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Question and Answer for Animal Cell Technology K61CNSHE

1. What is proteomic?
2. What are the applications of Proteomic?
3. Overview of Cytomic?
4. What is biochip and its application?
5. What are Nanorobots and their application?
6. What is the basic definition of animal cell culture?
7. What are the steps of Artificial Insemination Technique?
8. What is basic meaning of cloning process?
9. What is GMA and give some examples?
10.How does the gene therapy work?
11.What are the characteristics of animal cell in cultures?
12.Description about the basic concepts of cell lines?
13.What is name of the level culture? What are advantage and disadvantage
of the cell culture?
14.What are the requirements for designing the laboratory and describe these
requirements?
15.What are the components of an autoclave and how does it work?
16.Description about influences of environmental conditions on animal cell
culture?
17.Description about cultural medium component?
18.Description about advantages and disadvantages of media which have and
don’t have serum?
19.Description about techniques to prepare animal cell culture media?
20.Description about the notices when you prepare culture media
21.What is the MTT protocol?
22.What is plating efficiency and its formula
23.Describe the common features of disaggregation? How many kinds of
enzymes used in disaggregation?
24.Perform protocol of isolation chick embryos?
25. What is the growth curve? The main phage of the growth curve?
26.What are the advantages and disadvantages of serum - free media?
27.Describe the components of serum-free subculture?
28.Describe the definition of the primary culture? How many stages are there
for primary culture?
29.How does mechanical disaggregation proceed?
30.Describe the definition, objectives, protocol of collagenase disaggregation?
31.What are limitations of traditional method culture?
32.What is the different between 2D and 3D cell culture?
33.What are the limitations of tissue engineering scaffold?
34.Describe the scaffolding properties have been defined has being essential
for three-dimensional bone cell culture techniques?
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Question and Answer for Animal Cell Technology K61CNSHE

35.Describe about gas foaming method to fabricate scaffolds. What are the
advantages and disadvantages of this method?
36.Describe about fibre meshes/ fire bonding method to fabricate scaffolds.
What are advantages and disadvantages of this method?
37.What is the Solid Freeform Fabrication and its function in Tissue
Engineering?
38.What are the techniques of SFF in Tissue Engineering and its
characteristics?
39.What is the spinner flask and its characteristics?
40. What are the differences between the batch cultivation and the continuous
cultivation?
41.What is the major advantage of working in a laminar-flow hood? Compare
horizontal laminar flow hood with vertical laminar flow hood?
42.When applying aseptic techniques, what should we pay attention to
regarding personal hygiene and reagents and media?
43.Describe the wet heat method in sterilization?
44.What is chemical sterilization?
45.Describe the protocol of animal cell observation?
46.Describe the protocol for microbial decontamination treatment?
47.What are the general removal methods for contaminated cultures?
48.How can the cells be stored in cryopreservation?
49.How can the cells be recovered from frozen state?
50.Describe the thawing protocol?
51.What is the application of animal cell in biological compounds production?
52.What is the application of animal cell in the production virus kill insects?
53.Introduce briefly about creating organ from animal cells culture?
54.What is the application of animal cell culture in livestock-poultry farming
industry?
55.What is the process of pumping sperm into the uterus ovary in intrauterine
insemination (IUI) method?
56.What is genetic marker in biotechnology in Animal Breeding?
57.What are the advantages of QTL / ETL in breeding selection programs?
58.How the gene transfer method applies in cow?
59.Definition and basic procedure of transgenic in aquaculture?
60.What is the process Polyploidy in fish?

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Question and Answer for Animal Cell Technology K61CNSHE

1. What is proteomic?
- Proteomic
• Proteomics generally refers to the large-scale experimental analysis of
proteins and proteomes, but often is used specifically to refer to protein
purification and mass spectrometry.
• Proteomics has enabled the identification of ever increasing numbers of
protein. This varies with time and distinct requirements, or stresses, that a
cell or organism undergoes.
• Proteomics is an interdisciplinary domain that has benefitted greatly from the
genetic information of various genome projects, including the Human
Genome Project. It covers the exploration of proteomes from the overall level
of protein composition, structure, and activity. It is an important component
of functional genomics
• Proteomics can provide significant biological information for many
biological problems, such as:
 Which proteins interact with a particular protein of interest?
 Which proteins are localized to a subcellular compartment?
 Which proteins are involved in a biological process?
2. What are the applications of Proteomic?
- Post-translational modifications
• Proteomics studies involve certain unique features as the ability to analyze
post- translational modifications of proteins. These modifications can be
phosphorylation, glycosylation and sulphation as well as some other
modifications involved in the maintenance of the structure of a protein.
• These modifications are very important for the activity, solubility and
localization of proteins in the cell. Determination of protein modification is
much more difficult rather than the identification of proteins. As for
identification purpose, only few peptides are required for protease cleavages
followed by database alignment of a known sequence of a peptide.

- Protein-protein interactions
• The major attribution of proteomics towards the development of protein
interactions map of a cell is of immense value to understand the biology of a
cell. The knowledge about the time of expression of a particular protein, its
level of expression, and, finally, its interaction with another protein to form
an intermediate for the performance of a specific biological function is
currently available.
• These intermediates can be exploited for therapeutic purposes also. An
attractive way to study the protein-protein interactions is to purify the entire
multi-protein complex by affinity based methods using GST-fusion proteins,
antibodies, peptides etc.
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Question and Answer for Animal Cell Technology K61CNSHE

- Protein expression profiling

• The largest application of proteomics continues to be protein expression


profiling. The expression levels of a protein sample could be measured by 2-
DE or other novel technique such as isotope coded affinity tag (ICAT). Using
these approaches the varying levels of expression of two different protein
samples can also be analyzed.

- Protein expression profiling

• The largest application of proteomics continues to be protein expression


profiling. The expression levels of a protein sample could be measured by 2-
DE or other novel technique such as isotope coded affinity tag (ICAT). Using
these approaches the varying levels of expression of two different protein
samples can also be analyzed.

3. Overview of Cytomic?

- Cytomics is the study of cell biology (cytology) and biochemistry in cellular


systems at the single cell level. It combines all the bioinformatics knowledge to
attempt to understand the molecular architecture and functionality of the cell system
(Cytome). Much of this is achieved by using molecular and microscopic techniques
that allow the various components of a cell to be visualized as they interact in vivo

- The cytomics concept has been significantly advanced by a multitude of current


developments. Amongst them are confocal and laser scanning microscopy,
multiphoton fluorescence excitation, spectral imaging, fluorescence resonance
energy transfer (FRET), fast imaging in flow, optical stretching in flow, and
miniaturized flow and image cytometry within laboratories on a chip or laser
microdissection, as well as the use of bead arrays.

- In addition, biomolecular analysis techniques like tyramide signal amplification,


single‐cell polymerase chain reaction (PCR), and the labelling of biomolecules by
quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity
and molecular specificity at the single‐cell level. Data sieving or data mining of the
vast amounts of collected multiparameter data for exhaustive multilevel
bioinformatics knowledge extraction avoids the inadvertent loss of information
from unknown molecular relations being inaccessible to an a priori hypothesis.
4. What is biochip and its application?

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Question and Answer for Animal Cell Technology K61CNSHE

- In addition, biomolecular analysis techniques like tyramide signal amplification,


single‐cell polymerase chain reaction (PCR), and the labelling of biomolecules by
quantum dots, magnetic nanobeads, or aptamers open new horizons of sensitivity
and molecular specificity at the single‐cell level. Data sieving or data mining of the
vast amounts of collected multiparameter data for exhaustive multilevel
bioinformatics knowledge extraction avoids the inadvertent loss of information
from unknown molecular relations being inaccessible to an a priori hypothesis.
- Application of biochip are:By using this chip we can trace a person or animal
anywhere in the world.The biochip can be applicable in the medical field as a BP
sensor, glucose detector, and oxygen sensor.These chips are effective in restoring
the records of medical, cash, passport, etc.A biochip leads to safe E-commerce
systems. This chip is used to store and update the information of a person like
medical financial and demographics.

5. What are Nanorobots and their application?


- A nanorobot is an extremely small robot that is designed to perform specific tasks
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at the nanoscale dimension of few nanometers i.e. 1 nm = 10 meter. In healthcare
IT industry, this technology brings a great boom & helps in protecting &
maintaining the human body against viruses or bacteria. Researchers
in robotics industry will use the pollution-free process to build cheapest &
inexpensive nanorobots.
- Application of nanorobot: Treatment & Diagnosis of Diabetes: To maintain the
human metabolism, glucose molecules are carried through the bloodstream. To
determine the need for injecting insulin inside the body, glucose monitoring
nanorobots uses the chemosensor.
- Surgical nanorobots can act as a semi-autonomous onsite surgeon will perform
various functions such as diagnosing, detection of pathology, correcting lesions by
nanomanipulation etc.
- Cancer Detection & treatment: Nano robots are made up of a mixture of protein &
a polymer known as transferrin which is capable of detecting tumor cells. These
robots kill the cancer cells without damaging the healthy cells which will lead to
hair loss, nausea etc.
- Gene Therapy: Genetic disease can be treated by nanorobots by comparing the
molecular structure of both proteins & DNA found in the cell.
6. What is the basic definition of animal cell culture?
- Cell culture refers to cultures derived from dissociated cells taken from the original
tissue ('primary cell culture'). Cells are dispersed (mechanically and/or
enzymatically) into a cell suspension which may then be cultured as a monolayer
on a solid substrate, or as a suspension in the culture medium.

