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MIC-610 1 Advance Virology and Advance Virology and Tissue Culture Tissue Culture MIC-610 Course Incharge: Shazia Tabassum Hakim Assistant Professor & Chairperson, Department of Microbiology, Jinnah University for Women, Karachi, Pakistan (2006 AD)

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Page 1: Advance Virology and Tissue Culture

MIC-610 1

Advance Virology Advance Virology and Tissue Cultureand Tissue Culture

MIC-610Course Incharge: Shazia

Tabassum HakimAssistant Professor & Chairperson,

Department of Microbiology,

Jinnah University for Women, Karachi, Pakistan

(2006 AD)

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A long path from Viruses, A long path from Viruses, Virology,Vaccines and Drug Virology,Vaccines and Drug

discovery to Stem Cell discovery to Stem Cell ResearchResearch

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Cell biologyCell biology

Cell biology (also called cellular biology or cytology, from the Greek kytos, "container") is an academic discipline that studies cells. This includes their physiological properties, their structure, the organelles they contain, interactions with their environment, their life cycle, division and death. This is done both on a microscopic and molecular level. Cell biology research extends to both the great diversity of single-celled organisms like bacteria and the many specialized cells in multicellular organisms like humans.

Every cell typically contains hundreds of different kinds of macromolecules that function together to generate the behavior of the cell. Each type of protein is usually sent to a particular part of the cell. An important part of cell biology is investigation of molecular mechanisms by which proteins are moved to different places inside cells or secreted from cells.

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Cell biologyCell biology

Most proteins are synthesized by ribosomes in the cytoplasm. This process is also known as protein biosynthesis or simply protein translation.

Some proteins, such as those to be incorporated in membranes (membrane proteins), are transported into the ER during synthesis and further processed in the Golgi apparatus. From the Golgi, membrane proteins can move to the plasma membrane, to other subcellular compartments or they can be secreted from the cell.

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Cell biologyCell biology The ER and Golgi can be thought of as the "membrane

protein synthesis compartment" and the "membrane protein processing compartment", respectively.

There is a semi-constant flux of proteins through these compartments. ER and Golgi-resident proteins associate with other proteins but remain in their respective compartments.

Other proteins "flow" through the ER and Golgi to the plasma membrane. Motor proteins transport mebrane protein-containing vesicles along cytoskeletal tracks to distant parts of cells such as axon terminals.

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Cell biologyCell biology Some proteins that are made in the cytoplasm contain

structural features that target them for transport into mitochondria or the nucleus. Some mitochondrial proteins are made inside mitochondria and are coded for by mitochondrial DNA. In plants, chloroplasts also make some cell proteins.

Extracellular and cell surface proteins destined to be degraded can move back into intracellular compartments upon being incorporated into endocytosed vesicles. Some of these vesicles fuse with (lysosomes) where the proteins are broken down to their individual amino acids. The degradation of some membrane proteins begins while still at the cell surface when they are cleaved by secretases. Proteins that function in the cytoplasm are often degraded by proteasomes.

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Animal CellAnimal Cell

An animal cell is a form of eukaryotic cell which make up many tissues in animals.

The animal cell is distinct from other eukaryotes, most notably plant cells, as they lack cell walls and chloroplasts, and they have smaller vacuoles.

Due to the lack of a rigid cell wall, animal cells appear to be circular (though are often deformed by surrounding cells) under microscopes

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Cell OrganelleCell Organelle

The most significant organelles of an animal cell include: Cell membrane Cytoplasm Golgi apparatus Mitochondria Endoplasmic reticulum (including ribosomes) Lysosome Peroxisome Vacuole Microtubule Microfilament (nikheel) Vesicle

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Cell membraneCell membrane

A cell membrane, plasma membrane or plasmalemma is a selectively permeable lipid bilayer coated by proteins which comprises the outer layer of a cell.

It consists of, among other components, phospholipid and protein molecules which separate the cell interior from its surroundings within animal cells, and control the input and output of cell through the use of receptor and cell adhesion proteins, which also play a role in cell behavior and the organization of cells within tissues.

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Cell membraneCell membrane In animal cells, the cell membrane establishes this

separation alone, whereas in yeast, bacteria and plants an additional cell wall forms the outermost boundary, providing primarily mechanical support.

The plasma membrane is only about 10 nm thick and may be discerned only faintly with a transmission electron microscope. One of the key roles of the membrane is to maintain the cell potential.

Phospholipid molecules in the cell membrane are "fluid," in the sense that they are free to diffuse and exhibit rapid lateral diffusion. Lipid rafts and caveolae are examples of cholesterol-enriched microdomains in the cell membrane.

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Cell membraneCell membrane

Many proteins are not free to diffuse. The cytoskeleton undergirds the cell membrane and provides anchoring points for integral membrane proteins.

Anchoring restricts them to a particular cell face or surface – for example, the "apical" surface of epithelial cells that line the vertebrate gut – and limits how far they may diffuse within the bilayer. Rather than presenting always a formless and fluid contour, the plasma membrane surface of cells may show structure.

Returning to the example of epithelial cells in the gut, the apical surfaces of many such cells are dense with involutions, all similar in size. The finger-like projections, called microvilli, increase cell surface area and facilitate the absorption of molecules from the outside.

Synapses are another example of highly-structured membrane.

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Cell membraneCell membrane

New material is incorporated into the membrane, or deleted from it, by a variety of mechanisms.

Fusion of intracellular vesicles with the membrane not only excretes the contents of the vesicle, but also incorporates the vesicle membrane's components into the cell membrane. The membrane may form blebs that pinch off to become vesicles.

If a membrane is continuous with a tubular structure made of membrane material, then material from the tube can be drawn into the membrane continuously.

Although the concentration of membrane components in the aqueous phase is low (stable membrane components have low solubility in water), exchange of molecules with this small reservoir is possible.

In all cases, the mechanical tension in the membrane has an effect on the rate of exchange. In some cells, usually having a smooth shape, the membrane tension and area are interrelated by elastic and dynamical mechanical properties, and the time-dependent interrelation is sometimes called homeostasis, area regulation or tension regulation.

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Cell membraneCell membrane

Functions It attaches parts of the cytoskeleton to the cell membrane in order to

provide shape. It attaches cells to an extra-cellular matrix in grouping cells together to

form tissues. It transports molecules into and out of cells by such methods as ion

pumps, channel proteins and carrier proteins. It acts as receptor for the various chemical messages that pass between

cells such as nerve impulses and hormone activity. It takes part in enzyme activity which can be important in the

metabolism or as part of the body's defense mechanism.

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Endoplasmic reticulumEndoplasmic reticulum

The endoplasmic reticulum (endoplasmic meaning "within the cytoplasm," reticulum meaning "little net") or ER is an organelle found in all eukaryotic cells that is an interconnected network of tubules, vesicles and cisternae that is responsible for several specialized functions: Protein translation, folding, and transport (e.g., transmembrane receptors and other integral membrane proteins) of proteins to be used in the cell membrane, are to be secreted or "exocytosed" from the cell (e.g., digestive enzymes); sequestation of calcium; production and storage of glycogen, steroids, and other macromolecules; and creation and transport of cell membrane proteins. The endoplasmic reticulum is part of the endomembrane system. The basic structure and composition of the ER is similar to the plasma membrane, although it is actually an extension of the nuclear membrane.

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Endoplasmic reticulumEndoplasmic reticulum

The ER consists of an extensive membrane network of tubes and cisternae (sac-like structures) held together by the cytoskeleton. The membrane encloses a space, the cisternal space (or internal lumen) from the cytosol. Parts of the ER membrane are continuous with the outer membrane of the nuclear envelope, and the cisternal space of the ER is continuous with the space between the two layers of the nuclear envelope (the intermembrane space).

Parts of the ER are covered with ribosomes (which assemble amino acids into proteins based on instructions from the nucleus). Their rough appearance under electron microscopy led to their being called rough ER (rER), other parts are free of ribosomes and are called smooth ER (sER). The ribosomes on the surface of the rough ER insert the freshly produced proteins directly into the ER, which processes them and then passes them on to the Golgi apparatus

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Endoplasmic reticulumEndoplasmic reticulum

Rough ERThe rough ER contains protein-manufacturing ribosomes (the ribosomes on its surface are responsible for it being named "rough") and transports proteins destined for membranes and secretion and is connected to the nuclear envelope as well as linked to the cis cisternae of the Golgi complex by vesicles that shuttle between the two compartments. The rough ER works in concert with the Golgi apparatus to target new proteins to their proper destinations.

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Endoplasmic reticulumEndoplasmic reticulum Smooth ER The smooth ER has functions in

several metabolic processes, including synthesis of lipids, metabolism of carbohydrates, and is connected to the nuclear envelope. It is found in a variety of cell types and it serves different functions in each. It consists of tubules and vesicles that branch forming a network. In some cells there are dilated areas like the sacs of rough ER. The network of smooth endoplasmic reticulum allows increased surface area for the action or storage of key enzymes and the products of these enzymes. The smooth ER is known for its storage of Calcium ions in muscle cells.

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FunctionsFunctions of of Endoplasmic Endoplasmic

reticulumreticulum The endoplasmic reticulum serves many general functions, including

the facilitation of protein folding and the transport of synthesized proteins in sacs called cisternae.

Insertion of proteins into the ER membrane: Integral proteins must be inserted into the ER membrane after they are synthesized. Insertion into the ER membrane requires the correct topogenic sequences.

Glycosylation: Glycosylation involves the attachment of oligosaccharides.

