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u The nutrient media used for culture of animal cells and tissues
must be able to support their survival as well as growth, i.e.,
must provide nutritional, hormonal and stromal factors.

u Besides meeting the basic nutritional requirement of the cells,


the culture medium should also have any necessary growth
factors, regulate the pH and osmolality, and provide essential
gases (O2 and CO2)

u the culture medium consists of amino acids, vitamins,


minerals, and carbohydrates.

u These allow the cells to build new proteins and other


components essential for growth and function as well as
providing the energy necessary for metabolism
u The various types of media used for tissue culture may be
grouped into two broad categories:
1) natural media 2) artificial media.

u The choice of medium depends mainly on the type of cells to


be cultured (normal, immortalized or transformed), and the
objective of culture (growth, survival, differentiation,
production of desired proteins).

u Nontransformed or normal cells (finite life span) and primary


cultures from healthy tissues require defined quantities of
proteins, growth factors and hormones
u But immortalized cells (spontaneously or by transfection with
viral sequences) produce most of these factors, but may still
need some of the growth factors present in the serum.

u In contrast, transformed cells (autonomous growth control and


malignant properties) synthesize their own growth factors
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u These media consist of naturally occurring biological fluids


and are of the following three types:
(1)coagula or clots,
(2)biological fluids and
(3)tissue extracts.
u The natural biological fluids are generally used for organ
culture.
u For cell cultures, artificial media with or without serum are
used.
Clots
u The most commonly used clots are plasma clots, which have been
in use for a long time.
u Plasma is now commercially available either in liquid or
lyophilized state. It may also be prepared in the laboratory, usually
from the blood of male fowl, but blood clotting must be avoided
during the preparation.
ë olog l Flu s
u Of the various biological fluids used as culture medium, serum is
the most widely used.
u Serum may be obtained from adult human blood, placental cord
blood, horse blood or calf blood (foetal calf serum, newborn calf
serum, and calf serum); of these foetal calf serum is the most
commonly used.
u Serum is the liquid obtained from coagulating blood. Different
preparations of serum differ in their properties; they have to be
tested for sterility and toxicity before use.
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u Chick embryo extract is most commonly used

u Bovine embryo extract is also used

u They can be substituted by a mixture of amino acids and


certain other organic compounds
u | t   l Me  D
Different artificial media have been devised to serve one of the
following purposes:

(1) immediate survival (a balanced salt solution, with specified


pH and osmotic pressure is adequate),

(2) prolonged survival (a balanced salt solution supplemented


with serum, or with suitable formulation of organic compounds),

(3) indefinite growth, and

(4) specialized functions.


u The various artificial media developed for cell cultures may be
grouped into the following four classes:

(i) serum containing media,

(ii) serum free media,

(iii) chemically defined media, and

(iv) protein free media.


u èe um ont n ng me 

u arious defined media supplemented with 5-20% serum


u Serum provides various plasma proteins, peptides, lipids, carbohydrates,
minerals and some enzymes
u Funt ons:-
u Provides basic nutrients for cells; nutrients are present both in the solution
as well as are bound to the proteins
u Provides hormones- insulin, cortisone, testosterone etc
u Contains growth factors- PDGF( platelet derived growth factors),
endothelial growth factor etc
u Supply proteins D fibronectin(promote attachment of cells)
u Provides spreading factors that help the cells to spread out before they can
begin to divide
u Provides binding proteins- albumin, transferrin( carry other molecules in to
the cell)
u Increases the viscosity of medium and protects cells from
mechanical damage
u protease inhibitors present in the serum protect cells
especially trypsinised cells from proteolysis
u Provides several minerals- Na+, K+, Fe2+ etc
u also act as a buffer
u  è|||è
u Serum may inhibit growth of some cell types D epidermal
keratinocytes
u May contain some cytotoxic or potentially cytotoxic
constituents ( foetal calf serum contains the enzyme polyamine
oxidase which converts polyamines like spermidine and
spermine (secreted by fast growing cells)in to cytotoxic
polyamino aldehydes)
u arge variation in serum quality from one batch to another