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Question and Answer for Animal Cell Technology K61CNSHE

- Cell culture is one of the major tools used in cellular and molecular biology,
providing excellent model systems for studying the normal physiology and
biochemistry of cells (e.g., metabolic studies, aging), the effects of different toxic
compounds on the cells, and mutagenesis and carcinogenesis.
- Normal cells can be transformed into cancer cells by methods including radiation,
chemicals and viruses. These cells can then be used to study cancer more closely
and to test potential new treatments. Cell lines are also used in in vitro fertilization
(IVF) technology, recombinant protein and drug selection and improvement,
products vaccine.
7. What are the steps of Artificial Insemination Technique?
- Step 1: Restrain the animal to be inseminated. There are several things that should
be kept in mind when choosing a location for inseminating cattle. Some of these
include safety of both the animal and the inseminator, ease of use, and shelter from
adverse weather. A gentle pat on the animal’s rump or a soft spoken word as the
inseminator approaches will help to avoid startling or surprising the cow.
- Step 2: Raise the tail with the right hand and gently massage the rectum with the
lubricated glove on the left hand. Place the tail on the back side of the left forearm
so it will not interfere with the insemination process. Cup the fingers together in a
pointed fashion and insert the left hand in the rectum, up to the wrist.
- Step 3: Gently wipe the vulva with a paper towel to remove excess manure and
debris. Be careful not to apply excessive pressure which may smear or push manure
into the vulva and vagina. With the left hand, make a fist and press down directly
on top of the vulva. This will spread the vulva lips allowing clear access to insert
the gun tip several inches into the vagina before contacting the vaginal walls.
- Step 4: Insert the gun at a 30° upward angle to avoid entering the urethral opening
and bladder located on the floor of the vagina. With the gun about 6 to 8 inches
inside the vagina, raise the rear of the gun to a somewhat level position and slide it
forward.
To become a successful inseminator, it is very important to always know where the
tip of the insemination gun is located. The walls of the vagina consist of thin layered
muscle and loose connective tissue. The insemination gun can be easily felt with
the left hand in the rectum. As the breeding gun is inserted into the vagina, keep the
gloved hand even with the gun tip. Manure in the rectum can often interfere with
the inseminator’s ability to palpate the cervix and gun tip. However, it is seldom
necessary to remove all the manure from the bowel. Instead, keep the open hand
flat against the floor of the rectum, allowing the manure to pass over the top of the
hand and arm
With the hand in the rectum, the inseminator may notice colon constrictions or
"rings" attempting to force the left arm from the cow. To relax these rings, place
two fingers through the center of a ring and massage back and forth. The

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Question and Answer for Animal Cell Technology K61CNSHE

constriction ring will eventually relax, pass over the hand and arm, and the
inseminator can continue the palpation process
Because the reproductive tract is freely movable, cows that have strong rectal and
abdominal contractions in response to being palpated may actually push their
reproductive tract back into the pelvic cavity. This will cause many folds to form
in the vagina. In such cases, the insemination gun can get caught in these folds and
little or no progress will be made until they are removed. If the cervix can be
located, grasp it and gently push it forward. This will straighten the vagina and the
gun should pass freely up to the cervix. The inseminator will note a distinct gristly
sensation on the gun when it contacts the cervix.
The cervix consists of dense connective tissue and muscle and is the primary
landmark for inseminating cattle. It has often been described as having the size and
consistency of a turkey neck. The size will vary, however, with post partum interval
and age of the animal. The cervix usually has three or four annular rings or folds.
The opening into the cervix protrudes back into the vagina. In most cows, the cervix
will be located on the floor of the pelvic cavity near the anterior (front) end of the
pelvis. In older cows, the cervix may rest slightly over the pelvic bone and down
into the abdominal cavity.
- Step 5: Once the gun is in contact with the external surface of the cervix, the
inseminator is ready to begin threading the cervix over the end of the gun. Place the
cervix on or over the insemination gun; the gun is not passed through the cervix.
Excessive movement or probing with the insemination gun during this step is
seldom productive. The key to mastering this step of the insemination process is
knowing how to hold and manipulate the cervix and concentrating on doing the
work with the hand inside the cow, not the one holding the gun. When the gun first
contacts the cervix, the inseminator will usually find that the tip is in the fornix area
directly over the top of the opening of the cervix. If this happens, grasp the external
opening to the cervix with the thumb on top and forefingers underneath. This closes
the fornix at top and bottom. It is also still important to know the location of the
gun tip. This is accomplished by contacting the gun tip with the palm and 3rd and
4th fingers of the hand in the rectum. Use the palm and these two fingers to guide
the gun tip to the cervical opening located between the thumb and forefingers. With
gentle probing, the opening of the cervix should be located. The inseminator will
feel the gun slide forward until it contacts the second cervical ring.
- Step 6: Maintain gentle but steady forward pressure on the gun and slide the thumb
and forefingers just in front of the gun tip and re-grasp the cervix. Because the
cervix is composed of dense connective tissue and muscle, it is difficult to clearly
distinguish the gun tip when it is located within this structure. However, the
inseminator can determine the approximate location by bending the cervix. Using
the flexibility of the wrist, gently twist and bend the cervix until the second ring of
the cervix slides over the gun tip. Repeat the process until all the rings have been
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Question and Answer for Animal Cell Technology K61CNSHE

passed over the gun tip. Remember, the cervix is being placed over the gun, not the
gun through the cervix. For the most part, gentle forward pressure is all that is
necessary and gun movement should be minimal. When all rings of the cervix have
been cleared, the gun should slide forward freely with little resistance. Since the
uterine wall is very thin, the inseminator will once again be able to feel the tip of
the gun.
- Step 7: It is now time to check the gun placement and deposit the semen. Rotate
the gloved hand until it lies on top of the cervix. With the index finger of that hand,
locate the far end of the cervix. Pull back on the gun until the tip of it is directly
underneath the index finger near the internal opening of the cervix. Raise the finger
and slowly deposit the semen. Push the plunger slowly so that drops of semen fall
directly into the uterine body. With proper AI technique and gun placement, semen
will be deposited in the uterine body. Uterine contractions will then transport
spermatozoa forward to the horns and oviducts with a good distribution of both
sides. When the insemination gun is more than 1 inch through the cervix, all the
semen will be deposited in only one horn. Be sure to raise the index finger after
checking gun placement. Not doing so may obstruct one horn, creating a situation
of uneven semen distribution. When checking gun tip placement, be careful not to
apply excessive pressure. The delicate uterine lining is easily damaged, potentially
causing infections and reduced fertility. With proper AI technique and gun
placement, semen will be deposited in the uterine body. Uterine contractions will
then transport spermatozoa forward to the horns and oviducts with a good
distribution of both sides. When the insemination gun is more than 1 inch through
the cervix, all the semen will be deposited in only one horn. Be sure to raise the
index finger after checking gun placement. Not doing so may obstruct one horn,
creating a situation of uneven semen distribution. When checking gun tip
placement, be careful not to apply excessive pressure. The delicate uterine lining is
easily damaged, potentially causing infections and reduced fertility.
- Step 8: After properly depositing semen, slowly pull the gun from the reproductive
tract. Remove the gloved hand from the rectum. Check the gun tip for signs of
blood, infection or semen leakage inside the sheath. Make the necessary notes for
future reference and for the local veterinarian. Remove the sheath from the gun and
hold it in the gloved hand. Check again to see which bull was used. Remove the
glove starting at the top of the arm by turning it inside out trapping manure, the
sheath, and dirt inside. Dispose of the used glove in a proper receptacle. Wipe the
gun clean and dry and return it to the proper storage location.
8. What is basic meaning of cloning process?
- Cloning, the process of generating a genetically identical copy of a cell or an
organism. Cloning happens all the time in nature. For example, when a cell replicate
sit self asexually without any genetic alteration or recombination. Prokaryotic
organisms (organisms lacking a cell nucleus) such as bacteria create genetically
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Question and Answer for Animal Cell Technology K61CNSHE

identical duplicates of themselves using binary fission or budding.


In eukaryotic organisms (organisms possessing a cell nucleus) such as humans, all
the cells that undergo mitosis, such as skin cells and cells lining the gastrointestinal
tract, are clones; the only exceptions are gametes (eggs and sperm), which
undergo meiosis and genetic recombination.
- In biomedical research, cloning is broadly defined to mean the duplication of any
kind of biological material for scientific study, such as a piece of DNA or an
individual cell. For example, segments of DNA are replicated exponentially by a
process known as polymerase chain reaction, or PCR, a technique that is used
widely in basic biological research. The type of cloning that is the focus of
much ethical controversy involves the generation of cloned embryos, particularly
those of humans, which are genetically identical to the organisms from which they
are derived, and the subsequent use of these embryos for research, therapeutic, or
reproductive purposes.
9. What is GMA and give some examples?
- Genetic modification (GM) of farm animals started in the early 1980s. Most
research on GM farm animals (cattle, sheep, pigs, chickens and goats) has been
carried out in the United States, Australia, New Zealand and Japan. Some GM
animals have been produced to aid food production but there are also other uses.
- For example, researchers have produced GM farm animals to:increase meat
production through rapid growth or leaner animals, improve the amount and quality
of wool, alter the composition of milk to make better cheese, to reduce intolerance
to milk or to produce medicines and neutraceuticals, increase disease resistance,
reduce pollution from pig manure, produce strong, spider-silk fibres in goat milk
for military use.
- Animal welfare issues associated with GM animals: Only 1–3% of experiments to
genetically modify animals are successful.
- Cloning is often used with the genetic modification, which increases losses of
embryos and offspring at around the time of birth. GM and cloned animals are often
oversized, weak and susceptible to disease. In experiments, 50% of calves died
before weaning, and mice had greater than normal illness in later life plus
abnormalities in future generations. There is no information for GM farm animals
on what might happen in the future.
- Belgian Blue beef cattle have double-muscling genes producing muscles that
bodybuilders would envy. This may be good for beef production but not for
reproduction. As calves are so big, births are painful and most are by (repeated)
caesarian section.
- GM of gut microbes could make fodder more digestible for farm animals. However,
the GM microbes could unbalance the gut, cause disease to the animals and
contaminate food.

