Media used in animal cell culture

1. UNIT: 3- Animal Cell Culture- Media
Dr. Saraswathi, M.S.c, Ph.D
Assistant Professor,
Dept. of Life Sciences,
Assistant Professor,
Kristu Jayanti College, Bengaluru-77

2. MEDIA REQUIREMENTS FOR CELL CULTURE
Criteria for selecting culture media
• The choice of medium to be used for culture is dependent on the cell
type specifics which significantly affects the success of cell culture
experiments.
• The selection of the media also depends on the purpose of the culture
and 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.

8. Basic components in culture media
• The 10 basic components that make up most of the animal cell
culture media are as follows:
• inorganic salts (Ca 21, Mg 21, Na1, K1), nitrogen source
(amino acids), energy sources (glucose, fructose), vitamins, fat
and fat soluble component (fatty acids, cholesterols), nucleic
acid precursors, growth factors and hormones, antibiotics, pH
and buffering systems, and oxygen and carbon dioxide
concentrations.
• Complete formulation of media that supports growth and
maintenance of a mammalian cell culture is very complex.

9. Culture media:
• One of the most important factors in animal cell cul-ture is the medium
composition.
• In vitro growth and maintenance of animal cells require appropriate
nutritional, hormonal, and stromal factors.
• Important environmental factors are the medium in which the cells are
surrounded, the substratum upon which the cells grow.
• Temperature, oxygen and carbon dioxide concentration, pH, and osmolality.
• In addition, the cell requires chemical substances that cannot be
synthesized by the cells themselves.
• Any successful medium is composed of isotonic, low-molecular-
weight compounds known as basal medium and provides inorganic
salts, an energy source, amino acids, and various supplements.

10. Basic Components of Culture Media
• Culture media contain a mixture of amino acids,
glucose, salts, vitamins, and other nutrients, and available
either as a powder or as a liquid form from commercial
suppliers.
• The requirements for these components vary among cell
lines.
• These differences are responsible for the extensive
number of medium formulations.
• Each component performs a specific function, as
described below:

11. • Buffering systems: Regulating pH is critical for
optimum culture conditions and is generally achieved
by one of the two buffering systems:
• Natural buffering system : In a natural buffering
system, gaseous CO2 balances with the 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 a CO2 incubator.
• A natural buffering system is low-cost and non-toxic.

12. • HEPES : Chemical buffering using a zwitterion,
HEPES, has a superior buffering capacity in the pH
range 7.2-7.4
• Not require a controlled gaseous atmosphere.
• HEPES is relatively expensive and toxic at a higher
concentration for some cell types.
• Carbohydrates : Carbohydrates in the form of sugars
are the major source of energy.
• Most of the media contain glucose and galactose,
however, some contain maltose and fructose.

13. • Proteins and peptides : proteins and peptides are
albumin, transferrin, and fibronectin.
• They are particularly important in serum-free media.
• Serum is a rich source of proteins and includes
albumin, transferrin, aprotinin, fetuin, and
fibronectin.
• Albumin is the main protein in blood acting to bind
water, salts, free fatty acids, hormones.
• The binding capacity of albumin makes it a suitable
remover of toxic substances from the cell culture media.

14. • Each type of media has a recommended bicarbonate concentration and CO
2 tension to achieve the correct pH and osmolarity.
• The density of the culture may affect the CO2 requirement, however, in
general phenol red in the medium will indicate the state of the pH at any
given time.
• Glutamine and amino acids: Commonly the necessary amino acids
include cysteine and tyrosine, but some non-essential amino acids may be
needed.
• Glutamine is also required by most cell lines and it has been suggested that
cultured cells use glutamine as an energy and carbon source.
• Glutamine is usually added at a final concentration of 2 mM.
• The glutamine is only stable for about 3 weeks at 4 °C

15. • Fatty acids and lipids : Important in serum-free media as they
are generally present in serum.
• Vitamins: Many vitamins are essential for growth and
proliferation of cells.
• Vitamins cannot be synthesized in sufficient quantities by
cells.
• Therefore important supplements required in tissue culture.

16. • Trace elements: Trace elements like copper, zinc, selenium
and tricarboxylic acid intermediates.
• Chemical elements that 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.
• Media supplements: The complete growth media
recommended for certain cell lines requires additional
components which are not present in the basal media and serum.
• Supplements, help sustain proliferation and maintain normal
cell metabolism.

17. • Antibiotics: used to control the growth of bacterial
and fungal contaminants.
• Routine use of antibiotics for cell culture is not
recommended since antibiotics can mask contamination
by mycoplasma and resistant bacteria.
• Moreover, antibiotics can also interfere with the
metabolism of sensitive cells.

18. • Phenol red: Most of the commercially available culture
media include phenol red.
• pH indicator, which allows constant monitoring of pH.
• During the cell growth, the medium changes color 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.

19. • culture medium used for cell culture was based on biological fluids such
as plasma, lymph serum, and embryonic extracts.
• The nutritional requirements of cells can vary at different stages of the
culture cycle.
• Different cell types have highly specific requirements, and the most
suitable medium for each cell type must be determined experimentally.
Media may be classified into two cate-gories:
(1) Natural media
(2) Artificial media

20. Natural media
• Natural media consist of naturally occurring biological fluids sufficient for
the growth and proliferation of animals cells and tissues.
• Promoting cell growth are of the following three types.
1. Coagulant or clots: Plasma separated from heparinized blood from
chickens or other animals is commercially available in the form of
liquid plasma.
2. Biological fluids: Body fluids such as plasma
• These fluids are used as serum lymph, amniotic fluid, pleural fluid,
insect hemolymph, and fetal calf serum.
• Cell culture media after testing for toxicity and sterility.
3. Tissue extract: extracts of liver, spleen, bone marrow, and leucocytes are
used as cell culture media.
• Chicken embryo extract is the most common tissue extract used

21. • Natural media is derived from biological fluids such as
serum (i.e., fetal bovine serum) or plasm clots.
• Mainly used to promote cell adhesion and cell survival.
• It mainly contained essential cell growth proteins including
albumin, transferrin, globulin, insulin, growth factors, and
hormone.
• The exact amount and compositions of natural media
are difficult to define.
• This may vary from batch to batch.
• Used as a supplement for promoting cell growth in
combination with artificial media.

22. Artificial media
• The media contains partly or fully defined components that are
prepared artificially by adding several nutrients (organic and
inorganic).
• It contains a balanced salt solution with specific pH and osmotic
pressure designed for immediate survival of cells.
• Artificial media supplemented with serum or with suitable
formulations of organic compounds supports prolonged survival of the
cell culture.
• Contained essential amino acids and vitamins, balance inorganics salts
(i.e., Ca2+, Mg2+, Na+, and K+), energy sources (i.e., glucose or
fructose), fatty acids, nucleic acids precursor, hormones, and buffers.
• Artificial media is a comparatively low cost compared to serum

23. • The artificial media may be grouped into the following four
classes:
1. Serum-containing media,
2. Serum-free media,
3. Chemically defined media,
4. Protein-free media.