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ANIMAL CELL CULTURE. Ms. HALEEMA SHAHIN ASST PROFESSOR DEPT OF PHARMACOLOGY. ANIMAL CELL CULTURE: Animal culture includes cultivation of animal cells, tissue or organs.
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ANIMAL CELL CULTURE Ms. HALEEMA SHAHIN ASST PROFESSOR DEPT OF PHARMACOLOGY
ANIMAL CELL CULTURE: • Animal culture includes cultivation of animal cells, tissue or organs. • Animal cell culture relates to the cultivation of cells from a multicellular organism usually mammalian cell or even more commonly insect cell, in a suitable nutrient medium aseptically under carefully controlled conditions. • cell culture can be defined as the process of cultivating cells and tissues outside the body of an organism(invitro) in an artificial environment
Cell cultures-there are of four types of animal cell culture: • 1.Primary cell cultures This culture is derived from normal tissue taken from an animal such as mouse, hamster, chicken, monkey, or human. This tissue is treated with proteolytic enzymes to give suspension. These cells are then placed along with the growth medium in a vessel and incubated. The cells are observed to grow as a monolayer on the surface of the vessel.
2.Secondary cell culture or cell line • The primary culture or the original tissue contains many types of cells and also have variable growth functions. • Sub culturing a fraction of cells from the primary culture yields a more homogenous culture of cells. This culture is called a cell line which can be propagated, characterized and stored.
3.Diploid cell strain: • In the cell-line culture, still there may be several ‘cell lineages’. A particular cell lineage with specific properties is identified from the bulk of cells and then cultured. • This is then described as cell strain. In other words, cell strain is derived from a single cell type with specific stable properties like having chromosomes, marker enzymes & antigens.
4. continuous cell line: • All the above described cultures, if derived from a normal tissue can be sub-cultured only for a limited number of times with the cells generally dividing for 20-100 times and the cell in culture finally die out. This is called cellular senescence and may be connected to aging process. Thus, the culture from a normal tissue is finite.
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.
Apart from cell culture, animal culture also includes: • Tissue culture: wherein fragments of excised tissue are grown, in culture medium. • 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 • Organ culture: wherein the entire embryos, organs or tissues are excised from the body either vivisection or shortly after brain death and cultivated in vitro
Properties of the animal cell The animal cells are of 2 types : • Anchorage dependent: cells which remain viable and will proliferate when attached to a solid substrate. • E.g: Primary cultures, normal cell strains etc. • Anchorage independent: cells which will proliferate in suspension. • E.g: Transformed cells, hybridomas.
The techniques of animal cell culture: • The substrate • The culture medium • Explants: Source of tissue • Cultivation • Cell culture for production
The substrate: Most of the vertebrate cells need to spread out on a substrate or surface on to which they proliferate. The substrates used include • Glass E.g: slides, cover slips, flask or test tube. • Plastic E.g: sterilized disposable plastic ware. made of polystyrene, PVC, PTEE, polycarbonate etc. • Palladium and stainless steel discs. • Glass and specially treated plastics are the most commonly used substrates. • However, attachment factors, such as collagen, gelatin, fibronectinand laminin, can be used as substrate coatings to improve growth and function of normal cells derived from brain, blood vessels, kidney, liver, skin, etc.
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, Mg • Fat and Fat soluble components: Fatty acids,cholesterols • Antibiotics • Growth factors and hormones • Oxygen and CO2 concentration. • The physical environment includes the optimumpH, temperature, osmolality and gaseous environment, supporting surface and protecting the cells from chemical, physical, and mechanical stresses
The culture medium: There are 2 classes • Natural substances: these include, • Blood plasma: e.g: plasma from adult chicken. Plasma is used in culture medium to provide: • Nutritive substrate • Supporting structure. • Protection from traumatic damage of cells during sub culturing. • Localized pocket of medium around cells.
Blood serum: • e.g. Human placental cord serum, • chicken serum, • mammalian serum • 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.
Complex natural media: • These are made up of a number of natural occurring media of undefined composition, like beef embryo extract, horse serum, bovine amniotic fluid, yeast extract, bet albumin hydrosylate etc. • Biological fluids: • e.g. Amniotic fluid, ascetic and pleural fluids, aqueous humour(from eye),insect hem lymph.
Artificial media may be grouped into following types. • Serum-containing Media • Serum-free Media • Chemically defined Media 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. .
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.
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
Chemically defined culture media: • Though natural media are mostly used, they have the advantage of not giving reproducible results. • Almost all the chemically defined media contain: • Glucose as galactose. • Essential amino acids especially glutamine. • Vitamins of B-complex, cholineinositol. • Inorganic salts Na+,K+,Ca+2... Etc.
Explant: source of tissue • Embryo:consisting of cells termed as precursor or master cells. The cells are characterised by low level of specialization capacity and high survival rate. • Adult tissue:these may be of 3 types: • Stem cell containing. • Committed precursor cell containing. • Adult cells. • Neoplastic:these are characterised by their uncontrolled growth rate.
Cultivation: • Primary explant technique: • Slide or cover slip cultures • Corel flask cultures • Roller test tube culture.
Disaggregation of tissue: • Enzymatic disaggregation: • A tissue may be treated with certain enzymes such as trypsin or pepsin to break up into cells. • Mechanical disaggregation: This involves forcing the tissue repeatedly through • A stainless steel sieve followed by use of sieves with gradually reducing aperture size. • A needle in a disposable syringe • A pipette.
In vitro v/s in vivo environment: • Cell-cell interaction is reduced in vitro • Nutritional milieu is changed in vitro • Spreading is increased in vitro • Migration is increased in vitro • Proliferation is increased in vitro
Cell culture for production: • This pertains to the culturing of mammalian cells for producing commercially useful materials. • The culture may be setup at small scale in roller bottles of 100ml capacity or at large scale in a fermentors or bioreactors of 100 ltrs capacity.
Advantages: • There is absolute control of physical environment. • Homogenesity of sample • Less compound needed than in animal models. • Disadvantages : • Animal cells are hard to maintain in culture. • Only a small amount of tissue can be grown that too at high cost. • De-differentiation of animal tissue. • Instability and aneuploidy in animal cells. • Low productivity of desired product.
Products and applications: • Production of viral vaccines • Production of cell surface antigen • Culturing of immune cells • Production of monoclonal antibodies. • Production of therapeutic proteins • Production of A-interferons • Production of B-interferons • Production of human hormones • Production of enzymes • In vitro skin cell growth in clinical practice.
Monoclonal antibodies: Produced by hybridoma cell • Used for diagnostic assay systems (determine drugs, toxins & vitamin);therapeutic purposes & biologicalseparations – chromatographicseparations to purify protein molecules Immunobiological Regulators: • interferon – anticancer glycoprotein (secreted animal cell or recombinant bacteria) • Lymphokines • Interleukines (anticanceragent) Virus vaccines: Prophylactics Virus is collected, inactivated and used as vaccine • A weakened form will induce aprotective response but no disease
Hormons: • Large molecules: 50-200 amino acids • Produce by hormonesynthesizing organ ,May also produce By chemical synthesis. • Example: Erythropoietin Enzymes • Urokinase, rennin, asparaginase,collaginase, pepsin, trypsin, etc.. Insecticides • Production of some insect viruses that are highly specific and safe to envirionment. Whole cells and tissue Artificial organs and semi synthetic bone and dental Structure.