Disulfide bond formation and rearrangement: Disulfide bonds stabilize the tertiary and quaternary structure of many proteins.

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Golgi ApparatusGolgi Apparatus

In cell biology, the Golgi apparatus (also called a Golgi body, Golgi complex, or dictyosome) is an organelle found in most eukaryotic cells, including those of plants, animals, and fungi. The name comes from Italian anatomist Camillo Golgi, who identified it in 1898. The primary function of the Golgi apparatus is to process proteins targeted to the plasma membrane, lysosomes or endosomes, and those that will be formed from the cell, and sort them within vesicles. Thus, it functions as a central delivery system for the cell. It is part of the endomembrane system.

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Golgi ApparatusGolgi Apparatus

Vesicles that leave the endoplasmic reticulum (ER), specifically rough ER, are transported to the Golgi apparatus, where they are modified, sorted, and shipped towards their final destination. The Golgi apparatus is present in most eukaryotic cells, but tends to be more prominent where there are many substances, such as proteins, being secreted. For example, plasma B cells, the antibody-secreting cells of the immune system, have prominent Golgi complexes.

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Golgi ApparatusGolgi Apparatus The Golgi bodies are considered the "post office" of the cell. It handles all

incoming lipids, proteins, etc., and controls their export, as well. The transport vesicles from the Endoplasmic Reticulum (ER) fuse with

the cis face of the Golgi apparatus (to the cisternae) and empty their protein content into the Golgi lumen. The proteins are then transported through the medial region toward the trans face and are modified on their way. Possible modifications include glycosylation and phosphorylation. The proteins are also labeled with a sequence of molecules according to their final destination. For example, the Golgi apparatus adds a mannose-6-phosphate label to proteins destined for lysosomes.

The transport mechanism itself is not yet clear; it could happen by cisternae progression (the movement of the apparatus itself, building new cisternae at the cis face and destroying them at the trans face) or by vesicular transport (small vesicles transport the proteins from one cisterna to the next, while the cisternae remain unchanged). It is also proposed that the cisternae are interconnected, and the transport of cargo molecules within the Golgi is due to diffusion, while the localisation of Golgi-resident proteins is achieved by an unknown mechanism.

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MitochondrionMitochondrion

In cell biology, a mitochondrion (plural mitochondria) (from Greek μιτος or mitos, thread + κουδριον or khondrion, granule) is an organelle, variants of which are found in most eukaryotic cells.[1] Mitochondria are sometimes described as "cellular power plants," because they convert organic materials into energy in the form of ATP via the process of oxidative phosphorylation. Usually a cell has hundreds or thousands of mitochondria, which can occupy up to 25% of the cell's cytoplasm. Mitochondria have their own DNA and may, according to the endosymbiotic theory, be descended from free-living prokaryotes that were closely related to rickettsia bacteria.

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MitochondriMitochondrial Functionsal Functions Although it is well known that the

mitochondria convert organic materials into cellular energy in the form of ATP, mitochondria play an important role in many metabolic tasks, such as:

Apoptosis-programmed cell death Glutamate-mediated excitotoxic

neuronal injury Cellular proliferation Regulation of the cellular redox state Heme synthesis Steroid synthesis

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MitochondriMitochondrial Functionsal Functions

Some mitochondrial functions are performed only in specific types of cells. For example, mitochondria in liver cells contain enzymes that allow them to detoxify ammonia, a waste product of protein metabolism. A mutation in the genes regulating any of these functions can result in mitochondrial diseases.

Energy conversion Pyruvate: the citric acid cycle NADH and FADH2: the electron transport chain

Heat production Under certain conditions, protons can re-enter the mitochondrial matrix

without contributing to ATP synthesis. This process is known as proton leak or mitochondrial uncoupling and is due to the facilitated diffusion of protons into the matrix, mediated by a proton channel called thermogenin. This results in the unharnessed potential energy of the proton electrochemical gradient being released as heat. Thermogenin is found in brown adipose tissue (brown in colour due to high levels of mitochondria) where it is used to generate heat by non-shivering thermogenesis. Non-shivering thermogenesis is the primary means of heat generation in newborn or hibernating mammals.

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Cell nucleusCell nucleus

In cell biology, the nucleus (pl. nuclei; from Latin nucleus or nuculeus, kernel) is an organelle found in most eukaryotic cells. It contains the chromosomes that contain most of the cell's genetic material. The genes within these chromosomes make up the nuclear genome. The function of the nucleus is to maintain these genes and to control the activities of the cell by regulating when particular genes are copied for use.

The nucleus was the first organelle to be discovered, and was first described by Franz Bauer in 1802. It was later popularlized by Scottish botanist Robert Brown in 1831. While studying orchids microscopically, Brown observed an opaque area in the cells of the flower's outer layer, which he called the areola or nucleus.

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Cell nucleusCell nucleus

Structure The nucleus is the largest cellular organelle. It varies in diameter from 11 to

22 μm and occupies about 10% of the total cell volume. The viscous liquid within it is called nucleoplasm, and is similar to the cytoplasm found outside the nucleus.

Nuclear envelope and pores The eukaryotic nucleus and endomembrane system. The outer nuclear

membrane is continuous with the membrane of the rough endoplasmic reticulum, and is similarly studded by ribosomes. The space between the membranes is called the perinuclear space and is continuous with the lumen of the rough endoplasmic reticulum.

The nuclear envelope consists of two cellular membranes, an inner and an outer membrane, arranged parallel to one another and separated by 10 to 50 nm. One of the features that make the nuclear membranes unique are the large pores they contain. The nuclear envelope encloses the nucleus and separates the cell's genetic material from the surrounding cytoplasm, serving as a barrier to prevent macromolecules from diffusing freely between the nucleoplasm and the cytoplasm.

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Cell nucleusCell nucleus

The outer nuclear membrane is continuous with the membrane of the rough endoplasmic reticulum (RER), and is similarly studded with ribosomes. The space between the membranes is called the perinuclear space and is continuous with the RER lumen.

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Cell nucleusCell nucleus

Nuclear pores, which provide aqueous channels through the envelope, are composed of a number of different proteins, collectively referred to as nucleoporins. The pores are about 125 million daltons in molecular weight and consist of around 50 (in yeast) to 100 proteins (in vertebrates).

The pores are 100nm in diameter, however, after the annulus and other regulatory gating system molecules are present, the space left for molecules to enter is reduced to 9nm. This size allows the free passage of small water soluble molecules whilst excluding larger structures, such as DNA or proteins. Larger molecules can still enter the nucleus, but need to be transported. The nucleus of a typical mammalian cell will hold about 3000 to 4000 pores throughout its envelope.

Most proteins, ribosomal subunits, and some RNAs have their transport through the pore complexes mediated by karyopherins, a family of transport factors. Those karyopherins that mediate movement into the nucleus are also called importins, while those that mediate movement out of the nucleus are also called exportins. Most karyopherins interact directly with their cargo, although some use adaptor proteins

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Cell nucleus Cell nucleus

The entry and exit of large molecules from the nucleus is tightly controlled by the nuclear pore complexes. Although small molecules can enter the nucleus without regulation, macromolecules such as RNA and proteins require association karyopherins called importins to enter the nucleus and exportins to exit.

Proteins that must be imported to the nucleus from the cytoplasm carry nuclear localization signals (NLS) that are bound by importins. A NLS is a sequence of amino acids that acts as a tag. They are diverse in their composition and most commonly hydrophilic, although hydrophobic sequences have also been documented. Proteins, transfer RNA, and assembled ribosomal subunits are exported from the nucleus due to association with exportins, which bind signaling sequences called nuclear export signals (NES). The ability of both importins and exportins to transport their cargo is regulated by various GTPases.

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Cell nucleus Cell nucleus

GTPases are enzymes that bind to a molecule called guanosine triphosphate (GTP) which they then hydrolyze in order to release energy. These GTPases use the energy to allow the importins and exportins to release or bind the cargo, as well as restoring the transport protein to its original location. Ran is a GTPase involved in nuclear transport. It can exist in two states, depending on whether it is binding GTP (to form the complex Ran-GTP) or GDP (to form Ran-GDP). The dominant nucleotide binding state of Ran depends on whether it is located in the nucleus or the cytoplasm.

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During apoptosisDuring apoptosis

Apoptosis is a controlled process resulting in death of the cell. Various of the changes directly affect the nucleus and its contents, especially condensation of the chromatin, disintegration of the nuclear envelope and lamina. The progressive organisation of the nuclear lamina throughout apoptosis is used to initiate and regulate the various phases of apoptosis. The breakdown of the lamina is controlled by a group of proteins called caspases that cleave the individual lamins.

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RibosomeRibosome

A ribosome is an organelle composed of ribosomal RNA and ribosomal proteins (known as a Ribonucleoprotein or RNP). It translates Messenger RNA (mRNA) into a polypeptide chain (e.g., a protein). It can be thought of as a factory that builds a protein from a set of genetic instructions. Ribosomes can float freely in the cytoplasm (the internal fluid of the cell) or bind to the endoplasmic reticulum, or to the nuclear envelope. Since ribosomes are ribozymes, it is thought that they might be remnants of the RNA world.

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RibosomeRibosome

Ribosomes were first observed in the mid-1950s by cell biologist George Palade in the electron microscope as dense particles or granules for which he would win the Nobel Prize. The term ribosome was proposed by scientist Richard B. Roberts in 1958:

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RibosomeRibosome

Free ribosomes Free ribosomes occur in all cells, and also in mitochondria and

chloroplasts of eukaryotic cells. Free ribosomes usually produce proteins used in the cytosol or organelle in which they occur. As the name implies, they are free in solution and not bound to anything within the cell.

