Introduction to Animal Cell Culture

Cell culture has become one of the major tools used in the life sciences today.

Cell and Tissue Culture?

Tissue Culture is the general term for the removal of cells, tissues, or organs from an
animal or plant and their subsequent placement into an artificial environment
conducive to growth.

This environment usually consists of a suitable glass or plastic culture vessel

containing a liquid or semi-solid medium that supplies the nutrients essential for sur
vival and growth.

The culture of whole organs or intact organ fragments with the intent of studying their
continued function or development is called Organ Culture.

When the cells are removed from the organ fragments prior to, or during cultivation,
thus disrupting their normal relationships with neighboring cells, it is called Cell
Culture.

Although animal cell culture was first successfully undertaken by Ross Harrison in
1907, it was not until the late 1940's to early 1950's that several developments
occurred that made cell culture widely available as a tool for scientists.

First, there was the development of antibiotics that made it easier to avoid
many of the contamination problems that plagued earlier cell culture
attempts.

Second was the development of the techniques, such as the use of trypsin
to remove cells from culture vessels, necessary to obtain continuously
growing cell lines (such as HeLa cells).

Third, using these cell lines, scientists were able to develop standardized,
chemically defined culture media that made it far easier to grow cells.

These three areas combined to allow many more scientists to use cell,
tissue and organ culture in their research.

During the 1960's and 1970's, commercialization of this technology had

further impact on cell culture that continues to this day. Companies, such
as Corning, began to develop and sell disposable plastic and glass cell
culture products, improved filtration products and materials, liquid and
powdered tissue culture media, and laminar flow hoods. The overall result
of these and other continuing technological developments has been a
widespread increase in the number of laboratories and industries using
cell culture today.

TYPES OF CELL CULTURES

Primary Culture:
When cells are surgically removed from an organism and placed
into a suitable culture environment, they will attach, divide and
grow. This is called a Primary Culture.
There are two basic methods for doing this.
First, for Explant Cultures, small pieces of tissue are attached to a
glass or treated plastic culture vessel and bathed in culture
medium. After a few days, individual cells will move from the tissue
explant out onto the culture vessel surface or substrate where they
will begin to divide and grow.
The second, more widely used method, speeds up this process by
adding digesting (proteolytic) enzymes, such as trypsin or
collagenase, to the tissue fragments to dissolve the cement holding
the cells together. This creates a suspension of single cells that are
then placed into culture vessels containing culture medium and
allowed to grow and divide. This method is called Enzymatic
Dissociation.

 The primary cell culture could be of two types depending upon

the kind of cells in culture.
a) Anchorage Dependent /Adherent cells- Cells shown to require
attachment for growth are set to be Anchorage Dependent cells. The
Adherent cells are usually derived from tissues of organs such as
kidney where they are immobile and embedded in connective tissue.
They grow adhering to the cell culture.
b) Suspension Culture/Anchorage Independent cells - Cells
which do not require attachment for growth or do not attach to the
surface of the culture vessels are anchorage independent
cells/suspension cells. All suspension cultures are derived from
cells of the blood system because these cells are also suspended in
plasma in vitro e.g. lymphocytes.

Secondary cell cultures

When a primary culture is sub-cultured, it becomes known as
secondary culture or cell line. Subculture (or passage) refers to
the transfer of cells from one culture vessel to another culture
vessel.

Subculturing
Subculturing or splitting cells is required to periodically provide
fresh nutrients and growing space for continuously growing cell
lines. The process involves removing the growth media, washing the
plate, disassociating the adhered cells, usually enzymatically. Such
cultures may be called secondary cultures.
Cell Line

A Cell Line or Cell Strain may be finite or continuous depending

upon whether it has limited culture life span or it is immortal in
culture.