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Question and Answer for Animal Cell Technology K61CNSHE

- Developing GM (transgenic) pigs to produce donor organs for humans causes


public concern but this is mostly about the ethics of human use, rather than about
the welfare of the pigs. Research in GM mice with a gene involved in organ
rejection has shown vision impairment and increased susceptibility to blood
infections.
- The main benefits from GM animals are economic — more product at a lower
production cost. Because some people in developing countries can now afford more
food, it has been argued that GM is needed to increase production to meet demand
for meat, cheese and milk.
- The use of GM in farm animals has focused on high-value products, for example
medicines in milk. In the future, it is hoped that GM will focus on improving the
health and welfare of animals. There has been some success with GM disease
resistance and it is now possible to produce cattle without the gene for mad cow
disease, giving hope it can be eliminated.
10. How does the gene therapy work?
- Sometimes the whole or part of a gene is defective or missing from birth, or a gene
can change or mutate during adult life. Any of these variations can disrupt how
proteins are made, which can contribute to health problems or diseases.
- In gene therapy, scientists can do one of several things depending on the problem
that is present. They can replace a gene that causes a medical problem with one that
doesn’t, add genes to help the body to fight or treat disease, or turn off genes that
are causing problems.
- In order to insert new genes directly into cells, scientists use a vehicle called a
“vector” which is genetically engineered to deliver the gene.
- Viruses, for example, have a natural ability to deliver genetic material into cells,
and therefore, can be used as vectors. Before a virus can be used to carry therapeutic
genes into human cells, however, it is modified to remove its ability to cause an
infectious disease.
- Gene therapy can be used to modify cells inside or outside the body. When it’s done
inside the body, a doctor will inject the vector carrying the gene directly into the
part of the body that has defective cells.
- In gene therapy that is used to modify cells outside of the body, blood, bone
marrow, or another tissue can be taken from a patient, and specific types of cells
can be separated out in the lab. The vector containing the desired gene is intoduced
into these cells. The cells are left, to multiply in the laboratory, and are then injected
back into the patient, where they continue to multiply and eventually produce the
desired effect.
11. What are the characteristics of animal cell in cultures?
- Weak mechanical properties
Animal cells have weak mechanical properties, are very vulnerable to external
forces. Therefore, in the in vitro culture process, animal cells are very fragile when
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Question and Answer for Animal Cell Technology K61CNSHE

mixing, separating cells... Time to conduct operations with animal cells should also
try to be the shortest. In preserving and moving cell samples, it is also necessary to
be gentle.
- Growth and division slowly
Time to double the number of animal cells in physiological conditions is 20-40
hours. The mammalian cells have the shortest cycle in the culture medium is
intestinal carcinoma cells - about 9 to 10 hours. And stem cells in rat skin may
have a cycle of up to 200 hours.
- Negative feed-back mechanism
The mechanism of inhibiting the synthesis and secretion of a substance in the
environment, will be carried out by the increase of its concentration in the
environment. When cultured in the laboratory, the culture medium is fixed, the
concentration of some substances secreted by the cell is very high, leading to
negative feed-back. Negative feed-back mechanism can also cause cell damage,
even mass deaths. Therefore, it is necessary to replace the environment after a
certain culture period
- Need a rack to live and divide
Most animal tissues and cells need to cling to the rack to live and divide. Except
for blood cells and some stages of sexual cells. Normally cells grow well when
attached to solid surfaces. However, some cell lines such as cancer cells, or
continued cell lines from normal tissue can grow and divide in a suspended state,
without clinging to the substrate.
- Change genotype and phenotype
Animal cell can change genotype and phenotype through combining two cells with
different nucleus, forming hybrid cell or transformation process.
- Can be preserved long-term by deep cold method
Animal cell lines can be stored in liquid nitrogen (-196oC) for many years. When
activated, the cell restores its original growth and division.
12. Description about the basic concepts of cell lines?
- Cell line is the term used to refer to an identical cell population originating from
an original cell.
- The continued cell line (established cell line) is a cell line cultured in in vitro
conditions for many generations and can maintain cell division for a very long
time, sometimes permanently long without changing properties.
- Temporary cell line is a cell line that is only capable of living in culture conditions
at a limited time. These are usually normal cell lines (not cancer).
- Primary cells are the cells cultured in the first in vitro conditions (primary culture)
after being separated from the tissue.
- Secondary cells are cells that have passed through several subcultures (secondary
culture) after being separated from the tissue.

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Question and Answer for Animal Cell Technology K61CNSHE

13.What is name of the level culture? What are advantage and disadvantage
of the cell culture?
- There are three level cultures: organ culture, tissue culture, and cell culture
- The advantage and disadvantage of cell culture

Advantage Disadvantage
Cell lines often differ genetically and For primary cells, however, high serum
phenotypically from their tissue origin, levels can lead to differentiation or
whereas primary cells maintain many of promote growth of contaminating cells
the important markers and functions like fibroblasts. In addition, serum is
seen in vivo plagued by rising costs and lot to lot
variability

Cell culture studies provide a valuable


complement to in vivo experiments,
allowing for a more controlled
manipulation of cellular functions and
processes

14. What are the requirements for designing the laboratory and describe
these requirements?
- Number of users.
• How many people will work in the facility?
• These considerations determine how many laminar-flow hoods will be
required and whether a large area will be needed to handle bioreactors,
animal tissue dissections, or large numbers of cultures.
• The laboratories usually have 3 different sizes:
 The large laboratory is suitable for 20 to 30 persons.
 The medium is suitable for 5 to 6 persons.
 The small laboratory is suitable for 2 to 3 persons.
- Space.
• First, the largest area should be given to the culture operation. After that, the
second largest is for washup, preparation, and sterilization. The third is for
storage. Last but not least, the fourth is for incubation.
- Location of preparation area.
• Facilities for washing up and for sterilization should be located:
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Question and Answer for Animal Cell Technology K61CNSHE

 Close to the aseptic area that they service.


 On an outside wall to allow for the possibility of heat extraction from
ovens and steam vents from autoclaves.
- Storage.
• What is the scale of the work contemplated and how much storage space will
this require for disposable plastics, and so on?
• What proportion of the work will be cell line work, with its requirement for
storage in liquid nitrogen?
- Access.
• Make sure that doorways are wide enough and high enough and that ceilings
have sufficient clearance to allow the installation of equipment such as
laminar-flow hoods, incubators, and autoclaves.
- Containment and sterility.
• Containment requires that the contents of the tissue culture room not escape
to adjacent work areas.
• Sterility requires that none of the contamination of surrounding areas enters
the tissue culture.
15. What are the components of an autoclave and how does it work?
- Definition:
Sterilizer autoclaves are equipment used to sterilize and disinfect medical
laboratory instruments including: hospitals, microbiology, pharmaceutical research
laboratories and specialized laboratories that should be done daily, often to avoid
bacteria that can spread. There are many ways to disinfect tools such as using heat,
chemicals ... Heat sterilization methods use autoclaves with saturated steam under
high pressure. Sterilized autoclaves are widely used equipment.
- Principle of operation:
An autoclave is a pressure tank that operates according to the evaporation principle
of the protoplasm, which leads to breaking the shell to kill microorganisms.
Especially when we increase the temperature suddenly, the evaporation process
quickly breaks the shell immediately. And at a temperature of about 121oC for 5 -
15 minutes, the bacteria will be killed under the action of saturated steam under
high pressure.
- Function:
Sterilized autoclaves are used to kill bacteria and viruses. Thanks to the pressure
and the superheated steam, the autoclave will destroy any microorganisms on metal
tools, allowing the reuse of these tools without the risk of infection. Research and
medical laboratories also use autoclaves to disinfect the environment before
culturing bacteria in experiments.

16. Description about influences of environmental conditions on animal cell


culture?
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Question and Answer for Animal Cell Technology K61CNSHE

There are four factors:


- Temperature
• 37 °C is the optimal temperature for the development of most cell lines taken
from humans and mammals (Cells will die after rising temperature a few
hours, or very quick. However, cells are able to withstand temperatures
lower than their optimal temperature such as epithelial cells, sperm cells,
etc.).
• Avian cell lines grow well at 38.5℃.
• Insect cells grow at optimal temperatures of 26-28℃.
• While cold-blooded vertebrate cells normally grow well at 15-26℃.
=>Therefore, to maintain the culture temperature, incubators must often be
used.
- pH
• Optimal pH is critical to cellular function. Cultural media that are too acid or
base will reduce the growth of cells.
• Most mammalian cell lines proliferate at pH 7.4.
• Some normal fibroblasts proliferate well at a pH range between 7.4 and 7.7.
• Transformed cells have an optimal growth at a pH varying from 7.0 to 7.4.
• Insect cells show better proliferation at lower pH values, from 6.2 to 6.5.
=>Culture medium needs to be buffered to compensate for CO2 and lactic
acid derived from glucose metabolism. Most culture media employed for
animal cells are buffered with CO2 originating from the gaseous phase, in
equilibrium with sodium bicarbonate (NaHCO3) added to the culture
medium.
- Osmolality
• Most cell lines present a wide tolerance range to osmotic pressure. Given the
osmolality of human plasma (290 mOsm/kg), it may be reasonable to assume
that this is an optimal value for human cells cultivated in vitro.
• The optimal osmolality range for mammalian cells in culture is from 260 to
320 mOsm/kg.
• While for insect cells higher values are optimal, from 340 to 390 mOsm/kg.
=> Osmometer is essential device to check osmolality.
- Oxygen supply
• The most important components of the gaseous phase are oxygen and carbon
dioxide. Monitoring and controlling these gases in culture medium are
essential for in vitro animal cell culture.
• Oxygen is the important role in growth, proliferation and cell differentiation.
• Carbon dioxide concentration is used to be stable the pH medium
17. Description about cultural medium component?