Membrane bound ribosomes When certain proteins are synthesized by a ribosome they can become

"membrane-bound". The newly produced polypeptide chains are inserted directly into the endoplasmic reticulum by the ribosome and are then transported to their destinations. Bound ribosomes usually produce proteins that are used within the cell membrane or are expelled from the cell via exocytosis.

Free and membrane-bound ribosomes differ only in their spatial distribution; they are identical in structure and function. Whether the ribosome exists in a free or membrane-bound state depends on the presence of a ER-targeting signal sequence on the protein being synthesized.

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Structure and Structure and functionfunction of of RibosomeRibosome

The ribosomal subunits of prokaryotes and eukaryotes are quite similar. However, prokaryotes have 70S ribosomes, each consisting of a (small) 30S and a (large) 50S subunit, whereas eukaryotes have 80S ribosomes, each consisting of a (small) 40S and a bound (large) 60S subunit.

However, the ribosomes found in chloroplasts and mitochondria of eukaryotes are 70S, this being but one of the observations supporting the endosymbiotic theory. The unit "S" means Svedberg units, a measure of the rate of sedimentation of a particle in a centrifuge, where the sedimentation rate is associated with the size of the particle.

It is important to note that Svedberg units are not addable - two subunits together can have Svedberg values that do not add up to that of the entire ribosome. This is resulting from the loss of surface area when the two subunits are bound. In addition, the ungainly shape of the fully assembled ribosome has different aqua dynamic properties from the two unbound subunits.

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Structure and functionStructure and function of of RibosomeRibosome

The differences between the prokaryotic and eukaryotic ribosomes are exploited by humans since the 70S ribosomes are vulnerable to some antibiotics that the 80S ribosomes are not. This helps create drugs that can destroy a bacterial infection without harming the animal/human host's cells. Even though human mitochondria possess 70S ribosomes, mitochondria are rarely affected by these antibiotics because mitochondria are covered by a double membrane that does not easily admit these antibiotics into the organelle.

Ribosomes are the workhorses of protein synthesis, the process of translating messenger RNA (mRNA) into protein. The mRNA comprises a series of codons that dictate to the ribosome the amino acids needed to make the protein. Using the mRNA as a template, the ribosome traverses each codon of the mRNA, pairing it with the appropriate amino acid. This is done using molecules of transfer RNA (tRNA) containing a complementary anticodon on one end and the appropriate amino acid on the other.

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Structure and functionStructure and function of of RibosomeRibosome

Protein synthesis begins at a start codon near the 5' end of the mRNA. The small ribosomal subunit, typically bound to a tRNA containing the amino acid methionine, binds to an AUG codon on the mRNA and recruits the large ribosomal subunit. The large ribosomal subunit contains three tRNA binding sites, designated A, P, and E. The A site binds an aminoacyl-tRNA (a tRNA bound to an amino acid); the P site binds a peptidyl-tRNA (a tRNA bound to the peptide being synthesized); and the E site binds a free tRNA before it exits the ribosome

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Cell CycleCell Cycle

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Cell CycleCell Cycle

The cell cycle is an ordered set of events, culminating in cell growth and division into two daughter cells. Non-dividing cells not considered to be in the cell cycle.

The stages, pictured to the left, are G1-S-G2-M. The G1 stage stands for "GAP 1". The S stage stands for "Synthesis". This is the stage when DNA replication occurs. The G2 stage stands for "GAP 2".

The M stage stands for "mitosis", and is when nuclear (chromosomes separate) and cytoplasmic (cytokinesis) division occur. Mitosis is further divided into 4 phases

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Cell CycleCell Cycle

Regulation of the cell cycle How cell division (and thus tissue growth) is controlled is very complex. The following terms are some of the features that are important in regulation, and places where errors can lead to cancer. Cancer is a disease where regulation of the cell cycle goes awry and normal cell growth and behavior is lost.

Cdk (cyclin dependent kinase, adds phosphate to a protein), along with cyclins, are major control switches for the cell cycle, causing the cell to move from G1 to S or G2 to M.

MPF (Maturation Promoting Factor) includes the CdK and cyclins that triggers progression through the cell cycle.

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Cell CycleCell Cycle

p53 is a protein that functions to block the cell cycle if the DNA is damaged. If the damage is severe this protein can cause apoptosis (cell death).

1. p53 levels are increased in damaged cells. This allows time to repair DNA by blocking the cell cycle.

2. A p53 mutation is the most frequent mutation leading to cancer. An extreme case of this is Li Fraumeni syndrome, where a genetic a defect in p53 leads to a high frequency of cancer in affected individuals.

p27 is a protein that binds to cyclin and cdk blocking entry into S phase. Recent research (Nature Medicine 3, 152 (1997)) suggests that breast cancer prognosis is determined by p27 levels. Reduced levels of p27 predict a poor outcome for breast cancer patients.

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Cell CycleCell Cycle

What is (and is not) mitosis? Mitosis is nuclear division plus cytokinesis, and produces two identical daughter cells during prophase, prometaphase, metaphase, anaphase, and telophase. Interphase is often included in discussions of mitosis, but interphase is technically not part of mitosis, but rather encompasses stages G1, S, and G2 of the cell cycle.

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MitosisMitosis

Interphase: The cell is engaged in metabolic activity and performing its prepare for mitosis (the next four phases that lead up to and include nuclear division). Chromosomes are not clearly discerned in the nucleus, although a dark spot called the nucleolus may be visible. The cell may contain a pair of centrioles (or microtubule organizing centers in plants) both of which are organizational sites for microtubules.

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MitosisMitosis

Prophase: Chromatin in the nucleus begins to condense and becomes visible in the light microscope as chromosomes. The nucleolus disappears. Centrioles begin moving to opposite ends of the cell and fibers extend from the centromeres. Some fibers cross the cell to form the mitotic spindle.

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MitosisMitosis

Prometaphase: The nuclear membrane dissolves, marking the beginning of prometaphase. Proteins attach to the centromeres creating the kinetochores. Microtubules attach at the kinetochores and the chromosomes begin moving.

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MitosisMitosis

Metaphase: Spindle fibers align the chromosomes along the middle of the cell nucleus. This line is referred to as the metaphase plate. This organization helps to ensure that in the next phase, when the chromosomes are separated, each new nucleus will receive one copy of each chromosome.

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MitosisMitosis

Anaphase: The paired chromosomes separate at the kinetochores and move to opposite sides of the cell. Motion results from a combination of kinetochore movement along the spindle microtubules and through the physical interaction of polar

microtubules.

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MitosisMitosis

Telophase: Chromatids arrive at opposite poles of cell, and new membranes form around the daughter nuclei. The chromosomes disperse and are no longer visible under the light microscope. The spindle fibers disperse, and cytokinesis or the partitioning of the cell may also begin during this stage.

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MitosisMitosis

Cytokinesis/ Telophase 2:

In animal cells, cytokinesis results when a fiber ring composed of a protein called actin around the center of the cell contracts pinching the cell into two daughter cells, each with one nucleus. In plant cells, the rigid wall requires that a cell plate be synthesized

between the two daughter cells.

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Enzymes for Enzymes for Regulation Regulation

The class of enzymes that are involved in triggering events in the cell cycle are called:

A.kinases kinases add phosphate to molecules, and the modification can

serve as a "switch" to turn events in the cell on or off. Cdk or cyclin dependent kinases regulate the cell cycle.

Cell Cycle Sequence : (G1 to S to G2 to M to cytokinesis) The correct sequence has G1 as a preparation for S and G2 as the

time between the completion of S and entry into M. Cytokinesis occurs after the other stages to create two daughter cells.

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MeiosisMeiosis

Chromosomes in a Diploid Cell Summary of chromosome characteristics Diploid set for humans; 2n = 46 Autosomes; homologous chromosomes, one from each

parent (humans = 22 sets of 2) Sex chromosomes (humans have 1 set of 2)

1. Female-sex chromosomes are homologous (XX)

2. Male-sex chromosomes are non-homologous (XY)

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MeiosisMeiosis

Karyotyping Karyotype A pictorial display of metaphase chromosomes from a

mitotic cell Homologous chromosomes- pairs The traditional process for karyotyping involves adding a

dye to metaphasic chromosomes. Different dyes that affect different areas of the chomosomes are used for a range of identification purposes. One common dye used is Giemsa; That process is known as G-banding. This dye is effective because it markedly stains the bands on a chromosome; Each chromosome can then be identified by its banding pattern, but the resuls is similar overall gray values for each chromosome.

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MeiosisMeiosis

Spectral Karyotyping- a new method Ploidy: Number of sets of chromosomes in a cell Haploid (n)-- one set chromosomes Diploid (2n)-- two sets chromosomes Most plant and animal adults are diploid (2n) Eggs and sperm are haploid (n) The new karyotyping methods introduced by Schrock et al use fluorescent

dyes that bind to specific regions of chromosomes. By using a series of specific probes each with varying amounts of the dyes, different pairs of chromosomes have unique spectral characteristics. A unique feature of the technology is the use of an interferometer similar to ones used by astronomers for measuring light spectra emitted by stars. Slight variations in color, undetectable by the human eye, are detected by a computer program that then reassigns an easy-to-distinguish color to each pair of chromosomes. The result is a digital image rather than film, in full color. Pairing of the chromosomes is simpler because homologous pairs are the same color, and abberrations and cross-overs are more easily recognizable. In additional, the spectral karyotype has been used to detect translocations not recognizable by traditional banding analysis

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MeiosisMeiosisI & III & II

What is meiosis I? In meiosis I, chromosomes in a diploid cell resegregate, producing four haploid daughter cells. It is this step in meiosis that generates genetic diversity.

