Mammalian Cell Culture

Introduction
Tissue culture is the growth of tissues and/or
cells separate from the organism. This is
typically facilitated via use of a liquid, semi-
solid, or solid growth medium.
The term tissue culture is used for both cell
and organ culture.
What is cell culture, exactly?
Cells, previously growing in a human or
animals modified to grow in plastic or glass
In the body = in vivo
On plastic or glass = in vitro
Kept in an incubator to stay at body
temperature
We use special media with nutrients so the
cells can grow and divide
If we treat the cells right
They might act similar to those in vivo
Making the same proteins
Then scientists can change the environment

In-vitro cell and tissue system five
main categories will be considered.
1. Requirement of Cells and tissues.
2. In-vitro Culture conditions.
3. Handling and Maintenance.
4. Cryopreservation.
5. Microbial, Viral, and Cellular Cross-
contamination.
1. Cell and Tissue

Isolated organs or Tissues.
Primary cultures and early passage cultures.
Cell lines.

Isolated organs or Tissues.
Taken for direct use from animal or human
donors.
Perfused with physiological buffers.
Maintain or reconstruction of three-
dimensional structures, with some of the
structural and functional properties of the
original organ or tissue.
Primary cultures and early passage
cultures
The initial in vitro culture of harvested cells and
tissues taken directly from animals and humans is
called primary culture.
They have a limited life span and known to change
their differentiated characteristics with time in
culture.
Primary cultures are maintain in suspension or
monolayer culture.
Cell-lines
Cell lines comprise cells that are able to
multiply for extended periods in vitro and
can therefore be maintained by serial
subculture.
Cell-lines are three types.
1. Finite cell-lines
2. Continuous cell-lines.
3. Stem cells.
Finite cell lines
Finite cell lines are cultures of cells that
possess the ability to be subcultured
numerous times, but which eventually cease
replication and enter a state of senescence.
Continuous cell lines

Certain cell lines show an apparent ability to be subcultured
indefinitely, and are known as continuous cell lines.
They do not show the senescence experienced with finite cell lines.
typically derived from tumors or normal embryonic tissues.
Continuous cell lines may arise spontaneously, or can be produced by
using a variety of other methodologies, such as:
exposure of normal cells and tissues to irradiation and/or
treatment with chemical mutagens or carcinogens;
isolation from cultures infected with viruses (for example,
Epstein-Barr virus);
genetic modification of cells by transfection with cloned
genes (for example, SV40 large T-antigen, adenovirus E1,
telomerase); and
isolation from transgenic animals.
Stem cell lines
Stem cell lines, such as embryonic and germ
cell lines, are types of continuous cell lines
that retain the characteristics of stem cells
and can produce diverse differentiated cell
types.
2. In Vitro Culture Conditions
Basal medium
Serum
Nutritional status
Antibiotics
Cell culture surface/matrix

Basal medium
Phosphate Buffered Saline
Used to wash/remove excess serum
that inhibits the function of TRED.
Must be warmed in the water bath
before use so cells are not shocked by
cold liquid.
What is in the media?
F-12 Growth Media
Contains glucose, some proteins, and essential salts
Contains a pH indicator (phenol red) Media looks
pink/red at pH 7.2
Acidic -yellow or orange (cell growth, bacterial
growth)
Basic -purple (no cell growth, not enough CO
2
)
More media components
Antibiotics (penicillin, Gentamycin and
streptomycin)
Prevent bacterial contamination
Salts and buffers
To simulate in vivo environment
Serum
Portion of blood after the cells and fibers have clotted
From cow, horse, sheep
added to media as a nutrient source for growing
cells
Lipids, proteins
3. Handling and Maintenance
Temperature
Atmosphere
pH
Cell detachment and subculture

Temperature

For mammalian cell optimal temp. 37C.
Above 37C induced apoptosis.
Below 37C slow replication but also
enhanced the expression of certain cell
proteins.
Atmosphere
Oxygen and carbon dioxide are known to be
vital for cell growth.
High level are toxic and very low level will
inhibit cell growth.
For mammalian cell 5% carbon dioxide is
used.
pH

The optimal physiological pH for mammalian
cell culture is usually considered to be pH
7.2-7.4.


Cell detachment and subculture

Trypsin EDTA (TRED)
An enzyme used to detach the cells from a
culture dish.
EDTA binds calcium ions in the media that
would normally inhibit trypsin.
4. Cryopreservation
Cells and tissues can be cryopreserved in a
stable state for limited or prolonged periods.
The cryopreservation process includes
freezing, storage and recovery.

5. Microbial, Viral and Cellular
Cross-contamination

Contamination with bacteria, yeast and other fungi
can result in the complete loss of cultures.
There are various potential sources of viral
contamination, including the operator, cell culture
reagents of animal origin, and cells or tissues of
animal origin.
Cross-contamination of cell lines with other cell lines
is a real, but often neglected, problem.
Animals are complex
Many different cells
Many different proteins
Interacting continuously
Difficult to watch individual events in vivo
Animals are usually harmed to observe
biological events

Using cultures has advantages
Fewer animals are harmed
Can control all external factors
Can easily test what the cells are doing
Cells are easy to manipulate and propagate
All of the cells are the same
Results of experiments will be consistent
Cheaper to maintain
What can we do with cells?
Test pharmaceutical drugs
Watch disease mechanisms
Design potential treatments
Observe the regenerative process
How do cells and tissues repair themselves after
damage from illness or injury?
Observe the developmental process