u Some growth factors may be inadequate for specific cell types


& may need supplementation

u Interferes with downstream processing when cell cultures are


used for production of biochemicals

u The supply of serum is always lower than its demand


u èe um F ee Me  -
In view of the disadvantages due to serum, extensive
investigations have been made to develop serum-free
formulations of culture media. These efforts were mainly based
on the following three approaches:
(1) analytical approach based on the analysis of serum
constituents,
(2) synthetic approach to supplement basal media by various
combinations of growth factors, and
(3) limiting factor approach consisting of lowering the serum
level in the medium till growth stops and then supplementing the
medium with vitamins, amino acids, hormones, etc. till growth
resumes
.
u These approaches have resulted in several elaborate media
formulations in which serum is replaced by a mixture of amino
acids, vitamins, several other organic compounds, etc.;
hormones, growth factors and other proteins are supplemented
when required.
u However, addition of 5-20% of serum even in these media is
essential for optimum growth.
u |ntges o èe um F ee Me  -
1. Improved reproducibility of results from different laboratories
and over time since variation due to batch change of serum is
avoided.
2. Easier downstream processing of products from cultured cells.
3. Toxic effects of serum are avoided

4. Bioassays are free from interference due to serum proteins.


5. There is no danger of degradation of sensitive proteins by serum
proteases.
6. They permit selective culture of differentiated and producing cell
types from the heterogenous cultures.
u  sntges o èe um F ee Me 

1. Most serum free media are specific to one cell type. Therefore,
different media may be required for different cell lines.
2. Reliable serum free preparations, for most of the media
formulations are not available commercially. This necessitates
time consuming task of preparing the desired formulations in the
laboratory.
3. A greater control of pH, temperature, etc. is necessary as
compared to that with serum containing media.

4. Growth rate and the maximum cell density attained are lower
than those with serum containing media.
5. Cells tend to become fragile during prolonged agitated cultures
unless biopolymers or synthetic polymers are added.
u Several defined media have been evolved from the Eagle's
minimal essential medium (MEM),
u e.g., Dulbecco's enriched modification (DME),
u Ham's F12, :,pMR1O66, RPMIl640,
u McCoy's 5A and
u Iscove's modified Dulbecco¶s' (IMDM); all are commercially
available.
u Often a 1: 1 mixture of DME and F12 is used as a serum free
formulation.
u Chem lly e ne Me  D
These media contain contamination free ultra pure inorganic and
organic constituents, and may contain pure protein additives, like
insulin, epidermal growth factor, etc. that have been produced in
bacteria or yeast by genetic engineering.
u Ñ ote n F ee Me  -
In contrast, protein free media do not contain any protein; they
only contain non-protein constituents necessary for culture of the
cells. The formulations MEM, DME, RPM-1640, etc. are protein
free; where required, protein supplementation is provided.
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|ntges  sntges
1. Provides basic nutrient for cells, nutrients being present in the Inhibits growth of some cell types,
solution and also bound to proteins. e.g. epidermal keratinocytes.

2. Provides several hormones e.g. insulin, testosterone, prostaglandin, May contain some cytotoxic
etc., which are essential for cell growth constituents.

3. Contains several growth factors, e.g. platelet derived growth factor Serum quality varies from batch to
(PDGF). batch which requires costly
and time-consuming tests for
each batch.
4. Supplies proteins, e.g. fibronectin promoting attachment of cells and Some growth factors are inadequate
spreading. for specific cell types.
5. Provides several binding proteins, e.g. albumin and transferrin, which When cell cultures are used for
carry other molecules into the cell. production of biochemicals,
the serum interferes with
downstream processing.
6. Increases viscosity of the medium. thereby protecting cells from Supply of serum is always lower
mechanical damage. than its demand.
7. Protease inhibitors in the serum protect cells from proteolysis.

8. Provides several minerals e.g. Na+, K+, etc.


9. Acts as a buffer.
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|ntges
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