Tissue culture was first devised at the beginning of the

twentieth century as a method for studying the

behavior of animal cells free of systemic variations that
might arise in vivo both during normal homeostasis
and under the stress of an experiment.
The first cultured animal cell was Frog embryo nerve
fiber by Harrison in 1907

The frog was chosen as the source of tissue is

suggested that it was a cold-blooded animal, and
consequently, incubation was not required

Tissue regeneration is more common in lower

vertebrates, he perhaps felt that growth was more
likely to occur than with mammalian tissue
Why animal cell culture
Major roles of animal cell culture

1. Model systems for

Studying basic cell biology, interactions between
disease causing agents and cells, effects of drugs on
cells, process and triggering of aging and nutritional
studies
Major roles of animal cell culture

2. Toxicity testing
Study the effects of new drugs

3. Cancer research
Study the function of various chemicals, virus and
radiation to convert normal cultured cells to
cancerous cells
Major roles of animal cell culture

4. Gene therapy
Cells having a functional gene can be replaced to
cells which are having non-functional gene
Major roles of animal cell culture

5. Virology
Cultivation of virus for vaccine production, also used
to study there infectious cycle.

6. Genetic Engineering
Production of commercial proteins, large scale
production of viruses for use in vaccine production
e.g. polio, rabies, chicken pox, hepatitis B & measles
TYPES OF TISSUE CULTURE
Organ culture implies that the architecture
characteristic of the tissue in vivo is retained, at least in
part, in the culture. The tissue is cultured at the liquid–
gas interface (on a raft, grid, or gel), which favors the
retention of a spherical or three-dimensional shape
In primary explant culture, a fragment
of tissue is placed at a glass (or
plastic)–liquid interface, where, after
attachment, migration is promoted in
the plane of the solid substrate
Cell culture implies that the tissue, or
outgrowth from the primary explant, is
dispersed (mechanically or
enzymatically) into a cell suspension,
which may then be cultured as an
adherent monolayer on a solid
substrate or as a suspension in the
culture medium
Generation of cell culture
There are four stages to consider in establishing cell
culture:
(1) acquisition of the sample
(2) isolation of the tissue
(3) dissection and/or disaggregation of the tissue
(4) Culture after seeding into the culture vessel
Isolation of the tissue
(mouse embryo)
Tear the ventral skin transversely
at the median line just over the
diaphragm and, grasping the skin
on both sides of the tear, pull in
opposite directions to expose the
untouched ventral surface of the
abdominal wall
Cut longitudinally along the median
line of the exposed abdomen with
sterile scissors, revealing the viscera.
At this stage, the uterus, filled with
embryos, is obvious in the posterior
abdominal cavity
Take the intact uteri to the tissue culture
laboratory, and transfer them to a fresh
Petri dish of sterile DBSS.

Free the embryos from the membranes

and placenta and place them to one side
of the dish to bleed
Isolation of the tissue
(Chick embryo)
Swab the egg with 70% alcohol, and place it
with its blunt end facing up in a small
beaker

Crack the top of the shell and peel the shell

off to the edge of the air sac with sterile
forceps

Resterilize the forceps and then use the

forceps to peel off the white shell
membrane to reveal the chorioallantoic
membrane (CAM) below, with its blood
vessels
Pierce the CAM with sterile curved forceps
and lift out the embryo by grasping it gently
under the head. Do not close the forceps
completely, or else the neck will sever; place
the middle digit under the forceps and use the
finger pad to restrict the pressure of the
Forefinger

Transfer the embryo to a 9-cm Petri dish

containing 20 mL DBSS
Dissection and/or disaggregation of the tissue
Establishing primary explants
The tissue is chopped finely and rinsed, and the pieces are
seeded onto the surface of a culture flask or Petri dish in a
small volume of medium with a high concentration (i.e.,
40–50%) of serum, such that surface tension holds the
pieces in place until they adhere spontaneously to the
surface. Once this is achieved, outgrowth of cells usually
follows
Schematic diagram of stages
in dissection and seeding
primary explants.
 Primary explant culture from mouse squamous skin
carcinoma; explant and early stage of outgrowth about
3 days after explantation
Outgrowth after removal of explant, about 7 days after
explantation
Establishing primary culture through enzymatic disaggregation
The tissue is chopped and stirred in trypsin for a few hours. The
dissociated cells are collected every half hour, centrifuged, and
pooled in medium containing serum
Enzymatic disagregation
The enzymes used most frequently for tissue
disaggregation are crude preparations of trypsin,
collagenase, elastase, pronase, dispase, and
hyaluronidase, alone or in various combinations
Establishing primary culture through
Mechanical disagregation
The outgrowth of cells from primary explants is a relatively
slow process and can be highly selective.

Enzymatic digestion is labor intensive and there is a risk

of proteolytic damage to cells during enzymatic digestion

Many people have chosen to use the alternative of

mechanical disaggregation
Mechanical disagregation is carried out by :
Pressing the dissected tissue through a series of sieves
for which the mesh is gradually reduced in size, forcing
the tissue fragments through a syringe or simply
pipetting it repeatedly

Sieving is probably the gentlest mechanical methods as

pipetting and syringing generate shear that cause cell
damage
Primary cells are morphologically similar to the parent tissue.

These cultures are capable of only a limited number of cell

divisions, after which they enter a nonproliferative state called
senescence and eventually die out.
Primary cells are considered by many researchers to be more
physiologically similar to in vivo cells (have the same karyotype
as the parent tissue normal or abnormal)

Primary cell culture is generally more difficult than culture of

continuous cell lines
-Difficult to obtain
-Relatively short life span in culture.
Cell line :
secondary or subclone culture of primary cell culture

The cultures will proliferate for a limited number of cell

divisions, after which they will senesce, some cell line may be
Undergone between 60-70 doublings before senescence.

The factors which control the replication of such cells in vitro

are related to the degree of differentiation of the cell
Cell line :
Can obtain a large population of similar cells

Most cellular characteristics are maintained

Can transform cells to grow indefinitely

Cell line :
Cells have a tendency to differentiate over time in culture

Over time the culture tends to select for aberrant cell

Infinite Cell line :

Continuous cell line

Immortal cells line

Transformed cell line

A cell line that has demonstrated the potential to be

subcultured indefinitely

This alteration is commonly known as in vitro transformation

or immortalization and frequently correlates with
tumorigenicity
HeLa : human epithelial cells from a fatal cervical carcinoma
transformed by human papillomavirus 18 (HPV18).