Advance Virology and Tissue Culture

Advance Virology
and Tissue Culture
MIC-610
Course Incharge: Shazia
Tabassum Hakim
Assistant Professor & Chairperson,
Department of Microbiology,
Jinnah University for Women, Karachi, Pakistan
(2006 AD)
MIC-610 1
A long path from Viruses,
Virology,Vaccines and Drug
discovery to Stem Cell
Research
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Cell 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 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 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 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 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 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 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 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 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 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 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 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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 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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Rough ER
The 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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 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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Functions of Endoplasmic
reticulum
 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 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. MIC-610 19
Golgi 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 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
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localisation of Golgi-resident proteins is achieved by an unknown
Mitochondrion
 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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Mitochondrial 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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Mitochondrial 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 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
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nucleus.
Cell 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 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 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 MIC-610 28
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
 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 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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Ribosome
 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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Ribosome
 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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Ribosome
 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. MIC-610 34
Structure and
function of
Ribosome
 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 function of
Ribosome
 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 function of
Ribosome
 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 Cycle
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Cell 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 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 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).
 p53 levels are increased in damaged cells. This allows time to repair
DNA by blocking the cell cycle.
 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 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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Mitosis
 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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Mitosis
 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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Mitosis
 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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Mitosis
 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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Mitosis
 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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Mitosis
 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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Mitosis
 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
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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Meiosis
 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)
6. Female-sex chromosomes are homologous (XX)
7. Male-sex chromosomes are non-homologous (XY)
MIC-610 51
Meiosis
 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.
MIC-610 52
Meiosis
 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
MIC-610 53
Meiosis
I & 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.
MIC-610 54
Meiosis
 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.
MIC-610 55
Meiosis
 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.
MIC-610 56
Meiosis
 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.
MIC-610 57
Meiosis
 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.
MIC-610 58
Meiosis
 Telophase 1: Nuclear envelopes

may reform, or the cell may
quickly start meiosis II.
MIC-610 59
Meiosis
 Cytokinesis: Analogous to mitosis

where two complete daughter cells
form.
MIC-610 60
Meiosis
 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.
MIC-610 61
Comparing Meiosis and Mitosis
 Chromosome behavior
2. Mitosis: Homologous chromosomes independent
3. Meiosis: Homologous chromosomes pair forming bivalents until
anaphase I
 Chromosome number- reduction in meiosis
5. Mitosis- identical daughter cells
6. Meiosis- daughter cells haploid
 Genetic identity of progeny:
8. Mitosis: identical daughter cells
9. Meiosis: daughter cells have new assortment of parental chromosomes
10. Meiosis: chromatids not identical, crossing over
MIC-610 62
Meiotic errors
 Nondisjunction- homologues don't separate in meiosis 1
2. Results in aneuploidy
3. Usually embryo lethal
4. Trisomy 21, exception leading to Downs syndrome
5. Sex chromosomes
6. Turner syndrome: monosomy X
7. Klinefelter syndrome: XXY
 Translocation and deletion: transfer of a piece of one
chromosome to another or loss of fragment of a
chromosome.
MIC-610 63
Mitosis, 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
MIC-610 64
Nutrition & 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.
MIC-610 65
The Nutrient Requirements
of 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
MIC-610 66
Minimal 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
MIC-610 67
Ham's Tissue Culture Medium for
Mammalian Cells
(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-Tryptophan
0.6 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
MIC-610 68
E.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.
MIC-610 69
The list of ingredients needed to
grow human cells in culture is far
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.
MIC-610 70
Why does a mammalian cell
require such a complex broth
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.
MIC-610 71
Cell & Tissue culture
 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.
MIC-610 72
Animal cell culture became a routine laboratory technique
in the 1950s, but the concept of maintaining live cell lines
separated from their original tissue source was
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.
MIC-610 73
Cell culture techniques were advanced significantly in the
1940s and 1950s to support research in virology
 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.
MIC-610 74
Concept
 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.
MIC-610 75
Sometimes, it is possible to fuse normal cells with an
immortal cell line. An example is the way
monoclonal antibodies are made: Lymphocytes isolated
from the blood of an immunised animal are combined with
hybridoma cell lines in a selective growth medium: only
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.
MIC-610 76
 . 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.
MIC-610 77
 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
MIC-610 78
Tissue 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
MIC-610 79
Organ 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.
MIC-610 80
Primary 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 containingMIC-610
freshly prepared media. 81
Secondary 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.
MIC-610 82
Diploid 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.
MIC-610 83
Continuous 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.
MIC-610 84
Some 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
MIC-610 85
Cell 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.
 .
MIC-610 86
Cell 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
MIC-610 87
Cell 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.]
MIC-610 88
Cell 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.
MIC-610 89
Cell 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.
MIC-610 90
Cell 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.
MIC-610 91
Cell 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.
MIC-610 92
Cell 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.
MIC-610 93
Cell 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
MIC-610 94
Cell 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
MIC-610 95
Splitting 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.
MIC-610 96
WORK AREA AND
EQUIPMENT
 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.
MIC-610 97
WORK 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.
MIC-610 98
WORK AREA AND
EQUIPMENT
 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.
MIC-610 99
 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.
MIC-610 100
 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.
MIC-610 101
WORK AREA AND
EQUIPMENT
 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.
MIC-610 102
 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.
MIC-610 103
 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.
MIC-610 104
 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).
MIC-610 105
 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.
MIC-610 106
 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.
MIC-610 107
 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.
MIC-610 108
 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
MIC-610 109
WORK AREA AND
EQUIPMENT
 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 agent
c. 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.
MIC-610 110
Media and growth
requirements
 1. Physiological parameters
 A. temperature - 37C for cells from
homeother
 B. pH - 7.2-7.5 and osmolality of medium
must be maintained
 C. 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;
MIC-610 111
Media and growth
requirements
 2. Medium requirements: (often empirical)
 A. Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2
B. Trace elements - iron, zinc, selenium
C. sugars - glucose is the most common
D. amino acids - 13 essential
E. vitamins - B, etc.
F. choline, inositol
G. 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.
MIC-610 112
Media and growth
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.
MIC-610 113
SAFETY 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)
MIC-610 114
SAFETY 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
MIC-610 115
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.
MIC-610 116
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.
MIC-610 117
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.
MIC-610 118
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.
MIC-610 119
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.
MIC-610 120
Drug Evaluation Against Viruse
Using 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.
MIC-610 121
Using 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.
MIC-610 122
Using 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.
MIC-610 123
Plaque 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.