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MeiosisMeiosis

Prophase 1: DNA replication precedes the start of meiosis I. During prophase I, homologous chromosomes pair and form synapses, a step unique to meiosis. The paired chromosomes are called bivalents, and the formation of chiasmata caused by genetic recombination becomes apparent. Chromosomal condensation allows these to be viewed in the microscope. Note that the bivalent has two chromosomes and four chromatids, with one chromosome coming from each parent.

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MeiosisMeiosis

Prometaphase 1: The nuclear membrane disappears. One kinetochore forms per chromosome rather than one per chromatid, and the chromosomes attached to

spindle fibers begin to move.

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MeiosisMeiosis

Metaphase 1: Bivalents, each composed of two chromosomes (four chromatids) align at the metaphase plate. The orientation is random, with either parental homologue on a side. This means that there is a 50-50 chance for the daughter cells to get either the mother's or father's homologue for each

chromosome.

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MeiosisMeiosis

Anaphase 1: Chiasmata separate. Chromosomes, each with two chromatids, move to separate poles. Each of the daughter cells is now haploid (23 chromosomes), but each chromosome has two chromatids.

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MeiosisMeiosis

Telophase 1: Nuclear envelopes may reform, or the cell may quickly start meiosis II.

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MeiosisMeiosis

Cytokinesis: Analogous to mitosis where two complete daughter cells form.

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MeiosisMeiosis

Meiosis II is similar to mitosis. However, there is no "S" phase. The chromatids of each chromosome are no longer identical because of recombination. Meiosis II separates the chromatids producing two daughter cells each with 23 chromosomes (haploid), and each chromosome has only one chromatid.

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Comparing Meiosis and MitosisComparing Meiosis and Mitosis

Chromosome behavior

1. Mitosis: Homologous chromosomes independent

2. Meiosis: Homologous chromosomes pair forming bivalents until anaphase I

Chromosome number- reduction in meiosis

1. Mitosis- identical daughter cells

2. Meiosis- daughter cells haploid Genetic identity of progeny:

1. Mitosis: identical daughter cells

2. Meiosis: daughter cells have new assortment of parental chromosomes

3. Meiosis: chromatids not identical, crossing over

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Meiotic errorsMeiotic errors

Nondisjunction- homologues don't separate in meiosis 1

1. Results in aneuploidy

2. Usually embryo lethal

3. Trisomy 21, exception leading to Downs syndrome

4. Sex chromosomes

5. Turner syndrome: monosomy X

6. Klinefelter syndrome: XXY Translocation and deletion: transfer of a piece of one

chromosome to another or loss of fragment of a chromosome.

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Mitosis, Meiosis, and PloidyMitosis, Meiosis, and Ploidy

Mitosis can proceed independent of ploidy of cell, homologous chromosomes behave independently

Meiosis can only proceed if the nucleus contains an even number of chromosomes (diploid, tetraploid).

Trisomy 21 does not prevent meiosis

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Nutrition & GrowthNutrition & Growth

Animals obtain nutrients for cell growth by ingesting various types of food.

The digestion process in the stomach and small intestine, mediated by acids and hydrolytic enzymes, break down large macromolecules such as proteins, carbohydrates and lipids, into amino acids, simple sugars, and glycerol and acetyl coenzyme A, respectively.

These molecules, along with oxygen, vitamins and minerals, are then absorbed into the blood stream and distributed to individual cells throughout the body.

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The Nutrient Requirements The Nutrient Requirements of Cellsof Cells

In order to keep running, all living things need an unceasing supply of energy and materials. One useful way to discover the nutritional needs of cells is to attempt to culture them in the laboratory.

For example the difference of ingredients needed in the medium for culturing

the bacterium E. coli human cells a unicellular green alga

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Minimal Medium for E. coliMinimal Medium for E. coli

Glucose 5g/ liter Na2HPO4 6 g/ liter

KH2PO4 3 g/ liter

NH4Cl 1 g/liter

NaCl 0.5 g/ liter MgSO4 0.12 g/ liter

CaCl2 0.01 g/ liter

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Ham's Tissue Culture Medium for Ham's Tissue Culture Medium for Mammalian CellsMammalian Cells

(amounts dissolved in 1 liter of triple distilled water)(amounts dissolved in 1 liter of triple distilled water)

L-Arginine211 mg, Biotin 0.024 mg, L-Histidine21 mg, Calcium pantothenate 0.7 mg, L-Lysine29.3 mg, Choline chloride 0.69 mg, L-Methionine4.48 mg, i-inositol 0.54 mg, L-Phenylalanine4.96 mg, Niacinamide0.6 mg, L-Tryptophan0.6 mg, Pyridoxine hydrochloride 0.2 mg, L-Tyrosine1.81 mg, Riboflavin0.37 mg, L-Alanine8.91 mg, Thymidine 0.7 mg, Glycine7.51 mg, Cyanocobalamin

1.3 mg, L-Serine10.5 mg , Sodium pyruvate 110 mg, L-Threonine3.57 mg, Lipoic acid 0.2 mg, L-Aspartic acid13.3 mg, CaCl2 44 mg, L-Glutamic

acid14.7 mg, MgSO4.7H2O 153 mg, L-Asparagine15 mg, Glucose 1.1 g, L-

Glutamine146.2 mg, NaCl 7.4 g, L-Isoleucine2.6 mg, KCl 285 mg, L-Leucine13.1 mg, Na2HPO4,290 mg, L-Proline11.5 mg, KH2PO4 83 mg, L-

Valine3.5 mg, Phenol red 1.2 mg, L-Cysteine31.5 mg, FeSO4 0.83 mg,

Thiamine hydrochloride1 mg, CuSO4.5H2O 0.0025 mg, Hypoxanthine 4 mg,

ZnSO4.7H2O 0.028 mg, Folic acid1.3 mg, NaHCO3 1.2 g

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E.coli is ableE.coli is able

to supply all its energy needs from the potential energy stored in glucose and

manufactures all of its organic molecules using the carbon atoms in glucose.

NH4Cl and MgSO4 supply the nitrogen and sulfur atoms needed to

synthesize proteins. Na2HPO4 and KH2PO4 supply the phosphorus atoms needed for

nucleic acid synthesis (as well as being needed for the functioning of many proteins).

These ingredients also supply the Na+, K+, Mg2+, and Cl- needed by the cell.

CaCl2 supplies the needed Ca2+ ions.

All these ingredients are dissolved in water and are taken up from this solution.

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The list of ingredients needed to The list of ingredients needed to grow human cells in culture is far grow human cells in culture is far

longer. longer. all 20 of the amino acids from which proteins are synthesized; a purine (hypoxanthine) and a pyrimidine (thymidine) for the synthesis of

nucleotides, and their polymers DNA and RNA; 2 precursors (choline and inositol) needed to synthesize some of the

phospholipids in the cell; 8 vitamins, all of which serve as parts of coenzymes; the coenzyme lipoic acid; glucose as a source of energy and carbon atoms; the inorganic ions Na+, K+, Ca2+, Cu2+, Zn2+, and CO2+ (E. coli may need some

of these as well but in such tiny amounts that it can acquire them as impurities in the other ingredients of its medium.)

Even when all these ingredients have been mixed together, most mammalian cells still fail to grow unless some blood serum (e.g., from a human or a calf) is added. Just what metabolic need is met by this supplement is uncertain, but trace amounts of hormones in the serum are probably important.

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Why does a mammalian cell Why does a mammalian cell require such a complex broth require such a complex broth

compared to E. coli?compared to E. coli?

It is the price of multicellularity. A mammal is made up of hundreds of different cell types,

each specialized to perform one or a few functions. All the many other functions of life - including the

synthesis of many of the organic molecules it needs, it delegates to other cells.

The extracellular fluid, derived from the blood, supplies it with these. Tissue culture medium is an attempt to recreate

this extracellular fluid.

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Cell Cell & Tissue & Tissue cultureculture

Cell culture is the process by which either prokaryotic or eukaryotic cells are grown under controlled conditions. In practice the term "cell culture" has come to refer to the culturing of cells derived from multicellular eukaryotes, especially animal cells. The historical development and methods of cell culture are closely interrelated to those of tissue culture and organ culture.

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Animal cell culture became a routine Animal cell culture became a routine laboratorylaboratory technique technique in the 1950s, but the concept of maintaining live cell lines in the 1950s, but the concept of maintaining live cell lines separated from their original tissue source was separated from their original tissue source was discovered in the 19th Century.discovered in the 19th Century.

19th Century English physiologist Sydney Ringer developed salt solutions containing the chlorides of sodium, potassium, calcium and magnesium suitable for maintaining the beating of an isolated animal heart outside of the body. In 1885 Wilhelm Roux removed a portion of the medullary plate of an embryonic chicken and maintained it in a warm saline solution for several days, establishing the principle of tissue culture.[3] Ross Granville Harrison, working at Johns Hopkins Medical School and then at Yale University, published results of his experiments from 1907-1910, establishing the methodology of tissue culture.

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Cell culture techniques were advanced significantly in the Cell culture techniques were advanced significantly in the 1940s and 1950s to support research in 1940s and 1950s to support research in virologyvirology

Growing viruses in cell cultures allowed preparation of purified viruses for the manufacture of vaccines. The Salk polio vaccine was one of the first products mass-produced using cell culture techniques. This vaccine was made possible by the cell culture research of John Franklin Enders, Thomas Huckle Weller, and Frederick Chapman Robbins, who were awarded a Nobel Prize for their discovery of a method of growing the virus in monkey kidney cell cultures.

