Culture Media-ATC

The artificial environment created in the

laboratory is generally known as media. A media
comprises an appropriate source of energy for the
cells which they can easily utilize and compounds
which regulate the cell cycle.
The choice of media is cell type specific and often
empirical and there is no all purpose medium.
It should provide many nutrients, buffering
capacity, isotonic, and should be sterile.
Characteristics and compositions of the cell culture
media vary depending on the particular cellular
requirements.
Important parameters include osmolarity,
pH, and

Culture media
The culture media is prepared in such a way that it
provides
1. The optimum conditions of factors like pH, osmotic
pressure, etc.
2. It should contain chemical constituents which the
cells or tissues are incapable of synthesizing.

Generally the media contain inorganic salts and

other organic nutrients capable of sustaining:
Hormones, Growth factors, cytokined, adhering
proteins, amino acids, fatty acids, sugars, vitamins,
Glucose is added as energy
cofactors,
Phenol Red is added as a pH indicator of the medium.

Media components
Basic Components in the Culture Media
Most animal cell culture media are generally
having following 10 basic components and they
are as
follows:
1. Energy sources: Glucose, Fructose, Amino
acids
2. Nitrogen sources: Amino acids
3. Vitamins: Generally water soluble vitamines B
&C
4. Inorganic salts: Na+, K+, Ca2+, Mg2+
5. Fat and Fat soluble components: Fatty acids,
cholesterols
6. Nucleic acid precursors

Types of media
1. Natural media
2. Artificial media.
The choice of medium depends mainly on the
type of cells
Non transformed or normal cells (finite life
span) and primary cultures from healthy tissues
require defined quantities of proteins, growth
factors and hormones even in the best media
developed so far.
transformed cells (autonomous growth control
and malignant properties) synthesize their own
growth factors

Natural Media
Natural Media
1. Cagula or clots
2. Biological fluids
3. Tissue extracts
The natural biological fluids are
generally used for organ culture.

Clots

The most commonly used clots are plasma

clots, which have been in use for a long
time.

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.

Biological Fluids
Of the various biological fluids used as culture medium, serum is the
most widely used. Serum is one of the very important components of
animal cell culture.
Serum is the source of various
-amino acids,
-hormones,
-lipids,
-vitamins,
-polyamines,
-and salts containing ions such as calcium, ferrous, ferric, potassium etc.
-growth factors which promotes cell proliferation, cell attachment and
adhesion factors.
Serum may be obtained from adult human blood, placental cord
blood, horse
blood or calf blood (fetal calf serum, newborn calf serum, and calf
serum);
of these fetal calf serum is the most commonly used.

Tissue Extracts
Tissue or organ extracts and/or hydrolysates
-bovine pituitary extract (BPE),
-bovine brain extract,
-chick embryo extract and
-bovine embryo extract)

Artificial Media
Artificial Media
Different artificial media have been devised
to serve one ofthe 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
4. Specialized functions.

Artificial Media

Artificial media
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
(iv) Protein free media.

Artificial Media
SERUM
Liquid yellowish, clear content left over after
fibrin and cells are removed from the blood
is known as serum.
Calf (bovine) serum,
fetal bovine serum,
horse serum
Fetal bovine serum (FBS) (10-20% v/v)
is the most commonly applied
supplement in animal cell culture
media.

Serum

Functions of Serum in the Culture Medium

-several hormones, e.g., insulin, which is essential for
growth of nearly all cells in culture, cortisone,
testosterone, prostaglandin, etc.
-several growth factors, e.g., platelet derived
growth factor (PDGF), transforming growth factor
(TGF-), epidermal growth factor, etc
A given hormone or growth factor may stimulate
growth of one cell type, may have no effect on
another and may even be inhibitory to some others.
For example, PDGF induces proliferation in
fibroblasts, but induces differentiation of some types
of epithelia.
Further, proliferation of a single cell type may be
induced by more than one growth factor, e.g.,
fibroblasts respond to PDGF, epidermal growth factor,
fibroblast growth factor and somatomidins.

4. A major role of serum is to supply proteins, e.g., fibronectin,

which promote attachment of cells to the substrate.
It also provides spreading factors that help the cells to spread out
before they can
begin to divide.

5. It provides several binding proteins, e.g., albumin,

transferrin, etc., which carry other molecules into the
cell.
For example, albumin carries into cells lipids, vitamins,
hormones, etc.
Transferrin usually carries Fe in a nonbasic form, but
binding of transferrin to its receptor in cell membrane
is believed to be mitogenic.

Function of serum in culture

media
6. It increases the viscosity of medium and
thereby, protects cells from mechanical
damages, e.g.,
shear forces during agitation of suspension
cultures.
7. Protease inhibitors present in the serum
protect cells, especially trypsinised cells,
from proteolysis.
8. The serum also provides minerals, like
Na+, K+, Fe2+, Zn2+, etc.
9. It also acts as a buffer.

Culture Vessel

Roux bottle

Culture vessel

Roller bottle

Culture vessel

Multi well plates

Types of cells
1. Epithelial Cell
-Attached to a substrate
and appears flattened
and po- lygonal in
shape

Fig. . Epithelial Cell

Type of cells

PROCEDURE OF CELL
CULTURE
1. Primary Cell Culture
Primary cultures are derived directly from excised, normal animal tissue
and cultures either as an explant culture or following dissociation into
a single cell suspension by enzyme digestion.
Primary cultures are initially heterogeneous but later become dominated
by fi broblasts.
The preparation of primary cultures is labour intensive and they can be
maintained in vitro only for a limited period of time. During their relatively
limited lifespan primary cells usually retain many of the differentiated
characteristics of the cell in vivo.
Important Note: Primary cultures by defi nition have not been
passaged, as soon as they are passaged they become a cell line and are
no longer primary. Primary cells sourced from most suppliers are in fact
low-passage cell lines.

Cell line
Cell line:
The sub-culturing of the primary
culture gives rise to cell lines.
Infinite cell line: The term continuous cell
lines implies the indefinite growth of the
cells in the subsequent sub-culturing.
Finite cell line: On the other hand, finite
cell lines represent the death of cells after
several subcultures.

7.5. Type of Cell Culture

7.5.1. Anchorage Dependent Cell Culture
Cells shown to necessitate attachment for growth are
set to be anchorage dependent cells. The adherent cells
are typically derived from tissues of organs such as
kidney where they are immobile in addition to
embedded in con- nective tissue. They cultivate
adhering to the cell culture.
7.5.2. Anchorage Independent Cell Culture
Cells which make not required attachment for growth or
do not attach to the surface of the culture vessels are
an- chorage independent cells/suspension cells [5].
Each and every one suspension cultures are derived
from cells of the blood system for the reason that these
cells are fur- thermore suspended in plasma in vitro e.g.
lymphocytes