MIC-610 124
Developmental 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.
MIC-610 125
MIC-610 126
Stem 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.
MIC-610 127
Stem 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."
MIC-610 128
Stem Cells
 Thestudy of stem cells is attributed as

beginning in the 1960s after research by
Canadian scientists Ernest A. McCulloch
and James E. Till.
MIC-610 129
Stem 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.
MIC-610 130
Embryonic 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.
MIC-610 131
Embryonic 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.
MIC-610 132
Embryonic 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.
MIC-610 133
Embryonic 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.
MIC-610 134
Embryonic 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
MIC-610 135
MIC-610 136
Adult 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.
MIC-610 137
Adult 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.
MIC-610 138
Adult 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.
MIC-610 139
Adult 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
MIC-610 140
MIC-610 141
Cancer 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.
MIC-610 142
Cancer 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.
MIC-610 143
Cancer 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
MIC-610 144
Treatments
 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.
MIC-610 145
Controversy surrounding
stem 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.
MIC-610 146
Key events in stem cell
research
 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.
MIC-610 147
Key events in stem cell
research
 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.
MIC-610 148
Thankyou
MIC-610 149

Advance Virology and Tissue Culture

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Advance Virology

and Tissue Culture

 Most proteins are synthesized by ribosomes in the

 An animal cell is a form of eukaryotic cell which make up

 The most significant organelles of an animal cell include:

 A cell membrane, plasma membrane or plasmalemma is a

 The endoplasmic reticulum (endoplasmic meaning "within the

 The ER consists of an extensive membrane network of tubes and

 Vesicles that leave the endoplasmic

 In cell biology, a mitochondrion (plural mitochondria)

 The outer nuclear membrane is continuous with the

 GTPases are enzymes that bind to a molecule called

 Apoptosis is a controlled process resulting in death of the

 A ribosome is an organelle composed of

 Ribosomes were first observed in the mid-1950s by cell biologist George

 Prophase: Chromatin in the

 Prometaphase: The nuclear

 Metaphase: Spindle fibers align the

 Anaphase: The paired

 Telophase: Chromatids arrive at

 The class of enzymes that are involved in triggering events in the

 Chromosomes in a Diploid Cell

 Spectral Karyotyping- a new method

 What is meiosis I? In meiosis I,

 Prophase 1: DNA replication

 Prometaphase 1: The nuclear

 Metaphase 1: Bivalents, each

 Anaphase 1: Chiasmata separate.

 Telophase 1: Nuclear envelopes

 Cytokinesis: Analogous to mitosis

 Meiosis II is similar to mitosis. However, there is

 Mitosis can proceed independent of ploidy of cell, homologous

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

 Glucose 5g/ liter

 L-Arginine211 mg, Biotin 0.024 mg, L-Histidine21 mg, Calcium pantothenate

 It is the price of multicellularity.

 Cell culture is the process by which either prokaryotic or

 19th Century English physiologist Sydney Ringer developed

 Growing viruses in cell cultures allowed preparation of

 Cell culture methods can be applied to either

 The term tissue culture was original applied to explants of

 Organ culture may maintain their original

 The primary culture may contain a variety of cell types such as

 Primary cell lines can be passed through secondary and

 Certain culture cells, notably mouse embryo fibroblasts and human

 Amniotic fluid-derived cell line: Cells isolated from amniotic

 Smooth muscle: Non-striated muscle tissue in vertebrates

 Culture cells forming a confluent monolayer on the surface of their

 E. Vessels. Anchorage dependent cells require a

 To maximize recovery of the cells when

 A. Growth pattern. Cells will initially go

 B. Harvesting. Cells are harvested when the cells have

 1. Suspension culture. Suspension cultures are fed by

 When the cells become semi-confluent, several methods

 Proteolytic enzymes - Trypsin, collagenase, or pronase,

 autoclave all waste,

 A freezing medium of 90% fetal calf serum and 10%

 The hemacytometer has two chambers, each chamber

 The findings of developmental biology can help to

 Medical researchers believe stem cell research has the

 Thestudy of stem cells is attributed as

 Research history and developments

 Adult stem cells are undifferentiated cells found