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ConceptConcept

Culture conditions (for example growth media, pH, temperature) vary widely for each cell type, and variation of conditions for a particular cell type can result in different phenotypes being expressed.

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Sometimes, it is possible to fuse normal cells with an Sometimes, it is possible to fuse normal cells with an immortal cell line. An example is the way immortal cell line. An example is the way monoclonal antibodiesmonoclonal antibodies are made: are made: LymphocytesLymphocytes isolated isolated from the blood of an from the blood of an immunisedimmunised animal are combined with animal are combined with hybridomahybridoma cell lines in a cell lines in a selective growth mediumselective growth medium: only : only the fused cells survive.the fused cells survive.

Cell culture methods can be applied to either free-living organisms, such as bacteria or eukarytotic microorganisms, or to cells removed from a multi-cellular tissue. Related to cell culture are tissue culture and organ culture, which refer to methods for growing pieces of tissue or entire organs removed from an organism in an artificial environment.

The culture of viruses requires the culture of cells as hosts for the growth and replication of the virus.

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. In 1912 carrel began growing bits of chick heart in drops of horse plasma. The cells at the edge of the explant divided and grew out of the plasma clot. The explants died within a few days due to exhaustion of nutrients. Carrel (1912) reported that the cells from an explant could be maintained indefinitely provided they were periodically subcultured and provided with a sterile aqueous extract of whole chick embryos.

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In 1950’s Earle demonstrated a technique for dissociation of cells, from a whole chick embryo, from each other with trypsin. When this suspension of single cells was mixed with plasma and embryo extract and placed in a sterile glass container, the cells adhered to the glass and divided to form a primary culture

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Tissue CultureTissue Culture

The term tissue culture was original applied to explants of tissues embedded in plasma. however, later on this term became associated with the cultures of cells. It refers to tissues dissociated into a suspension of single cells, which after being washed and counted, are diluted in growth medium and allowed to settle on to the flat bottom surface of a specially treated plastic or glass container. Many cell types of cell adhere quickly and undergoes mitosis until the surface is covered with a confluent cell monolayer

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Organ CultureOrgan Culture

Organ culture may maintain their original architecture and functions up to weeks. Slices of organs such as respiratory epithelium have been used to study the histopathogenesis of virus infection using organ culture.

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Primary Cell linePrimary Cell line

The primary culture may contain a variety of cell types such as macrophages, lymphocytes, muscles fibers, etc. the cells grew to monolayer, a thin sheet of cells ( one layer in thickness) which covers the entire bottom surface of their culture vessel, and then stop dividing.

When cells are taken freshly from animals and placed in culture, the culture consist of a wide variety of cell types, which may be capable of very limited growth in vitro.

These cells retain their diploid karyotype, the chromosome number and morphology of their in vivo tissues of origin. They may also retain some of the differentiated characteristics which they possessed in vivo. These cells support the replication of a wide range viruses.

The cells may then be redispersed with trypsin solution and planted in new culture vessels containing freshly prepared media.

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Secondary Cell LinesSecondary Cell Lines

Secondary cell cultures contain fewer cell types than the primary cell cultures, as many of the differentiated primary cells are out competed and do not survive upon transfer. Secondary cultures are often composed of spindle shaped cells (fibroblasts). Cells derived from kidneys and from certain carcinomas have a polygonal appearance in culture. Because of their tissue of origin, those and other cells with similar morphology are called epithelial cells.

Primary cell lines can be passed through secondary and several subsequent sub cultures while retaining their original characteristics. After 20 – 25 passages in vitro, these diploid cell strains usually undergoes a crises in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production.

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          Diploid cell strainsDiploid cell strains

Primary cell lines can be passed through secondary and several subsequent sub cultures while retaining their original characteristics. After 20 – 25 passages in vitro, these diploid cell strains usually undergoes a crises in which their growth rate slows and they eventually die out. Diploid strains of fibroblasts derived from human embryos are widely used in diagnostic virology and vaccine production.

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Continuous cell linesContinuous cell lines

Certain culture cells, notably mouse embryo fibroblasts and human carcinoma cells are able to survive the growth crises and undergo indefinite in vitro propagation. After an initial slow growth, these continuous cell lines grow more rapidly than before their karyotype becomes abnormal (aneuploid) and other changes takes place, which makes the cells immortal.

The cells are now dedifferentiated, having lost the specialized morphology, and biochemical abilities, which they possessed as differentiated cells in vivo. Continuous cell lines such as KB and HeLa (Human epithelial cell lines)both derived from humans and others derived from mice and hamsters are widely used in experimental virology.

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Some Cell Line TypesSome Cell Line Types Amniotic fluid-derived cell line Chorionic villus-derived cell line Endothelial Epithelial Erythroleukemic cell line Fibroblast Hybridoma Keratinocyte Kidney-derived cell line Lymphocyte (B-lymphoctye) Lymphocyte (T-lymphoctye) Mesothelial Microcell hybrid Myeloma Smooth muscle Somatic cell hybrid Tumor-derived cell line

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Cell line typesCell line types

Amniotic fluid-derived cell line:    Cells isolated from amniotic fluid samples are commonly used in prenatal diagnosis. These cells are thought to be sloughed from the fetal amnion, skin, and alimentary, repiratory and urogenitory tracts. Consequently, both fibroblasts and epithelial cell types can be present in cultures derived from amniotic fluid.

Chorionic villus-derived cell line:    Chorionic villus cultures are established from the mesenchyme core cells of the villi after first removing the trophoblast layers by dissection followed by enzymatic dissociation of the core.

.

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Cell line typesCell line types

Endothelial Endothelial, arterial:    Derived from, or pertaining to an artery,

a blood vessel carrying oxygenated blood away from the heart; lined with a single layer of endothelial cells, the outer walls have smooth muscle and are innervated by the sympathetic nervous system.

Endothelial, venous:    Derived from or pertaining to a vein, a blood vessel that returns blood from the microvasculature (i.e., after release of oxygen to the tissues) to the heart; venous walls are thinner and less elastic than those of artery

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Cell line typesCell line types

Epithelial Epithelial, lens:    The cellular covering of the lens of the eye. Epithelial, mammary:    Derived from, or pertaining to the cells

of the ducts, lobules, and alveoli of the mammary (milk-producing) gland of female mammals.

Epithelial, pigmented (or pigmented retinal epithelial cell):    Melanin-bearing cells just posterior to the rods and cones, originating from the outer layer of the embryologic optic cup. [from "International Dictionary of Medicine and Biology", Sidney I. Landau, Editor-in-Chief. In three volumes. Volume 1, pp488-499, 1986. John Wiley and Sons, New York, NY.]

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Cell line typesCell line types Erythroleukemic cell:    Abnormal precursor (virally

transformed) of mouse erythrocytes that can be grown in culture and induced to differentiate by treatment with, for example, DMSO.

Fibroblast Cell Line:    A propagated culture of cells exhibiting fibroblast-like morphology after at least one subculture. Fibroblast cell lines may be established at CCR by outgrowth of undifferentiated mesodermal cells from a biopsy or identified by a submitter as a fibroblast cell line. Cell morphology of a fibroblast cell line will vary somewhat with the culture conditions and with the age of the culture or the age of the cell line, but generally the fibroblastic morphology is spindle shaped (bipolar) or stellate (multipolar); usually arranged in parallel arrays at confluence in contact-inhibited cultures. These cells are migratory with processes exceeding the nuclear diameter by threefold or more.

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Cell line typesCell line types Hybridoma:    A transformed cell line derived by fusing a

myeloma cell with a normal B-lymphocyte. Produces a single kind of antibody determined by the normal fusion partner. As a result, hybridomas are used to produce monoclonal antibodies.

Keratinocyte:    Skin cell, of the keratinized layer of epidermis; epithelial cells that express the characteristic intermediate filament proteins cytokeratins, and other skin-specific proteins to form a protective barrier.

Kidney-derived cell line:    Cell isolated from kidney tissue. Specific tissue type was not specified.

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Cell line typesCell line types Lymphocyte: B-Lymphocyte:    A propagated culture of cells exhibiting lymphoblast-

like morphology after at least one subculture. B-Lymphoblast cell lines are established at CCR by transformation of B-lymphocytes isolated as peripheral blood mononuclear cells with Epstein-Barr virus (EBV) using phytohemaglutinin as a mitogen or identified by a submitter as a B-lymphoblast cell line. These lines are usually polyclonal in derivation. The lymphoblastoid morphology is small (7-9 micron) round cells that grow as loose aggregates in suspension.

T-Lymphocyte:    A propagated culture of cells exhibiting lymphoblast-like morphology after at least one subculture. T-Lymphoblast cell lines are established at CCR by transformation of T-lymphocytes isolated as peripheral blood mononuclear cells with human T-cell leukemic virus (HTLV) using interleukin-2 as a mitogen or identified by a submitter as a T-lymphoblast cell line. These lines are usually polyclonal in derivation. The lymphoblastoid morphology is small (7-9 micron) round cells that grow as loose aggregates in suspension.

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Cell line typesCell line types

Mesothelial:    Derived from, or pertaining to the mesothelium, a simple squamous epithelium of mesodermal origin. It lines the peritoneal, pericardial and pleural cavities and the synovial space of joints. The cells may be phagocytic.

