GROUP#4SADDAF TARIQHIRA ABBAS

MAHNOOR IQTIDAR MIRLARAIB FATIMA

ANIMAL TISSUE CULTURE

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 .

• 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:

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 serum- free 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 usedto produce commercially important genetically engineered proteins suchas 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.

QUIZQ-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