14
Question and Answer for Animal Cell Technology K61CNSHE

The culture medium must contain nutrients essential to the synthesis of new cells
and substrates for accomplishing cell metabolism, besides compounds allowing
physiological and catalytic functions, or which act as cofactors.
To maintain the conditions found in the original tissue from which a particular cell
originated, it is necessary for a culture medium to contain: inorganic salts, sugars,
amino acids, vitamins, lipids, organic acids, proteins, hormones, carbon and
nitrogen sources, micronutrients (organic ions and minerals), and water as well as
cell-specific substances. Sometimes, blood serum and antibiotics are also required,
and in addition, the culture must be kept free from inhibiting or toxic compounds.
- Water
• One of the basic components of a culture medium, and also one of the most
critical, is water. Animal cells are extremely sensitive to water quality, since
this can be the source of contamination that can affect cell growth.
• Since the presence of any of these contaminants can prevent cell culture on
any scale, strict purity standards of water quality must be maintained.
• The water used in the preparation of industrial culture media should be
subjected to similar purification methods and should be continuously
monitored to ensure the physicochemical and microbio-logical standards
established by national pharmacopoeia.
• The standards for water employed for pharmaceutical use in different
countries both for water for injection (WFI) and for purified water (PW).
While PW is used for the preparation of products not requiring sterile or
apyrogenic water, WFI meets the standards for PW as well as for bacterial
endotoxins, conductivity, and total organic carbon.
- Glucose
• Glucose is usually the main carbohydrate for animal cell growth, acting as
source of both carbon and energy glucose is metabolized mainly through
glycolysis, forming pyruvate, which can be converted to lactate or acetyl that
can enter into the citric acid cycle, forming carbon dioxide and water.
- Amino acids
• Amino acids are necessary for protein, nucleotide, and lipid synthesis and, in
addition, may be used as an energy source.
• These compounds can be provided as a defined mixture or in the form of
protein hydrolysates, such as lactoalbumin, or plant-derived hydroly-sates
- Vitamins
• Vitamins are used in very low amounts as enzyme cofactors, essential for
general cell metabolism.
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Question and Answer for Animal Cell Technology K61CNSHE

- Salts
• The salts most commonly added to the culture medium are Naþ, Kþ, Mg2þ,
Ca2þ, Cl –, SO42–, PO 43–, and HCO3–. These ions are important in the
maintenance of the ionic balance and osmotic pressure, besides acting as
enzymatic cofactors.
• Salts are the components that contribute most to the increase in culture
medium osmolality.
- Serum
• Blood serum, usually bovine-derived (calf or fetal bovine), contains amino
acids, growth factors, vitamins, proteins, hormones, lipids, and minerals,
among other components.
• The main functions of serum are to stimulate growth and other cellular
activities through hormones and growth factors, to increase
• Cellular adhesion through specific proteins, and to supply proteins for the
transport of hormones, minerals, and lipids.
• Buffer system:
 Regulating pH in culture can be done by: the "natural" buffer system
of CO2 with CO32- / HCO3- in the environment and the chemical
buffer system using HEPES.
 Most media use bicarbonate buffers with CO2 as the main component.
In addition, there is a phosphate buffer system and other complex
organic cushions. Organic or serum buffers may also be used in a basic
environment.
 When using bicarbonate as the main buffer system in the environment,
the interaction of CO2 collected from the cells (or from the air) with
water, will lead to the adjustment of the environmental pH in
equilibrium of the equation:
H2O + CO2 = H2CO3 = H+ + HCO 3-
 Using bicarbonate / CO2 buffer system needs to maintain air with 5-
10% CO2 in the cabinet
 The most used organic buffer system is HEPES (N-2
hydroxyethylpiperazine-N-2-ethane sulphonic acid).They are not
sensitive to CO2, they provide a good system for strong metabolic cells
(producing lots of CO2) to stabilize pH.
 HEPES buffer:
 pH from 7.2 to 7.4
 Relatively expensive
 Toxic to some cells in high concentrations
 HEPES buffer does not need CO2 phase
16
Question and Answer for Animal Cell Technology K61CNSHE

 Most resistant cell lines are buffered with HEPES in the range
of 10-15 mM, or even higher
 It should only be used at a concentration of 20 mM
- Other components necessary for cell culture
Besides the compounds already mentioned, some adherent cell lines need proteins of
the extracellular matrix (ECM) for efficient adherence to the support and for cell
growth. Many ECM proteins, such as fibronectin, are present in serum, while others,
such as collagen, are secreted by cells constitutively or after their stimulation with a
growth factor. Another ECM protein, laminin, can be used as an alternative to
fibronectin, especially for epithelial cell cultures. Growth factors, present in very low
amounts, are usually small peptides.
18.Description about advantages and disadvantages of media which have
and don’t have serum?
Serum No serum
Provide hormone and essential growth Add hormone growth factor
factors for cell function

Provide adherent factor Cover petri dish by adherent factor or


polylysine

Act as a buffer pH Use buffer organics


Close, inactivated or release toxic as Use fresh water and prepare fresh media,
organic compounds which attach metal… add albumin

Contain protein which stick stabilize, help Add sticky protein as transferring
to transfer hormones, nutrients in cell eruloplasm…

Provide nutrients Use complex medium


Contain protease inhibitor Use trysin inhibitor when transplant and
add protease suitable inhibitor

Contain differentiation factors Add or don’t regulate growth cell and


differentiation

17
Question and Answer for Animal Cell Technology K61CNSHE

Contain factor which cause aging in Don’t effect in weak old cell, continuous
mouse normal cells growth cells

Comfortable for many growth type of Can select considerable type of cell
cell, including fibroblast cell

19. Description about techniques to prepare animal cell culture media?


- Main components of animal cell culture media
The culture medium must contain nutrients essential to the synthesis of new cells
and substrates for accomplishing cell metabolism, besides compounds allowing
physiological and catalytic functions, or which act as cofactors.
To maintain the conditions found in the original tissue from which a particular cell
originated, it is necessary for a culture medium to contain: inorganic salts, sugars,
amino acids, vitamins, lipids, organic acids, proteins, hormones, carbon and
nitrogen sources, micronutrients (organic ions and minerals), and water as well as
cell-specific substances. Sometimes, blood serum and antibiotics are also required,
and in addition, the culture must be kept free from inhibiting or toxic compounds.
- Water
• One of the basic components of a culture medium, and also one of the most
critical, is water. Animal cells are extremely sensitive to water quality, since
this can be the source of contamination that can affect cell growth.
• Since the presence of any of these contaminants can prevent cell culture on
any scale, strict purity standards of water quality must be maintained.
• The water used in the preparation of industrial culture media should be
subjected to similar purification methods and should be continuously
monitored to ensure the physicochemical and microbio-logical standards
established by national pharmacopoeia.
• The standards for water employed for pharmaceutical use in different
countries both for water for injection (WFI) and for purified water (PW).
While PW is used for the preparation of products not requiring sterile or
apyrogenic water, WFI meets the standards for PW as well as for bacterial
endotoxins, conductivity, and total organic carbon.
- Glucose
• Glucose is usually the main carbohydrate for animal cell growth, acting as
source of both carbon and energy glucose is metabolized mainly through
glycolysis, forming pyruvate, which can be converted to lactate or acetyl that
can enter into the citric acid cycle, forming carbon dioxide and water.
- Amino acids
• Amino acids are necessary for protein, nucleotide, and lipid synthesis and, in
addition, may be used as an energy source.

18
Question and Answer for Animal Cell Technology K61CNSHE

• These compounds can be provided as a defined mixture or in the form of


protein hydrolysates, such as lactoalbumin, or plant-derived hydroly-sates
- Vitamins
• Vitamins are used in very low amounts as enzyme cofactors, essential for
general cell metabolism.
- Salts
• The salts most commonly added to the culture medium are Naþ, Kþ, Mg2þ,
Ca2þ, Cl –, SO42–, PO 43–, and HCO3–. These ions are important in the
maintenance of the ionic balance and osmotic pressure, besides acting as
enzymatic cofactors.
• Salts are the components that contribute most to the increase in culture
medium osmolality.
- Serum
• Blood serum, usually bovine-derived (calf or fetal bovine), contains amino
acids, growth factors, vitamins, proteins, hormones, lipids, and minerals,
among other components.
• The main functions of serum are to stimulate growth and other cellular
activities through hormones and growth factors, to increase cellular adhesion
through specific proteins, and to supply proteins for the transport of
hormones, minerals, and lipids.
• Buffer system
- Other components necessary for cell culture
- Conditions culture
Temperature varies on the type of host cell. Most mammalian cells are maintained
at 37oC for optimal growth, while cells derived from cold- blooded animals tolerate
a wider temperature range (i.e. 15oC to 26oC). Actively growing cells of log phage
should be used which divide rapidly during culture.
The concentration of CO2 in the cabinet is usually 5%, especially when up to 95%.
Sterile is an extremely important factor in animal cell culture.
 There are two methods of disinfection commonly used when the
environmental phase: wet and filtered by millipore.
 Depending on the chemical nature of the environmental components to
choose the appropriate disinfection method.
 With heat-resistant substances, it is necessary to use wet steaming method,
whereas sterile filtration method is used with substances that are easily
modified by temperature such as antibiotics, amino acids ...
20. Description about the notices when you prepare culture media?
- Weigh accurate each component of media according to the formula
- Filter media
- Adjust environmental pH
- Disinfect the media
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Question and Answer for Animal Cell Technology K61CNSHE

• Disinfection by Pasteur method


• Disinfection by Tyldal method
• Sterilize tape pressure steamer
21.What is the MTT protocol?
- MTT assay: a colorimetric assay for assessing cell metabolic activity.
- MTT protocol : There are 6 steps for carrying out this assay
• Step1: Discard media from cell cultures:
 For adherent cells, carefully aspirate the media cells.
 For suspension cells, spin the 96 well plate at 1,000 xg, 4°C for 5
minutes in a microplate-compatible centrifuge and carefully aspirate
the media.
• Step2: Add 50 µL of serum-free media and 50 µL of MTT solution into each
well.
• Step3: Incubate the plates at 37°C for 3 hours.
• Step4: After incubation, add 150 µL of MTT solvent into each well.
• Step5: Wrap plate in foil and shake on an orbital shaker for 15 minutes.
Occasionally, pipetting of the liquid may be required to fully dissolve the
MTT formazan.
• Step 6: Read plate within 1 hour. Read absorbance at OD=590 nm.