Microcell hybrid:    A hybrid cell produced by the fusion of a micro cell with the cell of another species. Microcells contain only a portion of the genome and cytoplasm of the cell from which they are derived. Microcells are produced by colcemid treatment to promote nuclear fragmentation into micronuclei followed by cytochalasin B treatment to extrude these micronuclei which are finally sheared from the cell by centrifugal force during centrifugation. Consequently each microcell contains only one or a few human chromosomes. The subset of microcell hybrids with a chromosome that carries a selectable

marker may be then be isolated.

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Cell line typesCell line types

Myeloma cell:    Neoplastic plasma cell (a white blood cell that produces and secretes a specific antibody, or immunoglobulin protein). The proliferating plasma cells often replace all the others within the marrow, leading to immune deficiency, and frequently there is destruction of the bone cortex. Because they are monoclonal in origin they secrete a monoclonal immunoglobulin. Bence-Jones proteins are monoclonal immunoglobulin light chains overproduced by myeloma cells and excreted in the urine. Myeloma cell lines are used for producing hybridomas in raising monoclonal antibodies.

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Cell line typesCell line types

Smooth muscle:    Non-striated muscle tissue in vertebrates made up from long tapering cells that may be anything from 20-500 microns long. Smooth muscle is generally involuntary, and differs from striated muscle in the much higher actin/myosin ratio, the absence of conspicuous sarcomeres, and the ability to contract to a much smaller fraction of its resting length. Smooth muscle cells are found particularly in blood vessel walls (vascular smooth muscle), surrounding the intestine (particularly the gizzard in birds), and in the uterus. The contractile system and its control resemble those of motile tissue cells (e.g. fibroblasts, leucocytes), and antibodies against smooth muscle myosin will cross-react with myosin from tissue cells, whereas antibodies against skeletal muscle myosin will not

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Cell line typesCell line types Somatic cell hybrid:    A hybrid cell produced by the fusion of

two somatic cells. Somatic cell hybrids are commonly produced for three reasons: 1) determination whether two mutations are in the same or distinct complementation groups from two cell lines; 2) production of specialized anitbodies (hybridomas); 3) production of inter-species hybrids to isolate particular chromosomes or chromosome segments for the assignment of genes first to chromosomes (NIGMS Map 2 Monochromosomal Hybrids), subsequently to specific chromosome segments (NIGMS Chromosome Regional Mapping Panels Hybrids) and finally to relate gene structure to function by the correlation of deleted or duplicated chromosomal segments to altered phenotype using hybrids constructed from human parental lines from probands with specific genetic disorders.

Tumor-derived cell line:    Cells isolated from a mass of neoplastic cells, i.e., a growth formed by abnormal cellular proliferation

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Splitting CellsSplitting Cells

Culture cells forming a confluent monolayer on the surface of their culture vessel, may be removed from the surface, diluted, and seeded into new vessels. If the initial culture was primary, the new cultures derived from it are called secondary, and are likely to consist of fewer cell types. Cells from glass surface or plastic surface may be removed by physical methods (scraping with a sterile rubber policeman) or chemical methods, proteolytic enzymes or chealating agents or a combination of the two. After removal are pipetted up and down and diluted appropriately in fresh secondary culturing, and after one becomes familiar with growth characteristics of a certain cell types, counting can usually be dispensed with.

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WORK AREA AND WORK AREA AND EQUIPMENTEQUIPMENT

Laminar flow hoods. There are two types of laminar flow hoods, vertical and horizontal. The vertical hood, also known as a biology safety cabinet, is best for working with hazardous organisms since the aerosols that are generated in the hood are filtered out before they are released into the surrounding environment.

Horizontal hoods are designed such that the air flows directly at the operator hence they are not useful for working with hazardous organisms but are the best protection for your cultures. Both types of hoods have continuous displacement of air that passes through a HEPA (high efficiency particle) filter that removes particulates from the air. In a vertical hood, the filtered air blows down from the top of the cabinet; in a horizontal hood, the filtered air blows out at the operator in a horizontal fashion.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

NOTE: these are not fume hoods and should not be used for volatile or explosive chemicals. They should also never be used for bacterial or fungal work.

The hoods are equipped with a short-wave UV light that can be turned on for a few minutes to sterilize the surfaces of the hood, but be aware that only exposed surfaces will be accessible to the UV light.

Do not put your hands or face near the hood when the UV light is on as the short wave light can cause skin and eye damage. The hoods should be turned on about 10-20 minutes before being used. Wipe down all surfaces with ethanol before and after each use. Keep the hood as free of clutter as possible because this will interfere with the laminar flow air pattern.

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WORK AREA AND WORK AREA AND EQUIPMENTEQUIPMENT

B. CO2 Incubators. The cells are grown in an atmosphere of 5-10% CO2 because the medium used is buffered with sodium bicarbonate/carbonic acid and the pH must be strictly maintained. Culture flasks should have loosened caps to allow for sufficient gas exchange. Cells should be left out of the incubator for as little time as possible and the incubator doors should not be opened for very long. The humidity must also be maintained for those cells growing in tissue culture dishes so a pan of water is kept filled at all times.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT C. Microscopes. Inverted phase contrast

microscopes are used for visualizing the cells. Microscopes should be kept covered and the lights turned down when not in use. Before using the microscope or whenever an objective is changed, check that the phase rings are aligned.

D. Preservation. Cells are stored at –20C , -70 C or in liquid nitrogen.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

E. Vessels. Anchorage dependent cells require a nontoxic, biologically inert, and optically transparent surface that will allow cells to attach and allow movement for growth. The most convenient vessels are specially-treated polystyrene plastic that are supplied sterile and are disposable. These include petri dishes, multi-well plates, microtiter plates, roller bottles, and screwcap flasks - T-25, T-75, T-150 (cm2 of surface area). Suspension cells are either shaken, stirred, or grown in vessels identical to those used for anchorage-dependent cells.

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WORK AREA AND WORK AREA AND EQUIPMENTEQUIPMENT

III. PRESERVATION AND STORAGE. Liquid N2 is used to preserve tissue culture cells, either in the liquid phase (-196°C) or in the vapor phase (-156°C). Freezing can be lethal to cells due to the effects of damage by ice crystals, alterations in the concentration of electrolytes, dehydration, and changes in pH. To minimize the effects of freezing, several precautions are taken. First, a cryoprotective agent which lowers the freezing point, such as glycerol or DMSO, is added. A typical freezing medium is 90% serum, 10% DMSO. In addition, it is best to use healthy cells that are growing in log phase and to replace the medium 24 hours before freezing. Also, the cells are slowly cooled from room temperature to -80°C to allow the water to move out of the cells before it freezes. The optimal rate of cooling is 1°-3°C per minute. Some labs have fancy freezing chambers to regulate the freezing at the optimal rate by periodically pulsing in liquid nitrogen.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

To maximize recovery of the cells when thawing, the cells are warmed very quickly by placing the tube directly from the liquid nitrogen container into a 37°C water bath with moderate shaking. As soon as the last ice crystal is melted, the cells are immediately diluted into prewarmed medium.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

IV. MAINTENANCE Cultures should be examined daily, observing the

morphology, the color of the medium and the density of the cells. A tissue culture log should be maintained that is separate from your regular laboratory notebook. The log should contain: the name of the cell line, the medium components and any alterations to the standard medium, the dates on which the cells were split and/or fed, a calculation of the doubling time of the culture (this should be done at least once during the semester), and any observations relative to the morphology, etc.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

A. Growth pattern. Cells will initially go through a quiescent or lag phase that depends on the cell type, the seeding density, the media components, and previous handling. The cells will then go into exponential growth where they have the highest metabolic activity. The cells will then enter into stationary phase where the number of cells is constant, this is characteristic of a confluent population (where all growth surfaces are covered).

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

B. Harvesting. Cells are harvested when the cells have reached a population density which suppresses growth. Ideally, cells are harvested when they are in a semi-confluent state and are still in log phase. Cells that are not passaged and are allowed to grow to a confluent state can sometime lag for a long period of time and some may never recover. It is also essential to keep your cells as happy as possible to maximize the efficiency of transformation. Most cells are passaged (or at least fed) three times a week.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

1. Suspension culture. Suspension cultures are fed by dilution into fresh medium.

2. Adherent cultures. Adherent cultures that do not need to be divided can simply be fed by removing the old medium and replacing it with fresh medium.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

When the cells become semi-confluent, several methods are used to remove the cells from the growing surface so that they can be diluted:

Mechanical - A rubber spatula can be used to physically remove the cells from the growth surface. This method is quick and easy but is also disruptive to the cells and may result in significant cell death. This method is best when harvesting many different samples of cells for preparing extracts, i.e., when viability is not important.

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WORK AREA AND EQUIPMENTWORK AREA AND EQUIPMENT

Proteolytic enzymes - Trypsin, collagenase, or pronase, usually in combination with EDTA, causes cells to detach from the growth surface. This method is fast and reliable but can damage the cell surface by digesting exposed cell surface proteins. The proteolysis reaction can be quickly terminated by the addition of complete medium containing serum

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WORK AREA AND WORK AREA AND EQUIPMENTEQUIPMENT

EDTA - EDTA alone can also be used to detach cells and seems to be gentler on the cells than trypsin. The standard procedure for detaching adherent cells is as follows:

1. Visually inspect daily 2. Release cells from monolayer surface   a. wash once with a buffer solution

b. treat with dissociating agentc. observe cells under the microscope. Incubate until cells become rounded and loosen when flask is gently tapped with the side of the hand.d. Transfer cells to a culture tube and dilute with medium containing serum.e. Spin down cells, remove supernatant and replace with fresh medium.f. Count the cells in a hemacytometer, and dilute as appropriate into fresh medium.

