ANIMAL TISSUE
CULTURE
GROUP#4
SADDAF TARIQ
HIRA ABBAS
MAHNOOR IQTIDAR MIR
LARAIB FATIMA
INTRODUCTION TO 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.
IMPORTANCE
• Animal cells in culture are used in
• Recombinant DNA technology
• Genetic manipulations
• the areas of research which have a potential for
economic value and commercialization
• production of vaccines
• monoclonal antibodies
• pharmaceutical drugs
• cancer research
HISTORICAL BACKGROUND
• Jolly, (1903) showed for the first time that the cells can survive
and divide in vitro.
•
Ross Harrison, (1907) was able to show the development of
nerve fibers from frog embryo tissue, cultured in a blood clot.
•
Later, Alexis Carrel (1912) used tissue and embryo extracts as
cultural media to keep the fragments of chick embryo heart
alive.
•
In 1996, the first use of regenerative tissue was used to replace
a small distance of a urethra, which led to the understanding
that the technique of obtaining samples of tissue, growing it
outside the body without a scaffold, and reapplying it, can be
used for only small distances of less than 1 cm.
TYPES OF ANIMAL TISSUE CULTURE
• Primary cell culture
• The maintenance of growth of cells dissociated
from the parental tissue (such as kidney, liver)
using the mechanical or enzymatic methods, in
culture medium using suitable glass or plastic
containers is called Primary Cell Culture.
• 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.
• Sub culturing- 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 .
• 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:
 Finite cell lines
 Continuous cell lines
• The cell lines are characterized by their
•
a) growth rate
•
b) karyotyping.
FACTORS EFFECTING ANIMAL TISSUE CULTURE.
1.
Temperature: In most of the mammalian cell cultures, the
temperature is maintained at 37C in the incubators as the body
temperature of Homo sapiens is 37C.
2.
pH:
• Most media maintain the pH between 7 and 7.4. A pH below 6.8
inhibits cell growth.
• The optimum pH is essential to maintain the proper ion balance,
optimal functioning of cellular enzymes and binding of hormones
and growth factors to cell surface receptors in the cell cultures.
• The regulation of pH is done using a variety of buffering systems.
Most media use a bicarbonate-CO2 system as its major
component.
3) Osmolality-
•
A change in osmolality can affect cell growth and function.
•
Salt, Glucose and Amino acids in the growth media determine the osmolality of the
medium.
•
All commercial media are formulated in such a way that their final osmolality is
around 300 mOsm.
•
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 is the mixture of inorganic salts and other nutrients
capable of sustaining cells in culture such as amino acids, fatty acids, sugars, ions, trace
elements, vitamins, cofactors, and ions. Glucose is added as energy source-it’s
concentration varying depending on the requirement. Phenol Red is added as a pH indicator
of the medium.
•
There are two types of media used for culture of animal cells and tissues- the
natural media and the synthesized media.
•
Natural Media - The natural media are the natural sources of nutrient sufficient
for growth and proliferation of animal cells and tissues. The Natural Media used
to promote cell growth fall in three categories.
•
i) Coagulant, such as plasma clots. It is now commercially available in the form
of liquid plasma kept in silicon ampoules or lyophilized plasma. Plasma can also
be prepared in the laboratory taking out blood from male fowl and adding
heparin to prevent blood coagulation.
•
ii) Biological fluids such as serum. Serum is one of the very important
components of animal cell culture which is the source of various amino acids,
hormones, lipids, vitamins, polyamines, and salts containing ions such as
calcium, ferrous, ferric, potassium etc. It also contains the growth factors which
promotes cell proliferation, cell attachment and adhesion factors. Serum is
obtained from human adult blood, placental, cord blood, horse blood, calf blood.
The other forms of biological fluids used are coconut water, amniotic fluid,
pleural fluid, culture filtrate, aqueous humour, from eyes etc
.
iii) Tissue extracts for example Embryo extracts- Extracts from tissues such as embryo, liver,
spleen, leukocytes, tumor, bone marrow etc are also used for culture of animal cells.
Synthetic media
•
Synthetic media are prepared artificially by adding several organic and inorganic
nutrients, vitamins, salts, serum proteins, carbohydrates, cofactors etc. Different types of
synthetic media can be prepared for a variety of cells and tissues to be cultured. Synthetic
media are of two types- Serum containing media (media containing serum) and serumfree media (media with out serum). Examples of some media are: minimal essential
medium (MEM), RPMI 1640 medium, CMRL 1066, F12 etc.
ADVANTAGES
• The behavior and nature of the cells in
controlled conditions can be studied and
interpreted for further research.
• The cell types produced are of single type
and are homogenous and they can be
maintained as pure lines for research.
• This technique can be more economical and
convenient and preparation for animal houses
and animal feed can be eliminated.
• Economical, since smaller quantities of
reagents are needed than in vivo.
• Legal, moral and ethical questions of
animal experimentation are avoided.
DISADVANTAGES
• If large scale production has to be done, it is a labor
intensive and expensive process. The experiments
involved in tissue culture are expensive because of
the expensive machinery required.
• There is a loss of phenotypic characters typical of
the tissue from which the cells had been isolated.
• It needs controlled physiochemical environment.
Controlled and defined physiological conditions are
needed for animal tissue culture.
• As all the animals are genetically similar, there is
reduction is genetic diversity. There is no
introduction of any new genes in the gene pool of
the whole stock.
• Expertise is needed, so that behavior of cells in
culture can be interpreted and regulated. The
procedure needs special attention and diligently
done observation.
• Growth is slow.