22. What is plating efficiency and its formula


- Definition: Plating efficiency (PE) is a measure of the number
of colonies originating from single cells. It is a very sensitive test and is often used
for determining the nutritional requirements of cells, testing serum lots, measuring
the effects of growth factors, and for toxicity testing.
• Plating Efficiency is the number of cells that grow into colonies per 100 cells
inoculated.
• PE can be determined by the following formula:

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑜𝑛 𝑑𝑎𝑦 1


PE = ⅹ 100%
𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑝𝑙𝑎𝑡𝑒𝑑 𝑜𝑛 𝑑𝑎𝑦 0

20
Question and Answer for Animal Cell Technology K61CNSHE

23. Describe the common features of disaggregation? How many kinds of


enzymes used in disaggregation?
- Although each tissue may require a different set of conditions, certain
requirements are shared by most of them:
• Fat and necrotic tissues are best removed during dissection.
• The tissue should be chopped finely with sharp scalpels to cause minimum
damage.
• Enzymes used for disaggregation should be removed subsequently by gentle
centrifugation.
• The concentration of cells in the primary culture should be much higher than
that normally used for subculture because the proportion of cells from the
tissue that survives in primary culture may be quite low.
• A rich medium, such as if serum is required, fetal bovine often gives better
survival than does calf or horse.
• Embryonic tissue disaggregates more readily, yields more viable cells, and
proliferates more rapidly in primary culture than does adult tissue.

- The enzymes used most frequently for tissue disaggregation:


• Nonmammalian enzymes, such as Trypzean (Sigma).
• Crude preparations are often more successful than purified enzyme
preparations. Example: Trypsin and pronase give the most complete
disaggregation but may damage the cells.
24. Perform protocol of isolation chick embryos?
- Procedure
• Step 1. Incubate the eggs at 38.5◦C in a humid atmosphere, and turn the eggs
through 180◦ daily. Although hens’ eggs hatch at around 20 to 21 days, the
lengths of their developmental stages are different from those of mouse
embryos. For a culture of dispersed cells from the whole embryo, the egg
should be taken at about 8 days, and for isolated-organ rudiments, at about
10 to 13 days.
• Step 2. Swab the egg with 70% alcohol, and place it with its blunt end facing
up in a small beaker
• Step 3. Crack the top of the shell, and peel the shell off to the edge of the air
sac with sterile forceps
• Step 4. Resterilize the and then use the forceps to peel off the white shell
membrane to reveal the chorioallantoic membrane (CAM) below, with its
blood vessels
• Step 5. Pierce the CAM with sterile curved forceps, and lift out the embryo
by grasping it gently under the head. Do not close the forceps completely, or
else the neck will sever; place the middle digit under the forceps and use the
finger pad to restrict the pressure of the forefinger
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Question and Answer for Animal Cell Technology K61CNSHE

• Step 6. Transfer the embryo to a 9-cm Petri dish containing 20-mL DBSS.
25. What is the growth curve? The main phage of the growth curve?
- The growth curve was established to analyze the developmental characteristics of
cell types. Microbial population growth was studied by analyzing the growth curve
in medium in batch culture or a closed system. The increase in the number of cells
is used as a method of assessing the effects of hormones, nutrients and other factors
on a specific cell type. It is the best measure of biological response, because it is
determined and influenced by many different factors, including substances that
cause mitosis, changes in nutritional level, transport, membrane association,
adhesion factors and some other factors.
• Lag phage: This time, the cells recover from transplants, adhesion and
spread. In this stage the cells are not cleaved, but the volume and increase
markedly due to the increase in new components of the cell
• Log phage: In this stage, microorganisms grow and cleave maximum rate
with their genetic nature if they have the appropriate conditions and nutrient.
• Stationary and Death phage: After log phage, the growth of the population
will stop, the growth curve tends to go sideways or go down. The main reason
is the limitation of nutrients, oxygen.
26. What are the advantages and disadvantages of serum - free media?
- Advantages of serum - free media
• Definition of Standard Medium: Given pure constituents are used, a given
medium formulation can be standardized regardless of where it is used and by
whom.
• Selective Media: One of the major advantages of the control over growth-
promoting activity afforded by serum-free media is the ability to make a
medium selective for a particular cell type. (E.g. MCDB 153 for epidermal
keratinocytes, LHC-9 for bronchial epithelium…)
• Example:
 Melanocytes can be cultivated in the absence of fibroblasts and
keratinocytes.
 Separate lineages and even stages of development may be selected in
hematopoietic cells by choosing the correct growth factor or group of
growth factors.
• Regulation of Proliferation and Differentiation: add to the ability to select for a
specific cell type the possibility of switching from a growth-enhancing medium
for propagation to a differentiation-inducing medium by altering the
concentration and types of growth factors and other inducers.
- Disadvantages of serum - free media
• Multiplicity of media: Each cell type appears to require a different recipe, and
cultures. So, it presents a problem for laboratories initiating or maintaining cell
lines of several different origins.
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Question and Answer for Animal Cell Technology K61CNSHE

• Reagent purity: The native serum does possess some amount of protective and
detoxifying machinery that can offer a cleansing effect on the apparatus and
reagents. Therefore, in the absence of serum, pure grade reagents and completely
sterile apparatus should be used.
• Cell proliferation: Growth is often slower in serum-free media, and fewer
generations are achieved with finite cell lines types.
• Selectivity: Some media may select a sublineage that is not typical of the whole
population, and even in continuous cell lines, some degree of selection may still
be required.
27. Describe the components of serum-free subculture?
Serum-free subculture consist of:
- Adhesion factors
• When serum is removed, it may be necessary to treat the plastic growth surface
with fibronectin (25–50 μg/mL) or laminin (1–5 μg/mL), added directly or via
the medium
• Pretreating the plastic with poly-D-lysine (1 mg/mL) was shown to enhance the
survival of human diploid fibroblasts.
- Protease inhibitors
• After trypsin-mediated subculture, the addition of serum inhibits any residual
proteolytic activity.
• Consequently, protease inhibitors such as soya bean trypsin inhibitor or 0.1
mg/mL aprotinin (Sigma) must be added to serum-free media after subculture.
• Furthermore, because crude trypsin is a complex mixture of proteases, some of
which may require different inhibitors, it is preferable to use pure trypsin (e.g.,
Sigma Gr. III) followed by a trypsin inhibitor.
- Trypsin and other proteases
• As the cells are more fragile and may need purified crystalline trypsin, and to be
chilled to 4 ◦C, to reduce damage.
• Purified porcine trypsin may be replaced with recombinant trypsin
• Alternative sources of proteases are available to keep animal products from
coming in contact with cells.
• Non-mammalian proteases are also available (e.g., Accutase™ or Accumax™,
Sigma).
• Pronase, Dispase, Liberace (Roche), and collagenase are bacterial proteases not
neutralized by trypsin inhibitors and will require removal by centrifugation.
28.Describe the definition of the primary culture? How many stages are
there for primary culture?
Definition:
- Primary culture is that stage of the culture after isolation of the cells but before the
first subculture after which it becomes a cell line.

23
Question and Answer for Animal Cell Technology K61CNSHE

- In some cases they may be dissociated cells from a primary culture that will
become a cell line when the recipient seeds them into culture, but in other cases
they may be growing cultures already passaged from a primary culture and
technically a cell line (usually a finite cell line) or frozen cells from a primary
culture.
- There are four stages to consider
• Acquisition of the sample.
• Isolation of the tissue.
• Dissection and/or disaggregation.
• Culture after seeding into the culture vessel.
=> After isolation, a primary cell culture may be obtained either by allowing cells to
migrate out from fragments of tissue adhering to a suitable substrate or by
disaggregating the tissue mechanically or enzymatically to produce a suspension of
cells, some of which will ultimately attach to the substrate.
29. How does mechanical disaggregation proceed?
- The outgrowth of cells from primary explants is a relatively slow process and can
be highly selective.
- Enzymatic digestion is rather more labor intensive, although, potentially, it gives
a culture that is more representative of the cells in the tissue
=> As there is a risk of proteolytic damage to cells during enzymatic digestion,
many people have chosen to use mechanical disaggregation.
- Protocol (5 steps)
• Step 1. After washing and preliminary dissection of the tissue, chop the tissue
into pieces about 3 to 5 mm across.
• Step 2. Force the tissue through the mesh into the medium by applying gentle
pressure with the piston of a disposable plastic syring.
• Step 3: Pipette the partially disaggregated tissue from the Petri dish into a
sieve of finer porosity.
• Step 4. The suspension may be diluted and cultured at this stage. In general,
the more highly dispersed the cell suspension, the higher the shear stress
required and the lower the resulting viability.
• Step 5. Seed the culture flasks by diluting the cell suspension in medium

30. Describe the definition, objectives, protocol of collagenase


disaggregation?
- Definition:
• This technique is very simple and effective for many tissues: embryonic,
adult, normal, and malignant.
• It is of greatest benefit when the tissue is either too fibrous or too sensitive to
allow the successful use of trypsin.
- Objectives:
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Question and Answer for Animal Cell Technology K61CNSHE

• Place finely chopped tissue in a complete medium containing collagenase


and incubate.
• When tissue is disaggregated, remove collagenase by centrifugation, seed
cells at a high concentration, and culture.
- Protocol (7 steps)
• Step 1. Collect tissue sample in DBSS
• Step 2. Chop with crossed scalpels to 2–3 mm
• Step 3. Wash by resuspension and settling 2–3X
• Step 4. Incubate finely chopped pieces in collagenase in complete medium
• Step 5. Disperse by gentle pipetting
• Step 6. Transfer to tube, and allow to settle
• Step 7. Collect supernatant cells and centrifuge
=> Resuspend cells in growth medium and seed flasks.
31.What are limitations of traditional method culture?
- 2D cultured cells do not mimic the natural structures of tissues or tumours (In this
culture method, cell-cell and cell-extracellular environment interactions are not
represented as they would be in the tumour mass. The loss of diverse phenotype is
also a result of 2D culturing. The changed morphology of the cells can affect their
function, the organization of the structures inside the cell, secretion and cell
signaling
- Another drawback of 2D culture is that the cells in the monolayer have unlimited
access to the ingredients of the medium such as oxygen, nutrients, metabolites and
signal molecules. For cancer cells in vivo, the availability of nutrients, oxygen, and
so forth, is more variable because of the natural architecture of the tumour mass
- Furthermore, it has been observed that the 2D system changes the gene expression
and splicing, topology and biochemistry of the cell
- In addition, adherent cultures are usually monocultures and allow for the study of
only one cell type, which results in a lack of tumour microenvironment, or niches,
which in vivo are required by cancer-initiating cells.
32. What is the different between 2D and 3D cell culture?

