range 7.2-7.4 and does not require a
controlled gaseous atmosphere.
• HEPES is relatively expensive and toxic at a
higher concentration for some cell types.
• HEPES has also been shown to greatly
increase the sensitivity of media to phototoxic
effects induced by exposure to fluorescent
light.

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The essential amino acids are required by
cultured cells,
• Cysteine
• Arginine
• Glutamine and tyrosine
• Other non essential amino acids are
often added as well.
• The concentration of amino acids
usually limits the maximum cell
concentration attainable, and the
balance may influence cell survival
and growth rate.
Amino acids

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• Glutamine is required by most cells, although some cell lines
will utilize glutamate.
• Glutamine is also used by cultured cells as a source of energy
and carbon.
• Glutamine is unstable in culture medium with a half-life of
between 3 and 5 days, and it generates ammonia, which can be
toxic as a by-product.
• Glutamax (Invitrogen) is an alanyl-glutamine dipeptide that is
stable an bioavailable due to the action of dipeptidase
Glutamine

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• Eagle’s MEM contains only the water-soluble vitamins
excluding biotin.
• Biotin is present in most of the more complex media, including
the serum free recipes.
• p aminobenzoic acid (PABA) is present in M199, CMRL 1066
and RPMI 1640.
• All the fat-soluble vitamins (A, D, E, and K) are present only in
M199, whereas vitamin A is present in LHC-9 and vitamin E in
MCDB 110.
• Amino acids, vitamin requirements have been derived
empirically and often relate to the cell line originally used in
their development.
Vitamins

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SALTS
• The salts are chiefly those of Na+, K+, Mg2+, Ca2+, Cl−,SO4
2−,
PO4
3−, and HCO3− and are the major components contributing to
the osmolality of the medium.
• Ca2+ are required by some cell adhesion molecules, such as the
cadherins.
• Ca2+ also acts as an intermediary in signal transduction and the
concentration of Ca2+ in the medium can influence whether cells
will proliferate or differentiate.
• Calcium is reduced in suspension cultures in order to minimize
cell aggregation and attachment.

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• Na+, K+, and Cl− regulate membrane potential.
• SO4
2−, PO4
3−, and HCO3− have roles as anions required by
the matrix and nutritional precursors for macromolecules, as
well as regulators of intracellular charge.
• The sodium bicarbonate concentration is determined by the
concentration of CO2 in the gas phase and has a significant
role in buffering capability.

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• Glucose is included in most media as a source of energy.
• It is metabolized principally by glycolysis to form pyruvate,
which may be converted to lactate or acetoacetate and may enter
the citric acid cycle and be oxidized to form CO2 and water.
Glucose

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The most commonly used proteins are :
1) Albumin
• Albumin binds to water, salts, free fatty acids, hormones, and
vitamins, and transport them between tissues and cells.
• Also remover of toxic substance from media.
2) Aprotinin
• Aprotinin is a protective agent in cell culture systems, stable at
neutral and acidic pH and resistant to high temperatures and
degradation by proteolytic enzymes.
• Inhibit proteases such as trypsin
Proteins
3) Fibronectin
• Fibronectin in cell attachment.
4) Transferrin

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Trace elements are often supplemented to serum-free media
to replace those normally found in serum.
• Trace elements like copper, zinc, selenium are needed in
minute amounts for proper cell growth.
• These micronutrients are essential for many biological
processes, e.g. the maintenance of the functionality of
enzymes.
Trace elements

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Hormones and Growth Factors :
• Hormones and growth factors are not specified in the
formulas of most regular media, although they are frequently
added to serum-free media
Media supplements
Antibiotics:
• Antibiotics were originally introduced into culture media to
reduce the frequency of contamination.
• Antibiotics have a number of significant disadvantages:
(1) They encourage the development of antibiotic-resistant
organisms.

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(2) They hide the presence of low-level, cryptic contaminants that
can become fully operative if the antibiotics are removed, the
culture conditions change.
(3) They may hide mycoplasma infections.
(4) They have anti metabolic effects that can cross-react with
mammalian cells.

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• Most of the commercially available culture media include phenol
red as a pH indicator, which allows constant monitoring of pH.
• During the cell growth, the medium changes colour as pH is
changed due to the metabolites released by the cells.
• At low pH levels, phenol red turns the medium yellow, while at
higher pH levels it turns the medium purple.
• Medium is bright red for pH 7.4, the optimum pH value for cell
culture.
Reagents – Phenol Red

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Disadvantages of using Phenol red :
1) Phenol red mimics the action of some steroid hormones,
particularly estrogen .Thus it is advisable to use media
without phenol red for studies using estrogen-sensitive cells
like mammary tissue.
2) Presence of phenol red in some serum-free formulations
interferes with the sodium potassium homeostasis.
3) Phenol red interferes with detection in flow cytometric
studies.

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• The choice of cell culture media is extremely important, and
significantly affects the success of cell culture experiments.
• The selection of the media depends on the :
 Type of cells to be cultured
 The purpose of the culture
 Resources available in the laboratory
• Different cell types have highly specific growth requirements,
therefore, the most suitable media for each cell type must be
determined experimentally.
Eg : MEM for adherent cells and RPMI-1640 for suspension cells.
Criteria for selecting Media

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• Balanced salt solution (BSS) is composed of inorganic salts and
may include sodium bicarbonate and, in some cases glucose.
• HEPES buffer (5–20 mM) may be added to these solutions if
necessary and the equivalent amount of NaCl omitted to maintain
the correct osmolality.
• BSS forms the basis of many complete media.
• commercial suppliers will provide Eagle’s MEM with Hanks’s
salts or Eagle’s MEM with Earle’s salts, indicating which BSS
formulation was used.
Balanced Salt Solution (BSS)

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• BSS is also used as a diluent for concentrates of amino acids
and vitamins to make complete media.
• As an isotonic wash or dissection medium, and for short
incubations up to about 4 h (usually with glucose present).
• Used to maintain pH and osmatic pressure of the medium.
BSS Uses

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• Eagle’s Minimum Essential Medium (EMEM)
• Dulbecco’s Modified Eagle’s Medium (DMEM)
• RPMI-1640
• Ham’s Nutrient Mixtures
• DMEM/F12
• Iscove’s Modified Dulbecco’s Medium (IMDM)
Common cell culture media

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1. Switch on the laminar flow cabinet 20 min prior to start working.
2. Swab all bottle tops & necks with 70% ethanol.
3. If working on the bench use a Bunsen flame.
4. Flame all bottle necks & pipette by passing very quickly through
the hottest part of the flame.
5. Avoiding placing caps & pipettes down on the bench; practice
holding bottle tops with the little finger.
6. Work either left to right or vice versa, so that all material goes to
one side, once finished.
Aseptic Conditions

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7. Clean up spills immediately & always leave the work place neat
& tidy.
8. Never use the same media bottle for different cell lines.
9. If caps are dropped or bottles touched unconditionally touched,
replace them with new ones.
10. Necks of glass bottles prefer heat at least for 60 sec.
11. Never use stock of materials during handling of cells.

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THANK
YOU