NEHRU ARTS AND SCIENCE COLLEGE
DEPARTMENT OF COMPUTER SCIENCE
E-LEARNING
CLASS
SUBJECT
: III B Sc Biotechnology
: CORE PAPER: IX ANIMAL BIOTECHNOLOGY
UNIT-1
Animal cell culture: Fundamentals. facilities and applications. Media for Animal cells.
Biology of cultured cells, measurement of growth, cell synchronization, and Apoptosis.
Part – A
1. Animal cell culture
It can be described as in vitro maintenance and propagation of animal Cells using suitable
nutrient media. Animal cell culture is the complex process by which 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.
2. Media
Any liquid or solid preparation made specifically for the growth, storage, or transport of
of cells.
3. Serum
Blood serum, a component of blood which is collected after coagulation.
4. Buffer
Buffer solution, a solution which reduces the change of pH upon addition of small
amounts of acid or base, or upon dilution eg HEPES
5. HEPES
It (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) is a zwitterionic organic chemical
buffering agent; one of the twelve Good's buffers. HEPES is widely used in cell culture,
largely because it is better at maintaining physiological pH despite changes in carbon
dioxide concentration (produced by cellular respiration) when compared to bicarbonate
buffers, which are also commonly used in cell culture.
6. What is the role of Co2 in cell culture
 Carbon di oxide influences the pH of the media.
 Buffering action of bicarbonate helps maintain PH.
 HEPES containing media well regulates ph.
7. Adhersion
•
•
Majority Of Cells Adhere On Plastic (Treated) Provided They Are Not
Transformed
It Was Observed That Cells Prefer –vely Charged Glass Surface
•
•
Plastic (polystyrene) Is Tissue Culture Treated
– With High Energy Ionizing Radiation
– Electric Ion Discharge
Adhesion Is Mediated By Surface Receptors And Matrix
– Matrix Is Secreted By Cells, Adheres To Charged Plastic
– Receptors Bind to Matrix
8. What are the classes of Cell Surface Adhesion Molecules
Three Major Classes
• Cell-Cell Adhesion Molecules
• Cell-Substrate Molecules
• Proteoglycans
9. Cell cycle
•
4 Phases
– M Phase, mitosis occurs
• Chromatin condensation, sister chromatid separation
• Daughter cells
– G1 Phase
• Progression to DNA SYNTHESIS
• Alternatively Go OR differentiation
• Restriction Points
– S Phase
• DNA Synthesis
• Progression to G2
– G2 Phase
• Integrity of DNA Checkpoints
• Apoptosis is an option
– DNA fragmentation, cell shrinkage, formation of small
vesicles
10. Dedifferentiation
•
•
•
Inability To Express In Vivo Phenotype Is Attributed To Dedifferentiation
Still Not Clear If Dedifferentiation Occurs
– Wrong lineage expansion is a possibility
– Undifferentiated cells dominate
– Absence of appropriate inducers, hormones, matrix
Deadaptation vs Dedifferentiation
– Deadaptation-enviroment suppresses phenotype, reversible
– Dedifferentiation-conversion to primitive phenotype, irreversible
11. Cell synchronization
Its a process by which cells at different stages of the cell cycle in a culture are brought to
the same phase. "Cell synchrony" is required to study the progression of cells through the
cell cycle. The types of synchronizations are broadly categorized into two groups:
"Physical Fractionation" and "Chemical Blockade."
12. Senescence
Senescence or biological aging is the change in the biology of an organism as it ages
after its maturity. Such changes range from those affecting its cells and their function to
that of the whole organism. The word senescence is derived from the Latin word senex,
meaning old man, old age, or advanced in age.
13. Apoptosis
Its the process of programmed cell death (PCD) that may occur in multicellular
organisms.
Part – B
1. Write a note on Animal cell culture
Its the process of culture of animal cells outside the tissue (ex vivo) from which they
were obtained. The process of ACC is carried out under strict laboratory conditions of
asepsis, sterility and controlled environment involving temperature, gases and
pressure. It should mimic the in vivo environment successfully such that the cells are
capable of survival and proliferation in a controlled manner.
Epithelial cells in culture, stained for keratin (red) and DNA (green)
Cell culture is the process by which 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. Animal cell culture became a common 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.
2. Write a note on tissue culture
The term tissue culture refers to the culture of whole organs, tissue fragments as well as
dispersed cells on a suitable nutrient medium. It can be divided into
(1) organ culture and
(2) cell culture mainly on the basis of whether the tissue organisation is retained or not.
In organ cultures, whole embryonic organs or small tissue fragments are cultured in vitro
in such a manner that they retain their tissue architecture. In contrast, cell cultures are
obtained either by enzymatic or mechanical dispersal of tissues into individual cells or by
spontaneous migration of cells from explants; they are maintained as attached
monolayers or as cell suspensions.
Freshly isolated cell cultures are called primary cultures; they are usually heterogeneous
and slow growing, but are more representative of the tissue of their origin both in cell
type and properties. Once a primary culture is subcultured, it gives rise to cell lines,
which may either die after several subcultures (such cell lines are known as finite cell
lines) or may continue to grow indefinitely (these are called continuous cell lines).
Usually, normal tissues give rise to finite cell lines, while tumours give rise to continuous
cell lines. But there are several examples of continuous cell lines, which were derived
from normal tissues and are themselves nontumorigenic, e.g., MDCK dog kidney, 3T3
fibroblasts, etc.