25
Question and Answer for Animal Cell Technology K61CNSHE

2D vs. 3D 2D Cell Culture 3D Cell Culture


 Well established and fast  Substrates mimic the natural
for primary assessment extracellular matrix
 Economical  Mimic the in
vivo microenvironment, as well
 Easy and convenient for the
as cell-cell and cell-matrix
analysis set up
interactions
 High throughput capacity:
 Enable the assessment of tissue
Advantages feasible for mass screening
penetration ability and bystander
killing efficiency of drugs, as
well as the drug resistance of
cells.
 Low design-in vitro-in vivo-
redesign requirement: reduce
overall cost for the pre-
clinical in vivo test
 Stiff, plastic substrate  More expensive and laborious
for the establishment of culture
 Lack cell-cell & cell-matrix
models and set-up of analyzing
interaction information
experiments
 Unable to mimic in
 Relatively low throughput
vivo microenvironment
capacity
 Homogenous drug
Disadvantages distribution
 Unable to assess the tissue
penetration ability and
bystander killing efficiency
of drugs or the drug
resistance of cells
 High design-in vitro-in
vivo-redesign cycle
26
Question and Answer for Animal Cell Technology K61CNSHE

requirement: overall high


cost

33. What are the limitations of tissue engineering scaffold?


- Scaffold microenvironment is not ideal for tissue growth.
- The size and shape of the hole cannot be precisely controlled, the spatial distribution
of the holes and the structure of the scaffold internal channels
- Reduce the number of pioneering cells that migrate deep into the scaffold
- This method only produces in vitro growth cells with a surface area of less than
500µm from the surface. Almost all cells cannot migrate inside more than 500 µm
- The gas foaming method:
• Only 10-30% of pores are interconnected
• The mesh fibers have weak mechanical property Solvent backlog high risk
of toxins and)
- Solvent backlog high risk of toxins and cancer (except for air bubble method and
melting mold method).
- For skeletal tissue technology, fast metabolism and oxygen at the scaffold surface
stimulate the scaffold surface mineralization, limiting the transfer of mass into the
scaffold
- When cells have been distributed throughout large-scale scaffold, still need to
provide blood vessels to nourish cells deep inside the scaffold.
34. Describe the scaffolding properties have been defined has being
essential for three-dimensional bone cell culture techniques?
The following properties have been defined has being essential:
- Biocompatibility
27
Question and Answer for Animal Cell Technology K61CNSHE

Scaffolds should be well integrated in the host’s tissue without eliciting an immune
response.
- Porosity
Scaffolds must possess an open pore, fully interconnected geometry in a highly porous
structure with large surface to area volume ratios that will allow cell in-growth and an
accurate cell distribution throughout the porous structure, and will facilitate the
neovascularization of the construct from the surrounding tissue.
- Osteoinductivity
Osteoinduction is the process by which stem and osteoprogenitor cells are recruited to
a bone healing site, and stimulated to undergo the osteogenic differentiation pathway.
However, when the portion of bone to regenerate is large, natural osteoinduction
combined with a biodegradable scaffold may be not enough. Because of this the
scaffold should be osteoinductive by itself.
- Mechanical Properties and Biodegradability
In vitro, the scaffolds should have sufficient mechanical strength to withstand the
hydrostatic pressures and to maintain the spaces required for cell in-growth and matrix
production.
35. Describe about gas foaming method to fabricate scaffolds. What are the
advantages and disadvantages of this method?
- A “gas foaming” method was developed by Nam et al using an effervescent salt as
a gas foaming agent.
- Nam et al.168 synthesized PLA scaffolds using ammonium bicarbonate, which
acted as both a gas foaming agent and a solid salt porogen.
- Procedure:
• Sieved effervescent salt particles (ammonium bicarbonate) in the form of a
polymer gel.
• Casted in mold and subsequently immersed in hot water.
• Formed pores with high interconnectivity by the evolution of ammonia,
carbon dioxide and the leaching out of ammonium bicarbonate particles.
- Advantages:
• Gas foaming yields high porosities (up to 93%) and varying the temperature,
pressure.
• The significant advantage is no loss of bioactive molecules in the scaffold
matrix, given that there is no need for the leaching process and no residual
organic solvent.
- Disadvantages:

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Question and Answer for Animal Cell Technology K61CNSHE

• The presence of skimming film layers on the scaffold surface, resulting in


the process to remove this skin layer, and poor interconnectivity of the
porosity.
36. Describe about fibre meshes/ fire bonding method to fabricate scaffolds.
What are advantages and disadvantages of this method?
- The fiber bonding methods produce scaffold made of polyglycolic acid (PGA)
polymer which was available as sutures and thus in shape of long fiber.
- These fibers, if bonded together in three-dimensions, provide large surface area for
cell interaction and growth.
- Procedure:
• PGA fibers are immersed in a PLLA solution.
• When the solvent evaporates, the network of PGA fibers is embedded in
PLLA.
• The composite is then heated to above the melting temperature of both
polymers. The PLLA melts first and fills all voids left by the fibers.
• Fibers at the cross-points become "welded" (melted) together, forming a
highly porous foam.
• The PLLA is then removed by dissolution with methylene chloride.
- The main advantage of the fiber-bonding technique is the high surface area: volume
ratio, which makes them ideal for tissue engineering applications and high porosity,
which provides more surface area for cell attachment and sufficient space for the
regeneration of ECM (extracellular matrix).
- Disadvantages are poor mechanical integrity, residual organic solvents, lack of
structural stability
37. What is the Solid Freeform Fabrication and its function in Tissue
Engineering?
- Solid Freeform Fabrication: The technology transfer of solid freeform fabrication
(SFF) to tissue engineering may be the key to produce scaffolds with customised
external shape and predefined and reproducible internal morphology, which not
only can control pore size, porosity an pore distribution, but can also make
structures to increase the mass transport of oxygen and nutrients throughout the
scaffold.
• SFF is a manufacturing technique that produces complex 3D structures by
selectively adding materials
• SFF technologies involve building 3D objects using layered manufacturing
strategies.
• Although there are several commercial variants of SFF technology, the
general process involves producing a computer-generated model using
computer-aided design (CAD) software. This CAD model is then expressed
as a series of cross-sectional layers.

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Question and Answer for Animal Cell Technology K61CNSHE

• The data is then implemented to the SFF machine, which produces the
physical model. Starting from the bottom and building layers up, each newly
formed layer adheres to the previous. Each layer corresponds to a
crosssectional division. Post-processing may be required to remove
temporary support structures.
• Furthermore, data obtained from Computerised Tomoga raphy (CT) or
Magnetic Resonance Imaging (MRI) medical scans can be used to create a
customised CAD model and consequently a scaffold possessing the exact
external shape required to correct the damaged tissue site. Only the SFF
technologies that have been applied to fabrication of tissue engineering
scaffolds will be presented below. The reader is referred to Pham and Dimov
(2000) for a general review on SFF.
- The function of SFF in Tissue Engineering
• It can improve current scaffold design by controlling scaffold parameters
such as pore size, porosity and pore distribution, as well as incorporating an
artificial vascular system.
• The SFF has made it possible to fabricate scaffolds with very fine structures
and complex geometries using computer-aided design (CAD) data acquired
from medical images of patients.
• It can be used to fabricate customized scaffolds from medical image data
such as magnetic resonance imaging (MRI) and computerized tomography
(CT) because it is based on computer-aided design (CAD). In addition, it can
be used to fabricate scaffolds with high reproducibility. SFF is categorized
into stereolithography (SL), fused deposition modeling (FDM), and selective
laser sintering (SLS)
• The outer shape of the scaffold can be precisely reconstructed based on 3D
CAD data of the target tissue or organ, which are converted from medical CT
or MRI images.
• The layer-by-layer SFF enables control of the internal architecture of the
scaffold
38. What are the techniques of SFF in Tissuse Engineering and its
characteristics?
The techniques are mostly used for SFF:
- 3D printed poly (D,L-lactide-co-glycolide) (PLGA) scaffold
• 3DP was used to create an intricate network of channels running
longitudinally and radially through the length of the scaffold.
• In building scaffold-like structure where multiple internal channels are
present, there will be loose powder trapped inside the channels.
• In scaffolds used in a later study, the use of liquid CO2 extraction was found
to improve solvent removal; a residual chloroform of lower than 100 ppm
was achieved.
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Question and Answer for Animal Cell Technology K61CNSHE

