tissue culture

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Mammalian Cell Culture:

Mammalian Cell Culture

Introduction:

Introduction Tissue culture is the growth of tissues and/or cells separate from the organism. This is typically facilitated via use of a liquid, semi-solid, or solid growth medium. The term tissue culture is used for both cell and organ culture.

What is cell culture, exactly?:

What is cell culture, exactly? Cells, previously growing in a human or animals modified to grow in plastic or glass In the body = in vivo On plastic or glass = in vitro Kept in an incubator to stay at body temperature We use special media with nutrients so the cells can grow and divide

If we treat the cells right:

If we treat the cells right They might act similar to those in vivo Making the same proteins Then scientists can change the environment

In-vitro cell and tissue system five main categories will be considered.:

In-vitro cell and tissue system five main categories will be considered. Requirement of Cells and tissues. In-vitro Culture conditions. Handling and Maintenance. Cryopreservation. Microbial, Viral, and Cellular Cross-contamination.

Cell and Tissue :

Cell and Tissue Isolated organs or Tissues. Primary cultures and early passage cultures. Cell lines.

Isolated organs or Tissues.:

Isolated organs or Tissues. Taken for direct use from animal or human donors. Perfused with physiological buffers. Maintain or reconstruction of three-dimensional structures, with some of the structural and functional properties of the original organ or tissue.

Primary cultures and early passage cultures:

Primary cultures and early passage cultures The initial in vitro culture of harvested cells and tissues taken directly from animals and humans is called primary culture. They have a limited life span and known to change their differentiated characteristics with time in culture. Primary cultures are maintain in suspension or monolayer culture.

Cell-lines:

Cell-lines Cell lines comprise cells that are able to multiply for extended periods in vitro and can therefore be maintained by serial subculture. Cell-lines are three types. Finite cell-lines Continuous cell-lines. Stem cells.

Finite cell lines:

Finite cell lines Finite cell lines are cultures of cells that possess the ability to be subcultured numerous times, but which eventually cease replication and enter a state of senescence.

Continuous cell lines :

Continuous cell lines Certain cell lines show an apparent ability to be subcultured indefinitely, and are known as continuous cell lines. They do not show the senescence experienced with finite cell lines. typically derived from tumors or normal embryonic tissues. Continuous cell lines may arise spontaneously, or can be produced by using a variety of other methodologies, such as: exposure of normal cells and tissues to irradiation and/or treatment with chemical mutagens or carcinogens; isolation from cultures infected with viruses (for example, Epstein-Barr virus); genetic modification of cells by transfection with cloned genes (for example, SV40 large T-antigen, adenovirus E1, telomerase); and isolation from transgenic animals.

Stem cell lines:

Stem cell lines Stem cell lines, such as embryonic and germ cell lines, are types of continuous cell lines that retain the characteristics of stem cells and can produce diverse differentiated cell types.

2. In Vitro Culture Conditions :

2. In Vitro Culture Conditions Basal medium Serum Nutritional status Antibiotics Cell culture surface/matrix

Basal medium:

Basal medium Phosphate Buffered Saline Used to wash/remove excess serum that inhibits the function of TRED. Must be warmed in the water bath before use so cells are not shocked by cold liquid.

What is in the media?:

What is in the media? F-12 Growth Media Contains glucose, some proteins, and essential salts Contains a pH indicator ( phenol red ) Media looks pink/red at pH 7.2 Acidic -yellow or orange (cell growth, bacterial growth) Basic -purple (no cell growth, not enough CO 2 )

More media components:

More media components Antibiotics (penicillin, Gentamycin and streptomycin) Prevent bacterial contamination Salts and buffers To simulate in vivo environment Serum Portion of blood after the cells and fibers have clotted From cow, horse, sheep added to media as a nutrient source for growing cells Lipids, proteins

3. Handling and Maintenance:

3. Handling and Maintenance Temperature Atmosphere pH Cell detachment and subculture

Temperature :

Temperature For mammalian cell optimal temp. 37°C. Above 37°C induced apoptosis. Below 37°C slow replication but also enhanced the expression of certain cell proteins.

Atmosphere:

Atmosphere Oxygen and carbon dioxide are known to be vital for cell growth. High level are toxic and very low level will inhibit cell growth. For mammalian cell 5% carbon dioxide is used.

pH :

pH The optimal physiological pH for mammalian cell culture is usually considered to be pH 7.2-7.4.

Cell detachment and subculture:

Cell detachment and subculture Trypsin EDTA (TRED) An enzyme used to detach the cells from a culture dish. EDTA binds calcium ions in the media that would normally inhibit trypsin.

4. Cryopreservation:

4. Cryopreservation Cells and tissues can be cryopreserved in a stable state for limited or prolonged periods. The cryopreservation process includes freezing, storage and recovery.

5. Microbial, Viral and Cellular Cross-contamination :

5. Microbial, Viral and Cellular Cross-contamination Contamination with bacteria, yeast and other fungi can result in the complete loss of cultures. There are various potential sources of viral contamination, including the operator, cell culture reagents of animal origin, and cells or tissues of animal origin. Cross-contamination of cell lines with other cell lines is a real, but often neglected, problem.

Animals are complex:

Animals are complex Many different cells Many different proteins Interacting continuously Difficult to watch individual events in vivo Animals are usually harmed to observe biological events

Using cultures has advantages:

Using cultures has advantages Fewer animals are harmed Can control all external factors Can easily test what the cells are doing Cells are easy to manipulate and propagate All of the cells are the same Results of experiments will be consistent Cheaper to maintain

What can we do with cells?:

What can we do with cells? Test pharmaceutical drugs Watch disease mechanisms Design potential treatments Observe the regenerative process How do cells and tissues repair themselves after damage from illness or injury? Observe the developmental process

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