Dr: RAWIA BADRAssociate Professor of Microbiology&Immunology

Cell culture

Commonly refers to the culture of animal cells and

tissues , while the more specific term plant tissue

culture is used only for plants.

In cell culture , the cells are capable of dividing ,

increasing in size and in a batch culture , single

cells are allowed to act as independent units, such

as a bacterium or fungus until limited by some culture

variables such as nutrient depletion .

Cell culture-cont.

When the process is carried out in the laboratory, it is

called Cell Culture , It occurs in vitro ‟in glass‟ as

opposed to occur in vivo ‟in life‟ .

Tissue culture is often used interchangeably with cell

culture.

History

The history of cell culture dates back to early 20th century,

when the first development of cell culture was to study, under the microscope , normal physiological events such as nerve

development.

Animal cell culture didn‟t become routine laboratory technique untill the 1950s, where the need for cell culture, especially at large scales , became apparent with the need for viral vaccines when major epidemics of polio in the 1940s promoted a lot of efforts to develop an effective vaccine.

Cell culture-Types

Freshly isolated cultures from mammalian tissues are

known as primary cultures until subcultured . At this

stage cells are usually heterogeneous but still closely

represent the parent cell types as well as in the

expression of tissue specific properties.

Types-cont.

After several subcultures onto a fresh media, the cell

line will either die out or „transform‟ to become

a continuous cell line.

Such cell lines show many alterations from the primary

cultures including morphological changes and

chromosomal variations.

Cell Culture-Forms

Cells can be grown either in suspension or adherent cultures.

Some cells naturally live in suspension, without being

attached to a surface, such as cells found in the blood

stream . There are also cell lines that have been

modified to be able to survive in suspension cultures

so that they can be grown to a higher density than

adherent conditions would allow .

Forms-cont.

Adherent cells (organs) require a surface, such as tissue

culture plastic, which may be coated with extracellular

matrix components to increase adhesion properties

and to provide other signals needed for process of

growth and differentiation .

Cell Culture-Harvesting

“When to harvest cells“

Cells are harvested when the cells have reached a population

density which suppresses growth.

Ideally, cells are harvested when they are in a semi-confluent

state and are still in the log phase. Cells that are not passaged

and are allowed to grow to a confluent state can sometime lag

for a long period and some times may never recover.

This picture shows the

uninfected Vero cell line at

48 h after seeding of the

culture.

Several nucleoli are visible

inside each nucleus . Cells

have been stained with

hematoxylin and eosin.

This picture shows uninfected Vero cell line.

Complete monolayer sheet 48 -72 h after seeding of the culture.

Cell Culture-Applications

Investigation of the normal physiology or biochemistry of cells e.g. studies of cell metabolism.

Testing the efficacy of various chemical compounds or drugs on specific cell types (normal or cancerous cells).

Studying the sequential or parallel combination of various cell types to generate artificial tissues e.g. artificial skin. The possibility of generating artificial tissues is known as “tissue engineering”.

Applications-cont.

Production of Biological substances by recombinant

DNA ( rDNA) technology in cell cultures include

enzymes , synthetic hormones , immunobiologicals

(monoclonal antibodies , interleukins , lymphokines

and anticancer agents) and for studying oncogenesis .

Applications-cont.

Production of hybrid cells (hybridoma) from the fusion

of two or more cells capable of continuous production

of a single type of antibody. These antibodies have

diagnostic and therapeutic value and are now produced

commercially .

Applications-cont.

Expression of mammalian genes in bacteria, although

many simpler proteins can be produced using rDNA

in bacterial cultures, the more complex proteins that

are glycosylated ( carbohydrate - modified ) could not

be produced in bacteria , as they do not have

the appropriate metabolism to add sugar chains to

these proteins. These must be made in cell culture

e.g. the hormone erythropoietin.

Applications-cont.

Production of vaccines e.g. polio, measles, mumps, rubella and chickenpox are currently made in cell cultures.

Nowadays due to the H5N1pandemic threat, research into using cell culture for influenza vaccines is going ahead .

Recombinant DNA-based vaccines, such as that made using human adenovirus as a vector or as adjuvants.

Cell Culture-advantage

The major advantage of using cell culture for any of

these applications is:

the consistency and

the reproducibility of results obtained.

Cell Culture-disadvantage

The disadvantage is that after a period of

continuous growth when cells continue to divide

and to grow filling the available area or volume .

“Several obstacles are aroused ”

Disadvantage-cont.

Nutrient depletion in the growth media .

Accumulation of apoptotic/necrotic (dead) cells .

Cell-to-cell contact can stimulate cell cycle arrest,

causing cells to stop dividing “contact inhibition ” .

Cell-to-cell contact can stimulate cellular differentiation .

Disadvantage-cont.

Cells characteristics can change, and may become quite

different from those found in the starting

population.

Cells can adapt to different culture environments (e.g.

different nutrients , temperatures and salt

concentration ) by varying the activities of their

enzymes leading to mutants formation.

Others-Plant cell culture methods

Plant cells have been cultured to produce

many ingredients needed by the food industries.

Much progress has been made in recent years in

understanding the basics of plant metabolism.

A wide range of food ingredients including

flavors , essential oils, sweeteners and

antioxidants have been produced in culture.

Bacterial/Yeast cell culture methods

For bacteria and yeast , small quantities of cells are

usually grown on a solid support that contains

nutrients embedded in it ( agar) , while large-scale

cultures are grown with the cells suspended in a

nutrient broth.

Viral cell culture methods

The culture of viruses requires the culture of cells of mammalian , plant , fungal or bacterial origin as hosts for the growth and replication of the virus .

Whole wild type viruses, recombinant viruses or viral products may be generated in cell types other than their natural hosts under suitable conditions. Depending on the species , the infection and the replication, viral infections result in host cell lysis and formation of a viral plaque.

These pictures show CPE typical of herpes simplex

virus onVero cells. The small syncytia,or multinucleated

giant cells, result from fusion of cell membranes

bearing viral glycoproteins.

Alterations in the cell nuclei and cytoplasmic tails

between the cells are seen. The cell cultures have

been stained with hematoxylin and eosin.

72 h after virus inoculation.

Uninfected monolayer

sheet of Vero cells.

Phase contrast microscopy

× 40.

Inoculation with a clinical

specimen.

Typical cytopathic effect

(CPE) caused by HSV-1.

Phase contrast microscopy

× 200.

Shell vial culture positive for

HSV-1.

Brightly fluorescing (apple

green) HSV-1 infected cells.

Indirect immunofluorescence

assay, x 250

Shell vial culture positive for

HSV-1.

HSV-1 infected cells (stained

dark brown). Uninfected cells

appear bluish purple due to

counterstaining with H&E

Indirect immunoperoxidase

assay, × 500.

Tube culture positive for HSV-1.

HSV-1 infected cells (stained dark

brown).

Uninfected cells appear bluish

purple due to counterstaining

with haematoxylin.

Indirect immunoperoxidase assay

.

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