- Stereolithography (SLA).
• Suspensions of alumina,silicon nitride and silica particles loaded in
UVphotocurable monomer were made using SLA.
• Curing of the monomer resulted in binding of ceramic particles to from a
green body. The binder was removed by pyrolysis and the ceramic parts
sintered.
- Fused deposition modeling (FDM)
• FDM has also been adapted for tissue engineering scaffold production.
• The technology applies the melt extrusion method to fabricate a tissue
scaffold, making use of a layer-by-layer thermoplastic polymer
• The main advantages of the FDM method are that it does not require a toxic
solvent and offers flexibility in material handling and processing.
• Scaffolds with 48 to 77% porosity were made from filaments in the diameter
of 260 to 370 mm and spaced at between 160 and 700 mm
- FDM- The extrusion of Polycaprolactone (PCL) filaments to produce
honeycomb-like scaffolds:
• PCL is typically used on FDM for tissue engineering scaffold fabrication.
Cell culturing of these PCL scaffolds showed that human fibroblasts
colonized the struts and bars and formed a cell-to-cell and cell-to-
extracellular matrix interconnective network throughout the entire 3D
honeycomb-like architecture.
- 3D plotter
• Scaffolds have been made from PLA, PLGA and PCL using this system.
However, probably the most attractive feature of the 3D Plotter is in the
production of hydrogel scaffolds.
• Scaffold hydrogel is a more comprehensive system, capable of creating hot
melt forms, forming solutions, pastes, polymer dispersions as well as reactive
monomers and oligomers.
• Hydrogel scaffolds are used in particular to provide bulk and mechanical
structures to a tissue construct, whether cells are suspended within or adhered
to the 3D hydrogel framework.
• The delicate hydrogel strands were supported by dispensing the agar in a
liquid medium with matched density and polarity.
• The agar solution was heated to 70oC and then dispensed into an aqueous
gelatin solution kept at 20o C.
• Gelation of the agar occurs to form a stable gel with some mechanical
integrity.
- A rapid prototyping robotic dispensing (RPBOD)
• The concentration of sodium hydroxide was identified as important in
controlling the adhesion between layers.

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Question and Answer for Animal Cell Technology K61CNSHE

• The scaffolds were then hydrated, frozen and freeze-dried. Prior to cell
culturing with osteogenic cells, the scaffolds were seeded with fibrin glue.

39. What is the spinner flask and its characteristics?


- The spinner flask is a glass vessel, usually intended to be used for replicate cultures.
Sizes range from a few milliliters to 20 L. The stirring speed is between 10 and 300
rpm. The first scale-up step for cells growing in suspension or adherent cells on
micro carriers is the spinner flask. It can potentially be used with other support
materials (scaffolds).
- Spinner flasks are used as bioreactors for growing suspension cultures in liquid
media. These flasks are stirred tank bioreactors, in which paddle mixing maintains
the cells in suspension and the fluid movement helps in mass transport of nutrients
and wastes. There are 1 or 2 screw buttons to convenient for adding cells, changing
the environment, or providing O2 and C02 , middle above button attachs the agitator
mount. There are many types of paddle, high stirring effect, and smooth with cells.

40. What are the differences between the batch cultivation and the
continuous cultivation?

Continuous cultivation Batch cultivation


A technique used to grow A technique used for the production of
microorganisms in a limited supply microbes or microbial products in which
of nutrients, which declines when nutrients are continuously supplied to the
these are used to, or some other fermenter
factor becomes limiting
A closed system An open system
Internal environment is changed Environment is not changed during the
with the progression of the fermentation process
fermentation process

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Question and Answer for Animal Cell Technology K61CNSHE

Nutrients are added at the beginning Nutrients are continuously added


of the process; thus they are a throughout the process,; thus they are not
limiting factor a limiting factor
Whole process is stopped when the Process continues and the products are
products are formed continuously removed from the
fermenter
Yield is low Yield is high
Turnover rate is low Turnover rate is high
Suitable for the production of Suitable for the production of primary
secondary metabolites such as metabolites such as organic acids and
antibiotics amino acids
Labor demand is less Labor demand is more
Chance of contamination is less Chance of contamination is more
Large fermenters are used Small fermenters are used

41.What is the major advantage of working in a laminar-flow hood?


Compare horizontal laminar flow hood with vertical laminar flow hood?
- The major advantage of working in a laminar-flow hood is that the work space is
protected from dust and contamination by constant, stable flow of filtered air
passing over the work surface.
• Choose a horizontal laminar flow hood if:
 Your application requires less turbulence on the work surface.
 You will be using primarily small utensils and equipment that will not
cause airflow disturbance.
 You need the most control over contamination - Hands and gloves are
positioned downstream of the sample in a horizontal flow, therefore
greater contamination protection is possible with horizontal laminar
flow.
• Choose a vertical laminar flow hood if:
 Your application necessitates large equipment be used on the work
surface.
 Your application requires that soldering fumes or fine powders be
used.

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Question and Answer for Animal Cell Technology K61CNSHE

 You need a taller, larger work space - Because the filters are generally
positioned on top of a vertical laminar flow hood, there is greater
height available for work surface.
42. When applying aseptic techniques, what should we pay attention to
regarding personal hygiene and reagents and media?
- Washing will moisten the hands and remove dry skin that would otherwise be likely
to blow onto your culture. Washing will also reduce loosely adherent
microorganisms, which are the greatest risk to your culture. Surgical gloves may be
worn and swabbed frequently, but it may be preferable to work without them (if no
hazard is involved) and retain the extra sensitivity that this allows.
- Reagents and media obtained from commercial suppliers will already have
undergone strict quality control to ensure that they are sterile, but the outside
surface of the bottle they come in is not. Some manufacturers supply bottles
wrapped in polyethylene, which keeps them clean and allows them to be placed in
a water bath to be warmed or thawed. The wrapping should be removed outside the
hood. Unwrapped bottles should be swabbed in 70% alcohol when they come from
the refrigerator or from a water bath.
43. Describe the wet heat method in sterilization?
- Heat treatment under moist or wet conditions is a commonly used method of
sterilization. Many microorganisms, including bacteria and viruses, are relatively
easily inactivated at temperatures of 60–80◦C under these conditions, and this
process is termed pasteurization. In fact, lower temperatures of 55–60◦C are often
adequate.
- However, spore-forming bacteria and prions are more resistant to heat.
Pasteurization is used in the food industry for treating certain food products. These
procedures will kill vegetative microorganisms and any viruses that are likely to be
present, but not bacterial endospores.
44. What is chemical sterilization?
- Fumigation
• Gases such as formaldehyde or ethylene oxide can be used for fumigation.
Both are effective against all types of microorganisms including viruses,
although bacterial endospores are somewhat more resistant in the case of
ethylene oxide. Their activity is greatest at higher temperatures, and humidity
levels of 75–100%. However, conditions that reduce the accessibility of the
microorganisms to the gas, for example being dried in organic or inorganic
material, will decrease the effectiveness of fumigation.
- Filtration
• Membrane filtration using filters with a porosity of 0.2μm will remove
bacteria and fungi, and is the method commonly used for sterilizing solutions
that cannot be sterilized by methods such as autoclaving. Uses in the cell

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Question and Answer for Animal Cell Technology K61CNSHE

culture laboratory may include sterilizing culture media, sera and other cell
culture supplements, and any biological products made by the cells.
45. Describe the protocol of animal cell observation?
- Place a drop of physiological saline on a clean microscopic slide (central part of the
slide)
- Smear the cotton swab on to the center (part containing the saline drop) of the clean
slide for about 4 seconds to get the cells on to the center of the slide
- Add a drop of methylene blue solution on to the smear and gently place a cover slip
on top (to cover the stain and the cells)
- Place the slide on the microscope for observation using 4 x or 10 x objective to find
the cells
- Once the cells have been found, they can then be viewed at higher magnification
46. Describe the protocol for microbial decontamination treatment?
- Step 1: Collect the contaminated medium carefully. If possible, the organism should
be tested for sensitivity to a range of individual antibiotics. If not, autoclave the
medium or add hypochlorite.
- Step 2: Wash the cells in DBSS (Hanks BSS without bicarbonate, with Penicillin,
Streptomycin, Amphotericin B and Kanamycin or Gentamy- cin). For monolayers,
rinse the culture 3 times with DBSS, trypsinize, then wash the cells twice more in
DBSS by centrifugation and resuspension. For suspension cultures, wash the
culture five times (in DBSS) by centrifugation and resuspension.
- Step 3: Reseed a fresh flask at the lowest reasonable seeding density, depending on
cell type.
- Step 4: Add high-antibiotic medium and change the culture every 2 days.
- Step 5: Subculture in a high-antibiotic medium.
- Step 6: Repeat Steps 1 to 4 for three subcultures.
- Step 7: Remove the antibiotics, and culture the cells without them for a further three
subcultures.
- Step 8: Recheck the cultures (phase-contrast microscopy, Hoechst staining).
- Step 9: Culture the cells for a further two months without antibiotics, and check to
make sure that all contamination has been eliminated.
47. What are the general removal methods for contaminated cultures?
- If only one culture is contaminated, discard that culture and the source material
used.
- If the contamination is widespread, decontaminate the equipment and discard the
stock solutions. If you identify the cause of contamination, you should first check
the potential roots or causes of the contamination: aseptic techniques used, the
medium and reagents, the hood (e.g., last filter/pressure check), the incubator, the
refrigerator, the pipettes and other tools, the laboratory coats, the introduction of a
new cell line, the quality of the water, the autoclave, plastic disposable items
(pipettes, Petri plates, tips, etc). If the problem is affecting other people, check and
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Question and Answer for Animal Cell Technology K61CNSHE

decontaminate shared facilities (temperature, CO2, humidity, new plastic


disposables) and reagents (pH, improperly filtered water, new cell batch).
48. How can the cells be stored in cryopreservation?
- The temperature at which frozen cells are stored will affect their viability. Storage
at -80°C may permit slow chemical reactions (due to small amounts of unfrozen
water), which will eventually result in cell death. A temperature of less than -130°C
is required to completely stabilize cell preparations. This is usually achieved by
storage in liquid nitrogen (-196°C), liquid nitrogen vapor, or in a cryogenic freezer
(-150°C). All three methods are used with each presenting its own strengths.
- Liquid nitrogen is a non-mechanical method of cryopreserving cells. A large
thermos-like container is used to house either racks or sleeves that hold cryogenic
vials. Cells stored in nitrogen can be placed above the liquid in a cold vapor phase
or in the liquid nitrogen itself (-196°C).
- Cryogenic freezers are an alternative to the traditional methods of cryopreserving
animal cells. Cryogenic freezers use high efficiency compressors to reach
temperatures as low as -150°C.
49. How can the cells be recovered from frozen state?
- Unlike the freezing process, rapid thawing of frozen cells is necessary to maintain
viability. Certain precautions should be exercised when thawing cells. Vials stored
in liquid nitrogen, especially screw capped tubes, often fill with liquid nitrogen
while submersed. When these tubes are removed from the tank, the tubes may
pressurize and burst. Thus, a face shield or goggles should be worn while thawing
cells. Vials stored in cryogenic freezers are a reduced risk of bursting. Directly after
removal from storage, vials should be thawed with agitation (but not for fragile
hybridoma cells) in a 37°C water bath. As the last ice crystals are melting, the vial
is removed from the water. Wipe, spray, or submerse the vial with 70% ethanol
before opening it in a biosafety hood.
50. Describe the thawing protocol?

