Animal cell, tissue culture

Animal cell, tissue culture

1. MEDIA COMPOSITION OF ANIMAL CELL CULTURE

2. CELL CULTURE Cell culture is the process by which cells are grown under controlled conditions, generally outside of their natural environment. OR Tissue or cell Culture is the general term for the removal of cells, tissues , or organs from an animal or plant A nd their subsequent placement into an artificial environment for growth.

3. ANIMAL CELL CULTURE In vitro cultivation of animal organs , cells, & tissues at defined temperature using an incubator & supplemented with a medium containing cell nutrients and growth factors is collectively known as Animal Tissue culture And will continue to grow if supplied with appropriate conditions and nutrients. Culture process allows the single process to act as an independent unit. Cells are capable of dividing.

4. MEDIA COMPOSITION Most animal cell culture media are generally having following basic components and they are as follows: Energy sources: Glucose, Fructose, Amino acids Nitrogen sources: Amino acids Vitamins : Generally water soluble vitamins B & C. Inorganic salts: Na + , K +, Ca +2 , Mg +2

5. Fat and Fat soluble components: Fatty acids, cholesterols Antibiotics Growth factors and hormones Oxygen and CO2 concentration. The physical environment includes the optimum pH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses

6. CO2 incubators are used and designed to mimic the environmental conditions of the living cells. An inverted microscope is used for visualizing cell cultures invitro . For most animal cell cultures low speed centrifuges are needed Cryo preservation is storing of cells at very low temperature (-1800C to -196 0C) using liquid nitrogen. DMSO is a cryopreservative molecule which prevents damage to cells. Serum is essential for animal cell culture and contains growth factors which promote cell proliferation

7. Contd … Animal cell culture media vary in their complexity but most contain: Amino acids 0.1-0.2 mM Vitamins ca. 1 μM Salts NaCl 150mM KCl 4-6mM CaCl 1mM Glucose 5-10 mM

8. CHARACTERISTICS Animal cells can grow in simple glass or plastic containers in nutritive media but they grow only to limited generations.. The maintenance of growth of cells under laboratory conditions in suitable culture medium is known as PRIMARY CELL CULTURE . Cells can grow as adherent cells ( anchorage dependent-must be cultured while attached to a solid or semi-solid substrate ) O r as suspension cultures ( anchorage independent- can be grown floating in the culture medium ).

9. The primary culture is sub-cultured in fresh media to establish SECONDARY CULTURES . Cells are dissociated from tissues by mechanical means and by enzymatic digestion using proteolytic enzymes.

10. FINITE VS CONTINUOUS CELL LINE Normal cells usually divide only a limited number of times before losing their ability to proliferate, which is a genetically determined event known as senescence ; these cell lines are known as finite .  Some cell lines become immortal through a process called transformation , which can occur spontaneously or can be chemically or virally induced.  When a finite cell line undergoes transformation and acquires the ability to divide indefinitely, it becomes a continuous cell line .

11. CULTURE MEDIA Two types of culture media are used in Animal tissue Culture. Natural media Artificial media The type of medium depends mainly on the type of cells to be cultured and its objectives .

12. NATURAL MEDIA These media consist of naturally occurring biological fluids and are of the following three types: Cagula or Clots Biological Fluids Tissue Extracts

13. CLOTS: The most commonly used clots are Plasma Clots, which have been in use for a long time. Plasma is now commercially available in liquid state which can be prepared in the laboratory. Biological Fluids : Various biological fluids can be used as culture media e.g. amniotic fluid, aqueous humoral from eye, insect hemolymph , serum etc. Serum is the most widely used.

14. Tissue Extracts: Chick embryo extract is the most commonly used tissue extract , but bovine embryo extract is also used. Tissue extract can often be substituted by a mixture of amino acids and certain other organic compounds.

15. ARTIFICIAL MEDIA Different artificial media have been devised to serve one of the following purposes: Immediate survival (a balanced salt solution, with specified pH and osmotic pressure is adequate ), Prolonged survival (a balanced salt solution supplemented with serum, or with suitable formulation of organic compounds), Indefinite growth Specialized functions

16. Artificial media may be grouped into following types. Serum-containing Media Serum-free Media Chemically defined Media Protein-free media

17. SERUM Liquid yellowish, clear content left over after fibrin and cells are removed from the blood is known as serum. Fetal bovine serum (FBS ) is the most commonly applied supplement in animal cell culture media. Normal growth media often contain 2-10% of serum . It provides the basic nutrients for cells P rovides several hormones C ontains several growth factor And i t also acts as a buffer.

18. Advantages of serum in media Disadvantages of serum in media Serum contains various growth factors and hormones which stimulates cell growth and functions. Lack of uniformity in the composition of serum Helps in attachment of the cells Testing needs to be done to maintain the quality of each batch before using Acts as a spreading factor May contain some of the growth inhibiting factors Acts as a buffering agent which helps in maintaining the pH of the culture media Increase the risk of contamination Functions as a binding protein Presence of serum in media may interfere with the purification and isolation of cell culture products Minimizes mechanical damages or damages caused by viscosity

19. SERUM CONTAINING MEDIA Fetal bovine serum is the most common supplement in animal cell culture media . It is used as a low-cost supplement to provide an optimal culture medium. Serum provides carriers water-insoluble nutrients, hormones and growth factors, protease inhibitors, and binds and neutralizes toxic moieties.

20. SERUM FREE MEDIA (SFM) Presence of serum in media can lead to serious misinterpretations in immunological studies. These media are generally specifically formulated to support the culture of a single cell type and incorporate defined quantities of purified growth factors, lipoproteins, and other proteins, which are otherwise usually provided by the serum. And is also referred to as ‘defined culture media’ since the components in these media are known.

21. CHEMICALLY DEFINED MEDIA These media contain contamination-free ultra pure inorganic and organic ingredients, and may also contain pure protein additives, like growth factors. Their constituents are produced in bacteria or yeast by genetic engineering with the addition of vitamins, cholesterol, specific amino acids, and fatty acids

22. PROTEIN-FREE MEDIA Protein-free media do not contain any protein and only contain non-protein constituents. Compared to serum-supplemented media, use of protein-free media promotes superior cell growth and protein expression and facilitates downstream purification of any expressed product.

23. MORPHOLOGY OF CELLS IN CULTURE Cells in culture can be divided in to three basic categories based on their shape and appearance (i.e., morphology). Fibroblastic (or fibroblast-like) cells are bipolar or multipolar, have elongated shapes, and grow attached to a substrate.

24. 2. Epithelial-like cells are polygonal in shape with more regular dimensions, and grow attached to a substrate in discrete patches .

25. 3. Lymphoblast-like cells are spherical in shape and usually grown in suspension without attaching to a surface .

26. APPLICATIONS Production of antiviral vaccines Cancer research, which requires the study of uncontrolled cell division in cultures. Production of pharmaceutical drugs using cell lines. Study the function of the nerve cells. Many commercial proteins have been produced by animal cell culture and there medical application is being evaluated. Tissue Plasminogen activator (t-PA) was the first drug that was produced by the mammalian cell culture by using rDNA technology. The recombinant t-PA is safe and effective for dissolving blood clots in patients with heart diseases and thrombotic disorders