The evolution of continuous cell lines from primary cultures is supposed to involve a
mutation, which alters their properties as compared to those of finite lines.
3. Write a note on animal cell culture media
Culture Media - The nutrient media used for culture of animal cells and tissues must be
able to support their survival as well as growth, i.e., must provide nutritional, hormonal
and stromal factors.
The various types of media used for tissue culture may be grouped into two broad
categories:
(1) natural media and
(2) artificial media.
The choice of medium depends mainly on the type of cells to be cultured (normal,
immortalized or transformed), and the objective of culture (growth, survival,
differentiation, production of desired proteins).
Nontransformed or normal cells (finite life span) and primary cultures from healthy
tissues require defined quantities of proteins, growth factors and hormones even in the
best media developed so far. But immortalized cells (spontaneously or by transfection
with viral sequences) produce most of these factors, but may still need some of the
growth factors present in the serum.
In contrast, transformed cells (autonomous growth control and malignant properties)
synthesize their own growth factors; in fact, addition of growth factors may even be
detrimental in such cases. But even these cultures may require factors like insulin,
transferrin, silenite, lipids, etc
4. Cell Surface Adhesion Molecules
•
Three Major Classes
– Cell-Cell Adhesion Molecules
• CAMs (Ca2+ Independent)
• Cadherins (Ca2+ Dependent)
• Primarily Between Homologous Cells
• Signaling occurs
– Cell-Substrate Molecules
• Integrins
• Bind to fibronectin, entactin, laminin, collagen
• Bind the specific motif (RGD, arginine, glycine,aspratic)
• Comprised of  and  unit
- Proteoglycans
• Also Binds Matrix or Other Proteoglycans
• Not Via RGD Motif
• Low affinity Growth Factor Receptors
• May Aid Binding To Higher Affinity Receptors
• No Signaling Capacity
5. Write a note on cell senescence
Senescence or biological aging is the change in the biology of an organism as it ages
after its maturity. Such changes range from those affecting its cells and their function to
that of the whole organism. There are a number of theories as to why senescence occurs,
including ones that claim it is programmed by gene expression changes and that it is the
accumulative damage of biological processes. The word senescence is derived from the
Latin word senex, meaning old man, old age, or advanced in age.
Cellular senescence
Cellular senescence (upper) Primary mouse embryonic fibroblast cells (MEFs) before
senescence. Spindle-shaped. (lower) MEFs became senescent after passages. Cells grow
larger, flatten shape and expressed senescence-associated β-galactosidase (SABG, blue
areas), a marker of cellular senescence.
Cellular senescence is the phenomenon by which normal diploid cells lose the ability to
divide, normally after about 50 cell divisions in vitro. Some cells become senescent after
fewer replications cycles as a result of DNA double strand breaks, toxins, etc. This
phenomenon is also known as "replicative senescence", the "Hayflick phenomenon", or
the Hayflick limit in honour of Dr. Leonard Hayflick who was the first to publish this
information in 1965. In response to DNA damage (including shortened telomeres), cells
either age or self-destruct (apoptosis, programmed cell death) if the damage cannot be
easily repaired. In this 'cellular suicide', the death of one cell, or more, may benefit the
organism as a whole. For example, in plants the death of the water-conducting xylem
cells (tracheids and vessel elements) allows the cells to function more efficiently and so
deliver water to the upper parts of a plant. The ones that do not self-destruct remain until
destroyed by outside forces.
Aging of the whole organism
Organismal senescence is the aging of whole organisms. In general, aging is
characterized by the declining ability to respond to stress, increased homeostatic
imbalance, and increased risk of aging-associated diseases. Death is the ultimate
consequence of aging, though "old age" is not a scientifically recognized cause of death
because there is always a specific proximal cause, such as cancer, heart disease, or liver
failure. Aging of whole organisms is therefore a complex process that can be defined as
"a progressive deterioration of physiological function, an intrinsic age-related process of
loss of viability and increase in vulnerability".
Differences in maximum life span among species correspond to different "rates of aging".
For example, inherited differences in the rate of aging make a mouse elderly at 3 years
and a human elderly at 80 years. These genetic differences affect a variety of
physiological processes, including the efficiency of DNA repair, antioxidant enzymes,
and rates of free radical production.
Supercentenarian Ann Pouder (8 April 1807 – 10 July 1917) photographed on her 110th
birthday. A heavily lined face is common in human senescence.
Senescence of the organism gives rise to the Gompertz–Makeham law of mortality,
which says that mortality rate rises rapidly with age.
Some animals, such as some reptiles and fish, age slowly (negligible senescence) and
exhibit very long lifespans. Some even exhibit "negative senescence", in which mortality
falls with age, in disagreement with the Gompertz–Makeham "law".
Whether replicative senescence (Hayflick limit) plays a causative role in organismal
aging is at present an active area of investigation.
6. Write a note on Apoptosis
Programmed cell death is called apoptosis. The pattern of events in death by suicide is so
orderly that the process is often called programmed cell death or PCD. The cellular
machinery of programmed cell death turns out to be as intrinsic to the cell as, say,
mitosis.
Cells that are induced to commit suicide:









shrink;
develop bubble-like blebs on their surface;
have the chromatin (DNA and protein) in their nucleus degraded;
have their mitochondria break down with the release of cytochrome c;
break into small, membrane-wrapped, fragments;
release (at least in mammalian cells) ATP and UTP.
These nucleotides bind to receptors on wandering phagocytic cells like
macrophages and dendritic cells and attract them to the dying cells (a "find-me"
signal").
The phospholipid phosphatidylserine, which is normally hidden within the
plasma membrane, is exposed on the surface.
This "eat me" signal is bound by other receptors on the phagocytes which then
engulf the cell fragments.

The phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and
TGF-β)
7. Explain measurement of cell growth
The cell growth can be detected by a variety of methods. The cell size growth can be
visualized by Microscopy, using suitable stains. But the increase of cells number is
usually more significative. It can be measured by manual counting of cells under
microscopy observation, using the dye exclusion method (i.e. Trypan blue) to count only
viable cells. Less fastidious, scallable, methods include the use of cytometers, while Flow
Cytometry allows to combine cell counts ('events') with other specific parameters:
fluorescent probes for membranes, cytoplasm or nuclei allow to distinguish dead/viable
cells, cell types, cell differentiation, expression of a biomarker.
Beside the increasing number of cells, one can be assessed regarding the metabolic
activity growth. I.e. the CFDA and Calcein-AM mesure (fluorimetrically) not only the
membrane functionality (dye retention), but also the functionality of cytoplasmic
enzymes (esterases). The MTT assays (colorimetric) and the Resazurin assay
(fluorimetric) dose the mitochondrial redox potentiel.
Finally, all these assays may correlate well, or not depending on cell growth conditions
and desired aspects (activity, proliferation). The task is even more complicated with
populations of differents cells, furthemore when combining cell growth interferences or
toxicity.
Part – C
1. Explain Biology of cultured cells
CELL ADHESION
 CAM
 cytoskeleton
CELL PROLIFERATION
 Cell cycle
DIFFERENTIATION
 Maintenance of differentiation
 Dedifferentiation
2. Write a note on Fundamentals, facilities and applications animal cell culture.
Mass culture of animal cell lines is fundamental to the manufacture of viral vaccines and
many products of biotechnology. Biological products produced by recombinant DNA
(rDNA) technology in animal cell cultures include enzymes, synthetic hormones,
immunobiologicals (monoclonal antibodies, interleukins, lymphokines), and anticancer
agents. Although many simpler proteins can be produced using rDNA in bacterial
cultures, more complex proteins that are glycosylated (carbohydrate-modified), currently
must be made in animal cells. An important example of such a complex protein is the
hormone erythropoietin. The cost of growing mammalian cell cultures is high, so
research is underway to produce such complex proteins in insect cells or in higher plants.
Tissue culture and engineering
Cell culture is a fundamental component of tissue culture and tissue engineering, as it
establishes the basics of growing and maintaining cells ex vivo.
Animal cell culture has become one of the major tools used in cell and molecular biology.
Some of the important areas where:
 Model system
 Toxicity study
 Cancer research
 Virology
 Cell-based manufacturing
 Genetic counseling
 Gene therapy
 Drug screening and development
3. Write a note on role of serum in animal cell culture media