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Media and growth Media and growth requirements requirements

1. Physiological parametersA. temperature - 37C for cells from

homeotherB. pH - 7.2-7.5 and osmolality of medium

must be maintainedC. humidity is required D. gas phase - bicarbonate conc. and CO2 tension in

equilibrium E. visible light - can have an adverse effect

on cells; light induced production of toxic compounds can occur in some media; cells should be cultured in the dark and exposed to room light as little as possible;

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Media and growth Media and growth requirements requirements

2. Medium requirements: (often empirical) A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2

B. Trace elements - iron, zinc, seleniumC. sugars - glucose is the most commonD. amino acids - 13 essentialE. vitamins - B, etc.F. choline, inositolG. serum - contains a large number of growth promoting activities such as buffering toxic nutrients by binding them, neutralizes trypsin and other proteases, has undefined effects on the interaction between cells and substrate, and contains peptide hormones or hormone-like growth factors that promote healthy growth. H. antibiotics - although not required for cell growth, antibiotics are often used to control the growth of bacterial and fungal contaminants.

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Media and growth Media and growth requirements requirements

3. Feeding - 2-3 times/week. 4. Measurement of growth and viability. The

viability of cells can be observed visually using an inverted phase contrast microscope. Live cells are phase bright; suspension cells are typically rounded and somewhat symmetrical; adherent cells will form projections when they attach to the growth surface. Viability can also be assessed using the vital dye, trypan blue, which is excluded by live cells but accumulates in dead cells. Cell numbers are determined using a hemocytometer.

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SAFETY CONSIDERATIONSSAFETY CONSIDERATIONS

Assume all cultures are hazardous since they may harbor latent viruses or other organisms that are uncharacterized. The following safety precautions should also be observed:

pipetting: use pipette aids to prevent ingestion and

keep aerosols down to a minimum no eating, drinking, or smoking wash hands after handling cultures and before leaving

the lab decontaminate work surfaces with disinfectant (before

and after)

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SAFETY CONSIDERATIONSSAFETY CONSIDERATIONS

autoclave all waste, use biological safety cabinet (laminar flow hood) when

working with hazardous organisms. The cabinet protects worker by preventing airborne cells and viruses released during experimental activity from escaping the cabinet; there is an air barrier at the front opening and exhaust air is filtered with a HEPA filter make sure cabinet is not overloaded and leave exhaust grills in the front and the back clear (helps to maintain a uniform airflow),

use aseptic technique, dispose of all liquid waste after each experiment and

treat with bleach

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Changing media:Changing media: This should be done every couple of days between splits. The cell

culture media is changed regularly to prevent a build-up of waste products as the cells grow and divide. As always, use aseptic technique in your approach to this by cleaning the hood with Virkon and 70% Ethanol before use. Have pre-warmed 1X PBS and pre-warmed media on hand before entering hood. ALWAYS wear gloves. Maintain sterility!

For NIH3T3 and ND7/23 use Dulbecco’s modified eagle’s medium. For Neuro2a use Minimum essential medium eagle.

Remove old media from small flask into 15 mL centrifuge tube (50 mL tube for medium flask). Rinse cells with 1 volume of sterile 1X PBS. Remove PBS to centrifuge tube with old media. Spin at around 700 rpm (100 x g) for 5 min. Much higher g-forces and prolonged spin time may decrease viability of cells. Remove supernated old media and PBS to Virkon-containing waste container. Add 1 volume of pre-warmed media to pelleted cells. Resuspend cells in warmed media and return to in-use tissue culture flask for reincubation.

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Splitting cell cultures:Splitting cell cultures:

When confluence of cells is reached, the cell culture should be split. This can be accomplished by increasing the flask size or placing cells from one flask into two.

In addition to the pre-warmed PBS and media, a ½ volume aliquot of pre-warmed trypsin-EDTA (Invitrogen) will be needed for each flask. Brief treatment with trypsin-EDTA removes adherent cells from tissue culture flask bottom so that they can be seeded into a new flask. Each cell line will differ in the degree of adherence it has, and this will be described in the literature that accompanies it.

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Splitting cell cultures:Splitting cell cultures:

After removing old media and rinsing cells with PBS as described in changing media section, add ½ volume of sterile trypsin-EDTA to tissue culture flask still containing adherent cells. Place flask back into incubator for about 1-2 minutes. Check after a minute to see if cells have come off bottom of flask (prolonged treatment with trypsin may damage cells). This can be observed macroscopically as sheets of floating cells will be visible.

Immediately remove cells to respective centrifuge tube with media/PBS and rinse up and down with pipette to neutralise trypsin. Wash down sides of flask with a portion of the cell/PBS mixture. All cells should now be in centrifuge tube. Spin at around 700 rpm (100 x g) for 5 min. Remove supernatant to Virkon-containing waste container. Re-suspend cells in 2x volume of appropriate pre-warmed media and reseed into a larger size or 2x the previous number of flasks. Re-incubate flasks.

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Freezing cell cultures:Freezing cell cultures:

A freezing medium of 90% fetal calf serum and 10% DMSO should be used.

After washing cell pellet in sterile 1X PBS, re-suspend pellet in freeze medium to give a final cell concentration between 2 and 4 x 10[6] cells/mL.

Do cell count as follows: Take 10 ul of cell suspension from each of the 5 mL just prepared and dilute with 990 ul of PBS to make a 1:100 dilution. Use a 20 ul pipettor to fill each side of a hemacytometer chamber with this dilution. Use one tube for each side of the chamber.

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Freezing cell cultures:Freezing cell cultures:

The hemacytometer has two chambers, each chamber inscribed with a grid. There are 9 large squares on this grid. The center square is divided into 25 smaller squares. The cells counted in each large square corresponds to a count of [no. of cells in one large square x 10[4] = no. of cells/mL]. An easy way to derive this count is to count the 4 large corner squares of the grid, divide by 4 to average, and then multiply by the dilution factor (10[2]) and then 10[4]. This will give you the number of cells/ mL. Pipette 1 mL of suspension into Nalgene cryovial. Individually wrap each cryovial to be frozen in bubblewrap and freeze slowly over-night in -80C freezer. Transfer vials to liquid nitrogen for long-term storage.

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Drug Evaluation Against Viruse Drug Evaluation Against Viruse Using Cell LinesUsing Cell Lines

Antibiotics are substances produced by certain microorganisms that kill or inhibit the growth of other microorganisms. Penicillin and cephalosporin are antibiotics that inhibit the synthesis of bacterial cell walls. Streptomycin, tetracycline, and erythromycin inhibit certain aspects of bacterial protein synthesis. The use of antibiotics to treat bacterial infection in vitro is possible because of the property of selective toxicity. The antibiotic’s effect is greater on prokaryotic cellular metabolism than on eukaryotic cellular metabolism.

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Drug Evaluation Against Viruse Drug Evaluation Against Viruse Using Cell LinesUsing Cell Lines

The cell wall structure of bacteria is not found in any structure of eukaryotic cells, so penicillin can be administered in very high doses with little effect on the eukaryotic cells. Antibiotics that affect protein synthesis in prokaryotic cells have a much greater affinity for bacterial ribosome than for eukaryotic ribosomes and kill or inhibit the growth of bacteria without affecting the eukaryotic cells. This is why penicillin and streptomycin are used in eukaryotic cell culture medium.

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Drug Evaluation Against Viruse Drug Evaluation Against Viruse Using Cell LinesUsing Cell Lines

Certain chemicals have been found to have an inhibitory effect on the replication of certain types of viruses infected cell cultures. Viruses initiate infection by adsorbing to host cell receptor sites. Enveloped viruses have virus coded glycoproteins inserted into the envelope that specifically binds to the host cell receptors. If glycoprotein synthesis is interfered with during the viral infection process, virus particle may be formed that are not infectious because of lack of viral glycoproteins in envelope.

The effect of viral replication inhibitors may be measured by either the virus yield reduction assay or plaque

reduction assay.

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Plaque AssayPlaque Assay

Plaque: an area of cells in a monolayer which display a cytopathic effect, e.g. appearing round and darker than other cells under the microscope, or as white spots when visualized by eye; the plaque center may lack cells due to virus-induced lysis.

Plaque-forming unit (PFU): a virus or group of viruses which cause a plaque.

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Developmental biologyDevelopmental biology

The findings of developmental biology can help to understand developmental malfunctions such as chromosomal aberrations, for example, Down syndrome. An understanding of the specialization of cells during embryogenesis may yield information on how to specialize stem cells to specific tissues and organs, which could lead to the specific cloning of organs for medical purposes. Another biologically important process that occurs during development is apoptosis - cell "suicide". For this reason, many developmental models are used to elucidate the physiology and molecular basis of this cellular process.

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Stem CellsStem Cells

Stem cells in people are primal undifferentiated cells that retain the ability to produce an identical copy of themselves when they divide (clone) and differentiate into other cell types. In higher animals this function is the defining property of the deleted cells. Stem cells have the ability to act as a repair system for the body, because they can divide and differentiate, replenishing other cells as long as the host organism is alive.

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Stem CellsStem Cells

Medical researchers believe stem cell research has the potential to change the face of human disease by being used to repair specific tissues or to grow organs. Yet there is general agreement that, "significant technical hurdles remain that will only be overcome through years of intensive research."

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Stem CellsStem Cells

The study of stem cells is attributed as beginning in the 1960s after research by Canadian scientists Ernest A. McCulloch and James E. Till.