On the basis of the life span of culture, the cell lines are categorized
into two types:
a) Finite cell Lines - The cell lines which have a limited life span
and go through a limited number of cell generations (usually 20-80
population doublings) are known as Finite cell lines. These cell lines
exhibit the property of contact inhibition, density limitation and
anchorage dependence. The growth rate is slow and doubling time is
around 24-96 hours.

 b) Continuous Cell Lines - Cell lines transformed under laboratory

conditions or in vitro culture conditions give rise to continuous cell
lines. The cell lines show the property of ploidy (aneupliody or
heteroploidy), absence of contact inhibition and anchorage
dependence. They grow in monolayer or suspension form. The
growth rate is rapid and doubling time is 12-24 hours.
Monolayer cultures:
When the bottom of the culture vessel is covered with a continuous layer of
cells, usually one cell in thickness, they are referred to as monolayer
cultures.

Monolayer cultures are usually grown in tissue culture treated dishes,

T-flasks, roller bottles, Culture Chambers, or multiple well plates, the
choice being based on the number of cells needed, the nature of the
culture environment, cost and personal preference.
Suspension cultures:

Majority of continuous cell lines grow as monolayers. Some of the

cells which are non-adhesive e.g. cells of leukemia or certain cells
which can be mechanically kept in suspension, can be propagated
in suspension. There are certain advantages in propagation of cells
by suspension culture method.

 Suspension cultures are usually grown either:

In magnetically rotated spinner flasks or shaken Erlenmeyer flasks

where the cells are kept actively suspended in the medium.

In stationary culture vessels such as T-flasks and bottles where,

although the cells are not kept agitated, they are unable to attach
firmly to the substrate.

These advantages are:

The process of propagation is much faster.,

The frequent replacement of the medium is not required.,

Suspension cultures have a short lag period,

treatment with trypsin is not required,

a homogenous suspension of cells is obtained,

the maintenance of suspension cultures is easy and bulk

production of the cells is easily achieved.,

scale-up is also very convenient.

DIAGRAM SHOWING THE ROLLER BOTTLE CELL CULTURE

Erlenmeyer flasks are often

used for growing cells in
suspension.

microplates

The cell lines are known by:

A code e.g. NHB for Normal Human Brain.
A cell line number- This is applicable when several cell lines are derived from
the same cell culture source e.g. NHB1, NHB2.
Number of population doublings, the cell line has already undergone e.g.
NHB2/2 means two doublings.

Figure
showing
the
salient
features of cell
culture
with
evolution of a
cell line.

CHARACHTERIZATION OF CELL LINES

The cell lines are characterized by their

growth rate and
Karyotyping

Growth Rate:

A growth curve of a particular cell line is established taking into

consideration the population doubling time, a lag time, and a
saturation density of a particular cell line. A growth curve
consist of:
Lag Phase: The time the cell population takes to recover from such
sub culture, attach to the culture vessel and spread.
Log Phase: In this phase the cell number begins to increase
exponentially.
Plateau Phase: During this phase, the growth rate slows or stops
due to exhaustion of growth medium or confluency.

Karyotyping
Karyotyping is important as it determines the species of origin and
determine the extent of gross chromosomal changes in the line.
The cell lines with abnormal karyotype are also used if they
continue to perform normal function.
Karyotype is affected by the growth conditions used, the way in
which the cells are subcultured and whether or not the cells are
frozen.

There are certain terms that are associated with the cell lines.
These are as follows:
(i) Split ratio- The divisor of the dilution ratio of a cell culture at
subculture.
(ii) Passage number- It is the number of times that the culture has
been cultured.,
(iii) Generation number- It refers to the number of doublings that a
cell population has undergone.

SOME ANIMAL CELL LINES AND THE PRODUCTS OBTAINED FROM THEM
Cell line

Product

Human tumour

Angiogenic factor

Human leucocytes

Interferon

Mouse fibroblasts

Interferon

Human Kidney

Urokinase

Transformed human kidney cell line, Single chain urokinase-type plasminogen

TCL-598

activator (scu-PA)

Human kidney cell (293)

Human protein (HPC)

Dog kidney

Canine distemper vaccine

Cow kidney

Foot and Mouth disease (FMD) vaccine

Chick embryo fluid

Duck embryo fluid

Vaccines

for

influenza,

measles

and

mumps
Vaccines for rabies and rubella
1. Tissue-type plasminogen activator (t-

Chinese hamster ovary (CHO) cells

PA)
2. B-and gamma interferons
3. Factor VIII