It provides the basic nutrients for cells; the nutrients are present both in the
solution as well as are bound to the proteins.

It provides several hormones, e.g., insulin, which is essential for growth of nearly
all cells in culture, cortisone, testosterone, prostaglandin, etc.

It contains several growth factors, e.g., platelet derived growth factor (PDGF),
transforming growth factor β (TGF- β), epidermal growth factor, etc.; these are
present in concentrations of µg/l. Both hormones and growth factors are involved
in growth promotion and specialized cell function. A given hormone or growth
factor may stimulate growth of one cell type, may have no effect on another and
may even be inhibitory to some others. For example, PDGF induces proliferation
in fibroblasts, but induces differentiation of some types of epithelia.
Further, proliferation of a single cell type may be induced by more than one
growth factor, e.g., fibroblasts respond to PDGF, epidermal growth factor,
fibroblast growth factor and somatomidins

A major role of serum is to supply proteins, e.g., fibronectin, which promote
attachment of cells to the substrate. It also provides spreading factors that help the
cells to spread out before they can begin to divide. Although cells do produce
these factors, but trypsinized cells are usually unable to attach to the substrate.

It provides several binding proteins, e.g., albumin, transferrin, etc., which carry
other molecules into the cell. For example, albumin carries into cells lipids,
vitamins, hormones, etc. Transferrin usually carries Fe in a nonbasic form, but
binding of transferrin to its receptor in cell membrane is believed to be mitogenic.

It increases the viscosity of medium and, thereby, protects cells from mechanical
damages, e.g., shear forces during agitation of suspension cultures.

Protease inhibitors present in the serum protect cells, especially trypsinised cells,
from proteolysis.

The serum also provides minerals, like Na+, K+, Fe2+, Zn2+, etc.

It also acts as a buffer.
4. Explain cell synchronization
Cell Synchronization is a process by which cells at different stages of the cell cycle in a
culture are brought to the same phase. "Cell synchrony" is required to study the
progression of cells through the cell cycle. The types of synchronizations are broadly
categorized into two groups: "Physical Fractionation" and "Chemical Blockade." Cell
separation by physical means
Physical fractionation or cell separation techniques, based on the following
characteristics are in use.