- Remove vials from the cryogenic freezer. If cells are stored in liquid nitrogen, use
tongs and insulated gloves, keeping in mind that pressure will build up inside the
vials as the nitrogen expands into a gas. The vial may shatter, thus wear goggles
and a lab coat.
- Thaw in a 37°C water bath with constant gentle shaking until completely thawed
(<1 minute). Carefully observe whether the glass vials have cracked during freezing
or thawing.
- Wash the vial with 70% ethanol. Open the cryogenic tube or snap the ampoule
aseptically. Plate cells immediately into pre-warmed medium.
- After attachment of monolayer cells, usually 1 to 10 hours, change the medium to
remove the cryoprotectant. If the cells are non-adherent, allow a sufficient recovery

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Question and Answer for Animal Cell Technology K61CNSHE

time (about 6 hours), then gently pellet (5 minutes at 400 X g) and resuspend in
fresh medium.

51.What is the application of animal cell in biological compounds


production?
- Virus vaccine
• The vaccine helps promote the immune system of animals and humans to
produce antibodies against pathogens.
• The vaccine produced from animal cells has contributed significantly to
modern medicine, typically in preventing smallpox and poliomyelitis.
- Production of interferon
• Interferon helps prevent virus infection in surrounding cells or inhibits the
growth of tumors …
• Nowadays, people produce IFN by recombining in E.coli cells - In the
interferon people divided into 2 groups:
 Acid-stable group I, including 3 types of interferon alpha, beta and
omega
 Group II is less stable in acid, including 1 type of gamma interferon.
- Monoclonal antibodies
• Combining two types of cells: lymphocytes B and cancer cells forming
hybrid cells. The hybrid cells have the ability to produce antibodies and
continuous division in culture in vitro.
52. What is the application of animal cell in the production virus kill
insects?
- Baculoviruses are arthropod‐specific, enveloped viruses with circular, supercoiled
double‐ stranded DNA genomes.
- While many viruses are studied because of their damaging effects, the study of
baculoviruses was stimulated by their potential utility to control insect pests.
- Later, the utility of baculovirus as gene expression vectors was evidenced and a
new research area emerged.
- Insecticide viruses can be produced by cultivating insect cells and for viral
infection, then collecting inoculants.
- Now, the use of the expression system by to Bacolovirus has been able to produce
insecticidal virus through insect cell culture.
- Eg: Bio-pesticide for Baculovirus
• In recent years several systems were developed with the goal of producing
viral proteins. The vaccine against hepatitis B is an important milestone in
the use of VLPs as a vaccination strategy, leading to replacement of the
previously existing inactivated virus vaccine.
53. Introduce briefly about creating organ from animal cells culture?
- Use of cultured animal cells for organ formation is increasingly popular trend.
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Question and Answer for Animal Cell Technology K61CNSHE

- The biggest prospect in this area is the use of embryonic stem cells pregnancy
- For example:
• Successful production of natural blood vessels by culturing cow's aorta cells,
including muscle cells, endothelial cells on a polymer substrate.
• Use of cells as replacement tissues and organs. Artificial skin for use in
treating burns and ulcers is the first commercially available product
- Current methods proposed for in vitro hematopoietic stem cell expansion still
represent a complex problem, since induction of proliferation is associated with the
loss of stemness. By contrast, skin therapies are performed with a small healthy
biopsy from the patient, since dissociated, isolated cells can be expanded ex vivo.
These expanded cells are useful for regeneration or fulfilling other therapeutic
procedures.
54. What is the application of animal cell culture in livestock-poultry
farming industry?
- One of the important impacts is improving animal genetics. Results in genetic
improvement through three main ways are as follows:
• Application of assisted reproductive technology: Biotechnology techniques
can affect reproductive efficiency and thus affect selection programs such as
insemination, IVF, ICSI, embryo culture, embryo transfer, gender selection.
Calculating, creating lines and applying stem cells.
• Application of genome research results: Biotechnology techniques can
improve the identification of genetic values of animals such as genetic
markers, candidate genes (selection) and related techniques.
• Application of genetic techniques: Biotechnology can transform artificial
genomes at DNA level such as genetic engineering, gene transfer and related
techniques.
55. What is the process of pumping sperm into the uterus ovary in
intrauterine insemination (IUI) method?
- When the follicle has matured, it means that the egg is enough to conceive and will
be injected with ovulation according to the doctor's appointment and then return the
sperm after 35-40 hours.
- After being pumped, many women will have reactions to stimulant drugs that make
the stomach bulge or not urinate. Drinking plenty of water to help the body stabilize
again, monitor the feeling.
- After a sperm pump, the wife can take a break and leave the hospital only 30
minutes later. Note that it is necessary to limit heavy work so that health effects are
weak.
- 2 weeks after sperm pumping is a golden time to receive results. Tests are conducted
to check the health and condition of the fetus if the wife has successfully conceived.
On the contrary, if the first IUI fails, you can continue to retry IUI for the second
time after 2-3 months.
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Question and Answer for Animal Cell Technology K61CNSHE

56. What is genetic marker in biotechnology in Animal Breeding?


- A genetic marker is a gene or DNA sequence with a known location on a
chromosome and associated with a particular gene or trait. It can be described as a
variation, which may arise due to mutation or alteration in the genomic loci that can
be observed.
- A genetic marker may be a short DNA sequence, such as a sequence surrounding a
single base-pair change (single nucleotide polymorphism, SNP), or a long one, like
mini & micro satellites
- The term “Smart Breeding” is used to describe marker supported breeding
strategies.
- The main aim of breeder is to select animal with superior genetic potential as a
parent for the next generation.
• The first attempt to improve animals used the phenotype of animal for a
specific trait as a tool for selection.
• The second type of markers represent by using of cytological markers.
• The third type of markers is biochemical markers such as blood type and
isozymes.
• The fourth type is molecular markers are based on the nucleotide sequence.
- DNA markers can be divided into the following groups:
• Classic Marker: RFLP and mtDNA.
• PCR-based marker.
• Hybridization based marker.
• Sequencing based marker.
57. What are the advantages of QTL / ETL in breeding selection programs?
- Accurately increase selection through additional information directly related to
genotype.
- The ability to reduce generation gap by adding an early selective state, because
QTL / ETL allows observations that are not related to sex, depending on the age of
the animal.
- The final applications of molecular genetics in the breeding program depend on
development in the following four important areas:
• Molecular genetics: identifying and mapping genetic and genetic
polymorphisms.
• Detection of QTL / ETL: helps detect and estimate the combination of
identified traits and genetic markers with economic traits.
• Genetic assessment: combination of phenotype and genotypic data in
statistical methods, to estimate breeding values of individual animals in a
breeding population.
• Selection combined with markers: development of breeding strategies and
programs using genetic information in selection and mating programs.
58. How the gene transfer method applies in cow?
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Question and Answer for Animal Cell Technology K61CNSHE

- The following basic steps:


• Collect eggs from slaughtered cows.
• Breeding ripe eggs.
• In vitro fertilization with bulls semen.
• Mindful, filtering fertilized eggs to concentrate yolk (inside Normal yolk
eggs prevent male predecessors from getting ready).
• Insert DNA into male precursor by injecting.Breeding eggs to blastocyst
stage.
• Embryo transfer.
• DNA screening of offspring to identify individuals carrying transgene.
59. Definition and basic procedure of transgenic in aquaculture?
- Transgenic fish are produced by the artificial transfer of rearranged genes into
newly fertilized eggs. Currently microinjection is the preferred method, although
the integration rates of transgenes are generally low.
- The basic procedure to generate transgenic fish for aquaculture includes:
• Design and construction of transgenic DNA;
• Transfer of the gene construct into fish germ cells;
• Screening for transgenic fish;
• Determination of transgene expression and phenotype;
• Study of inheritance;
• Selection of stable lines of transgenics.
60. What is the process Polyploidy in fish?
- Mollusc eggs are released, they are arrested at the prophase or metaphase of meiosis
I.
- Fish eggs are at the metaphase stage of Meiosis II on release. Further development
of the eggs is induced by the entry of the spermatozoon, leading to the resumption
of meiosis I selfish or of meiosis II in fish.
- Physical or chemical shock applied during meiosis I or meiosis II can suppress cell
division and prevent the extrusion of a polar body (either the first or the second in
the case of shellfish but only the second case in the case of fish), while allowing
chromosomal division, thus producing triploids.
- Preventing the extrusion of the first (shellfish) or the second (fish and shellfish)
polar body is thus key to be artificial induction of triploid.

40