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Stem Cell typesStem Cell types Potency The potency specifies the differentiation potential (the potential to

differentiate into different cell types) of the stem cell. Totipotent stem cells are produced from the fusion of an egg and

sperm cell. Cells produced by the first few divisions of the fertilized egg cell are also totipotent. These cells can differentiate into embryonic and extraembryonic cell types.

Pluripotent stem cells are the descendants of totipotent cells and can differentiate into cells derived from the three germ layers.

Multipotent stem cells can produce only cells of a closely related family of cells (e.g. hematopoietic stem cells differentiate into red blood cells, white blood cells, platelets, etc.).

Unipotent cells can produce only one cell type, but have the property

of self-renewal which distinguishes them from non-stem cells.

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Embryonic stem cellEmbryonic stem cell

Embryonic stem cells (ESCs) are stem cells derived from the inner cell mass of a blastocyst, which is an early stage embryo - approximately 4 to 5 days old in humans - consisting of 50-150 cells. Embryonic stem cells are pluripotent, meaning they are able to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of the more than 200 cell types of the adult body when given sufficient and necessary stimulation for a specific cell type. When given no stimuli for differentiation, ESCs will continue to divide in vitro and each daughter cell will remain pluripotent. The pluripotency of ESCs distinguishes them from adult stem cells or progenitor cells, the latter two only having the capacity to form a more limited number of different cell types.

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Embryonic stem cellEmbryonic stem cell

Because of their unique combined abilities of unlimited expansion and pluripotency, embryonic stem cells are a potential source for regenerative medicine and tissue replacement after injury or disease. To date, no approved medical treatments have been derived from embryonic stem cell research. This is not unusual for a new medical research field; in this case, the first human embryonic stem cell line was only reported in 1998.

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Embryonic stem cellEmbryonic stem cell

Research history and developments Embryonic stem cells were first derived from mouse

embryos in 1981 by two independent research groups (Evans & Kaufman and Martin). A breakthrough in human embryonic stem cell research came in November 1998 when a group led by James Thomson at the University of Wisconsin-Madison first developed a technique to isolate and grow the cells when derived from human blastocysts.

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Embryonic stem cellEmbryonic stem cell

Researchers at the Whitehead Institute announced in 2003 that they had successfully used embryonic stem cells to produce haploid, male gametes. They found embryonic stem cells that had begun to differentiate into embryonic germ cells and then further differentiated into the male haploid cells. When injected into oocytes, these haploid cells restored the somatic diploid complement of chromosomes and formed blastocysts in vitro.

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Embryonic stem cellEmbryonic stem cell

A study was published in the online edition of Lancet Medical Journal on March 8, 2005 that detailed information about a new stem cell line which was derived from human embryos under completely cell- and serum-free conditions. After more than 6 months of undifferentiated proliferation, these cells demonstrated the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem-cell lines. [3]

Recently, in California, researchers have injected embryonic stem cells into mice as they developed in the womb. Upon maturing, it was found that some of the human ESCs had survived and two months after injection, the researchers found that the HESCs had undertaken "the characteristics of mouse cells

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Adult stem cellAdult stem cell

Adult stem cells are undifferentiated cells found throughout the body that divide to replenish dying cells and regenerate damaged tissues. Also known as somatic (from Greek Σωματικóς, of the body) stem cells, they can be found in children, as well as adults.

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Adult stem cellAdult stem cell

Research into adult stem cells has been fueled by their abilities to divide or self-renew indefinitely and generate all the cell types of the organ from which they originate — potentially regenerating the entire organ from a few cells. The use of adult stem cells in research and therapy is not as controversial as embryonic stem cells, because the production of adult stem cells does not require the destruction of an embryo. In contrast with the adult stem cell research, very little US government funding has been provided for the embryonic stem cell research. Adult stem cells can be isolated from a tissue sample obtained from an adult. They have mainly been studied in humans and model organisms such as mice and rats.

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Adult stem cellAdult stem cell

Defining properties The rigorous definition of a stem cell requires that it possesses two

properties: Self-renewal - the ability to go through numerous cycles of cell division

while maintaining the undifferentiated state. Multipotency or multidifferentiative potential - the ability to generate

progeny of several distinct cell types, for example both glial cells and neurons, opposed to unipotency - restriction to a single-cell type. Some researchers do not consider this property essential and believe that unipotent self-renewing stem cells can exist.

These properties can be illustrated with relative ease in vitro, using methods such as clonogenic assays, where the progeny of single cell is characterized. However, in vitro cell culture conditions can alter the behavior of cells. Proving that a particular subpopulation of cells possesses stem cell properties in vivo is challenging. Considerable debate exists whether some proposed cell populations in the adult are indeed stem cells.

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Adult stem cellAdult stem cell

Types Adipose derived adult stem cells: Adipose-derived stem cells (ASCs) have also been isolated from

human fat, usually by method of liposuction Haematopoietic stem cells Mesenchymal stem cells Mammary stem cells: Mammary stem cells have been isolated from human and mouse tissue

as well as from cell lines derived from the mammary gland Neural stem cells Olfactory adult stem cells: Olfactory adult stem cells have been successfully from the cells

harvested from the human olfactory mucosa, the lining of the nose involved in the sense of smell

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Cancer stem cellCancer stem cell

Cancer stem cell theory is the theory that tumors arise from cells termed cancer stem cells that have properties of normal stem cells, particularly the abilities to self-renew and differentiate into multiple cell types, and that these cells persist in tumors as a distinct population that likely causes disease relapse and metastasis.

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Cancer stem cellCancer stem cell

The main question posed by proponents of the theory is "Are we targeting the right kind of cell?" Most existing cancer treatments were developed on animal models, where therapies able to promote tumor shrinkage were deemed effective. However, animals could not provide a complete model of human disease. In particular, in mice, whose life spans do not exceed two years, tumor relapse is exceptionally difficult to study and was largely neglected by most researchers. The theory suggests that conventional chemotherapies kill differentiated or differentiating cells, which form the bulk of the tumor but are unable to generate a new one. A population of cancer stem cells, which gave rise to it, remains untouched and may cause a relapse of the disease.

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Cancer stem cellCancer stem cell

In cancer research experiments, tumor cells are sometimes injected into an experimental animal to establish a tumor. Disease progression is then followed in time and novel drugs can be tested for their ability to inhibit it. However, efficient tumor formation requires thousands or tens of thousands of cells to be introduced. Classically, this has been explained by poor methodology (i.e. the tumor cells lose their viability during transfer) or the critical importance of the microenvironment, the particular biochemical surroundings of the injected cells. Supporters of the cancer stem cell paradigm argue that only a small fraction of the injected cells, the cancer stem cells, have the potential to generate a tumor. In human acute myeloid leukemia the frequency of these cells is less than 1 in 10,000

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TreatmentsTreatments

Medical researchers believe that stem cell research has the potential to change the face of human disease. A number of current treatments already exist, although the majority of them are not commonly used because they tend to be experimental and not very cost-effective. Medical researchers anticipate being able to use technologies derived from stem cell research to treat cancer, spinal cord injuries, and muscle damage, amongst a number of other diseases, impairments and conditions. However, there still exists a great deal of social and scientific uncertainty surrounding stem cell research, which could possibly be overcome by gaining the acceptance of the public and through years of intensive research.

Stem cells, however, are already used extensively in research, and some scientists do not see cell therapy as the first goal of the research, but see the stem cells as a tool worthy in itself.

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Controversy surrounding Controversy surrounding stem cell researchstem cell research

There exists a widespread controversy over stem cell research that emanates from the techniques used in the creation and usage of stem cells. Embryonic stem cell research is particularly controversial because, with the present state of technology, starting a stem cell 'line' requires the destruction of a human embryo and/or therapeutic cloning. Opponents of the research argue that this practice is a slippery slope to reproductive cloning and tantamount to the instrumentalization of a potential human being. Contrarily, medical researchers in the field argue that it is necessary to pursue embryonic stem cell research because the resultant technologies are expected to have significant medical potential. The ensuing debate has prompted authorities throughout the World to seek suitable regulatory frameworks and highlighted the fact that stem cell research represents a social and ethical challenge.

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Key events in stem cell Key events in stem cell researchresearch

1960s - Joseph Altman and Gopal Das present evidence of adult neurogenesis, ongoing stem cell activity in the brain; their reports contradict Cajal's "no new neurons" dogma and are largely ignored

1963 - McCulloch and Till illustrate the presence of self-renewing stem cells in mouse bone marrow

1968 - bone marrow transplant between two siblings successfully treats SCID

1978 - haematopoietic stem cells are discovered in human cord blood 1981 - mouse embryonic stem cells are derived from the

inner cell mass 1992 - neural stem cells are cultured in vitro as neurospheres 1995 - President Bill Clinton signs into law the Dickey Amendment

which makes it illegal for Federal money to be used for research where stem cells are derived from the destruction of the embryo.

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Key events in stem cell Key events in stem cell researchresearch

1997 - leukemia is shown to originate from a haematopoietic stem cell, the first direct evidence for cancer stem cells

1998 - James Thomson and coworkers derive the first human embryonic stem cell line at the University of Wisconsin-Madison.

2000s - several reports of adult stem cell plasticity are published 2003 - Dr. Songtao Shi of NIH discovers new source of adult stem

cells in children's primary teeth[2] 2004-2005 - Hwang Woo-Suk claims to have created several human

embryonic stem cell lines from unfertilised human oocytes. The lines are later shown to be fabricated

July 19, 2006 - President George W. Bush vetoes a bill which would have allowed Federal money to be used for research where stem cells

are derived from the destruction of the embryo.

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Thankyou