APPLICATIONS
• Toxicity Testing:
Animal tissue culture is used to study the effect of new drugs,
cosmetics and chemicals on survival and growth of a number of
types of cells, especially liver and kidney cells. Cultured animal
cells are also used to determine the maximum permissible dosage
of new drugs.
• Genetically Engineered Protein
Animal cell cultures are used
to produce commercially important
genetically engineered proteins such
as monoclonal antibodies, insulin,
hormones and much more.
• Replacement Tissue or Organ:
Animal tissue culture can be used as replacement
tissue or organs. For instance, artificial skin can be
produced using this technique to treat patients with
burns and ulcers.
• Genetic Counseling:
Fetal cell culture extracted from pregnant women can
be used to study or examine the abnormalities of
chromosomes, genes using karyotyping, and these
findings can be used in early detection of fetal
disorders.
• Genetic Engineering:
Cultured animal cells can be used to introduce new
genetic material DNA or RNA into the cell. These
can be used to study the expression of new genes
and its effect on the health of the cell.
• Gene Therapy:
Cultured animal cells can be genetically altered and
can be used in gene therapy technique. First cells
are removed from the patient lacking or missing a
functional gene. These genes are replaced by
functional genes and altered cells are cultured and
grown in laboratory conditions.
Production of commercial proteins:
Large scale production of viruses for use in vaccine
production e.g. polio, rabies, chicken pox, hepatitis B &
measles.
Insect cells are also used to produce commercially
important proteins.
RECENT DISCOVERIES IN ATC.
TISSUE CULTURE &
ENGINEERING
• Tissue culture and engineering Cell culture is a
fundamental component of tissue culture and
tissue engineering, as it establishes the basics
of growing and maintaining cells ex vivo.
Tissue engineering has been defined as
"understanding the principles of tissue growth,
and applying this to produce functional
replacement tissue for clinical use."
RECENT DISCOVERIES
• Bio artificial liver device — several research efforts have produced
hepatic assist devices utilizing living hepatocytes.
• Artificial pancreas — research involves using islet cells to produce
and regulate insulin, particularly in cases of diabetes.
• Artificial bladders — Anthony Atala (Wake Forest University) has
successfully implanted artificially grown bladders into seven out of
approximately 20 human test subjects as part of a long-term
experiment.
• Cartilage — lab-grown tissue was successfully used to repair knee
cartilage.
• Tissue-engineered airway
• Artificial skin constructed from human skin cells embedded in
collagen
• Artificial bone marrow
VACCINES
• Vaccines for polio, measles, mumps, rubella,
and chickenpox are currently made in cell
cultures. Due to the H5N1 pandemic threat,
research into using cell culture for influenza
vaccines is being funded by the United States
government. Novel ideas in the field include
recombinant DNA-based vaccines, such as one
made using human adenovirus (a common cold
virus) as a vector, or the use of adjuvants.
STEM CELLS
• Stem cells are undifferentiated biological that can differentiate into
specialized cells and can divide (through mitosis) to produce more
stem cells. They are found in multi-cellular organisms. In mammals,
there are two broad types of stem cells: embryonic stem cells, which
are isolated from the inner cell mass of blastocysts, and adult stem
cells, which are found in various tissues.
• Adult stem cells are frequently used in medical therapies, for example
in bone marrow transplantation. Stem cells can now be artificially
grown and transformed (differentiated) into specialized cell types
with characteristics consistent with cells of various tissues such as
muscles or nerves.
CONCLUSION
• In conclusion, animal tissue culture is a
very advanced and beneficial
technology for the human welfare.
• Many discoveries have been made and
further research is being carried out.
PATENTING, COPYRIGHTS &
TRADEMARK
• Some people confuse patents,
copyrights, and trademarks. Although
there may be some similarities among
these kinds of intellectual property
protection, they are different and serve
different purposes.
COPYRIGHT
• What Is a Copyright?
Copyright is a form of protection provided to the
authors of "original works of authorship" including
literary, dramatic, musical, artistic, and certain other
intellectual works, both published and unpublished.
• The copyright protects the form of expression rather
than the subject matter of the writing. For example, a
description of a machine could be copyrighted, but this
would only prevent others from copying the description;
it would not prevent others from writing a description
of their own or from making and using the machine.
TRADEMARK OR SERVICE MARK
•
What Is a Trademark or Service mark?
A trademark is a word, name, symbol or device which is
used in trade with goods to indicate the source of the
goods and to distinguish them from the goods of others.
• A service mark, however, identifies and distinguishes the
source of a service rather than goods.
• Trademark rights may be used to prevent others from
using a confusingly similar mark, but not to prevent others
from making the same goods or from selling the same
goods.
PATENTING
• What Is a Patent?
A patent is a limited duration property right relating to an
invention, granted by the United States Patent and
Trademark Office in exchange for public disclosure of the
invention.
• The right conferred by the patent grant is, "the right to
exclude others from making, using, offering for sale, or
selling" the invention or “importing” the invention.
QUIZ
Q-1: In what year was the in vitro technique
discovered?
A.1890
B.1900
C.1903
D.1911
Q-2: Write any two advantages of animal tissue
culture.
Q-3: Do suspension cells require attachment for
growth?
A.YES
B.No.
Q-4: What pH range is used for best working conditions
in animal cell culture?
A.9-9.5
B.11-11.5
C.4-4.5
D.7-7.5
Q-5: Which indicator is added in the Animal tissue
culture media?
A.Iodine.
B.Phenol Red.
C.Methyl Orange.
D.Phenolphthalein.
Q-6 Write the name of one recent discovery made
possible by using Animal cell culture.
Q-7: Write the names of the two different types of animal
cell cultures.
Q-8: Write any two disadvantages of using animal tissue
culture.
Q-9: Write two applications of animal cell culture.
THE END