Cell density
Cell size
Affinity of antibodies on cell surface epitopes.
Light scatter or fluorescent emission by labeled cells.
The two commonly used techniques are:
Centrifugal separation
The physical characteristics - cell size and sedimentation velocity - are operative in the
technique of centrifugal elutriation. Centrifugal elutriator (from Beckman) is an advanced
device for increasing the sedimentation rate so that the yield and resolution of cells is
better. The cell separation is carried out in a specially designed centrifuge and rotor.
Fluorescence-activated cell sorting
Fluorescence-activated cell sorting (FACS) is a technique for sorting out the cells based
on the differences that can be detected by light scatter (eg. cell size) or fluorescence
emission (by penetrated DNA, RNA, proteins, antigens). The procedure involves passing
of a single stream of cells through a laser beam so that the scattered light from the cells
can be detected and recorded. There are two instruments in use based on its principle:
a) Flow cytometer
b) Fluorescence-activated cell sorter
Cell separation by chemical blockade
The cells can be separated by blocking metabolic reactions. Two types of metabolic
blockades are in use:
Inhibition of DNA synthesis
During the S phase of cell cycle, DNA synthesis can be inhibited by using inhibitors such
as thymidine, aminopterin, hydroxyurea and cytosine arabinoside. The effects of these
inhibitors are variable. The cell cycle is predominantly blocked in S phase that results in
viable cells.
Nutritional deprivation
Elimination of serum from the culture medium for about 24 hours results in the
accumulation of cells at G1 phase. This effect of nutritional deprivation can be restored
by their addition by which time the cell synchrony occurs.
5. Explain the event of Apoptosis with diagram
Apoptosis is the process of programmed cell death (PCD) that may occur in multicellular
organisms. Biochemical events lead to characteristic cell changes (morphology) and
death. These changes include blebbing, loss of cell membrane asymmetry and
attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and
chromosomal DNA fragmentation. (See also Apoptosis DNA fragmentation.) Unlike
necrosis, apoptosis produces cell fragments called apoptotic bodies that surrounding cells
are able to engulf and quickly remove before the contents of the cell can spill out onto
surrounding cells and cause damage.
Process
The process of apoptosis is controlled by a diverse range of cell signals, which may
originate either extracellularly (extrinsic inducers) or intracellularly (intrinsic inducers).
Extracellular signals may include toxins, hormones, growth factors, nitric oxide or
cytokines, that must either cross the plasma membrane or transduce to effect a response.
These signals may positively (i.e., trigger) or negatively (i.e., repress, inhibit, or dampen)
affect apoptosis. (Binding and subsequent trigger of apoptosis by a molecule is termed
positive induction, whereas the active repression or inhibition of apoptosis by a molecule
is termed negative induction.)
A cell initiates intracellular apoptotic signalling in response to a stress, which may bring
about cell suicide. The binding of nuclear receptors by glucocorticoids, heat, radiation,
nutrient deprivation, viral infection, hypoxia and increased intracellular calcium
concentration, for example, by damage to the membrane, can all trigger the release of
intracellular apoptotic signals by a damaged cell. A number of cellular components, such
as poly ADP ribose polymerase, may also help regulate apoptosis.
Before the actual process of cell death is precipitated by enzymes, apoptotic signals must
cause regulatory proteins to initiate the apoptosis pathway. This step allows apoptotic
signals to cause cell death, or the process to be stopped, should the cell no longer need to
die. Several proteins are involved, but two main methods of regulation have been
identified: targeting mitochondria functionality, or directly transducing the signal via
adaptor proteins to the apoptotic mechanisms. Another extrinsic pathway for initiation
identified in several toxin studies is an increase in calcium concentration within a cell
caused by drug activity, which also can cause apoptosis via a calcium binding protease
calpain.