The document provides a history of the development of animal cell culture from the late 19th century to the late 20th century. It describes key developments like the use of antibiotics to prevent contamination, techniques for subculture of adherent cells using trypsin, and the development of standardized culture media. The requirements for an animal cell culture laboratory are outlined, including sterile work areas, incubation facilities, microscopes, tissue culture supplies, and various equipment. The types of culture media, including natural and synthetic options, are also summarized.

1. B.Sc.III, Sem-VI:Unit-I
Animal Cell Culture: History, Types and Applications
Authored By: Dr. Rajendra Chavhan, Department of Zoology, RMG College Nagbhid
History of Animal Cell Culture:
Although animal cell culture was first successfully undertaken by Ross Harrison in 1907, it
was not until the late 1940’s to early 1950’s that several developments occurred that made cell
culture widely available as a tool for scientists.
First, there was the development of antibiotics that made it easier to avoid many of the
contamination problems that plagued earlier cell culture attempts.
Second was the development of the techniques, such as the use of trypsin to remove cells from
culture vessels, necessary to obtain continuously growing cell lines (such as HeLa cells).
Third, using these cell lines, scientists were able to develop standardized, chemically defined
culture media that made it far easier to grow cells.
Historical Events in the Development of Cell Culture:
1878:
Claude Bernard proposed that physiological systems of an organism can be maintained in a
living system after the death of an organism.
1885:
Roux maintained embryonic chick cells in a saline culture.
1897:
Loeb demonstrated the survival of cells isolated from blood and connective tissue in serum and
plasma.
1903:
Jolly observed cell division of salamander leucocytes in vitro.
1907:
Harrison cultivated frog nerve cells in a lymph clot held by the ‘hanging drop’ method and
observed the growth of nerve fibres in vitro for several weeks. He was considered by some as
the father of cell culture.
1910:
Burrows succeeded in long-term cultivation of chicken embryo cell in plasma clots. He made
detailed observation of mitosis.

2. 1911:
Lewis and Lewis made the first liquid media consisted of sea water, serum, embryo extract,
salts and peptones. They observed limited monolayer growth.
1913:
Carrel introduced strict aseptic techniques so that cells could be cultured for long periods.
1916:
Rous and Jones introduced proteolytic enzyme trypsin for the subculture of adherent cells.
1923:
Carrel and Baker developed ‘Carrel’ or T-flask as the first specifically designed cell culture
vessel. They employed microscopic evaluation of cells in culture.
1927:
Carrel and Rivera produced the first viral vaccine – Vaccinia.
1933:
Gey developed the roller tube technique.
1940s:
The use of the antibiotics penicillin and streptomycin in culture medium decreased the problem
of contamination in cell culture.
1948:
Earle isolated mouse L fibroblasts which formed clones from single cells. Fischer developed a
chemically defined medium, CMRL 1066.
1949:
Enders reported that polio virus could be grown on human embryonic cells in culture.
1952:
Gey established a continuous cell line from a human cervical carcinoma known as HeLa (Helen
Lane) cells. Dulbecco developed plaque assay for animal viruses using confluent monolayers
of cultured cells.
1954:
Abercrombie observed contract inhibition: motility of diploid cells in monolayer culture ceases
when contact is made with adjacent cells.

3. 1955:
Eagle studied the nutrient requirements of selected cells in culture and established the first
widely used chemically defined medium.
1961:
Hay flick and Moorhead isolated human fibroblasts (WI-38) and showed that they have a finite
life-span in culture.
1964:
Littlefield introduced the HAT medium for cell selection.
1965:
Ham introduced the first serum-free medium which was able to support the growth of some
cells.
1965:
Harris and Watkins were able to fuse human and mouse cells by the use of a virus.
1975:
Kohler and Milstein produced the first hybridoma capable of secreting a monoclonal antibody.
1978:
Sato established the basis for the development of serum-free media from cocktails of hormones
and growth factors.
1982:
Human insulin became the first recombinant protein to be licensed as a therapeutic agent.
1985:
Human growth hormones produced from recombinant bacteria was accepted for therapeutic
use.
1986:
Lymphoblastoidy γlFN licensed.
1987:
Tissue-type plasminogen activator (tPA) from recombinant animal cells became commercially
available.
1989:

4. Recombinant erythropoietin in trial.
1990:
Recombinant products in clinical trial (HBsAG, factor VIII, HIVgp120, CD4, GM-CSF, EGF,
mAbs, IL-2).
Requirements for Animal Cell Culture:
a. Sterile Work Area:
Where possible, a separate room should be made available for clean cell culture work. This
room should be free of “through traffic” and, if possible, equipped with an air flow cabinet
which supplies filtered air around the work surface.
A HEPA (High Efficiency Particle Air Filter) filtered air supply is desirable but not always
affordable. Primary animal tissue and micro-organisms must not be cultured in or near the cell
culture laboratory and the laboratory must be specifically designated for clean cell culture
work.
b. Incubation Facilities:
In addition to an airflow cabinet and benching which can be easily cleaned, the cell culture
laboratory will need to be furnished with an incubator or hot room to maintain the cells at 30-
40°C. The incubation temperature will depend on the type of cells being cultivated. Insect cells
will grow best at around 30°C while mammalian cells require a temperature of 37°C.
c. Refrigerators and Freezer:
Both items are very important for storage of liquid media at 4°C and for enzymes (e.g., trypsin)
and some media components (e.g., glutamine and serum) at -20°C. A refrigerator or cold room
is required to store medium and buffers.
A freezer will be needed for keeping pre aliquot stocks of serum, nutrients and antibiotics.
Reagents may be stored at a temperature of -20°C but if cells are to be preserved it may be
necessary to provide liquid nitrogen or a -70°C freezer.
d. Microscopes:
A simple inverted microscope is essential so that cultures can be examined in flasks and dishes.
It is vital to be able to recognize morphological changes in cultures since these may be the first
indication of deterioration of a culture.
A very simple light microscope with x 100 magnification will suffice for routine cell counts in
a haemocytometer, although a microscope of much better quality will be required for chro-
mosome analysis or autoradiography work.

5. e. Tissue Culture Ware:
A variety of tissue culture plastic-ware is available, the most common being specially treated
polystyrene. Although all tissue culture plastic-ware should support cell growth adequately, it
is essential when using a new supplier or type of dish to ensure that cultures grow happily in
it.
The tests to ensure this, such as growth curves and time of reaching a confluent monolayer, are
similar, to those used to ensure that serum batches are satisfactory. Cells can be maintained in
Petri dishes or flasks (25 cm2
or 75 cm2
) which have the added advantage that the flasks can
be gassed and then sealed so that a CO2 incubator need not be used. This is particularly useful
if incubators fail. Tissue culture ware is always chosen to match the procedure.
f. Washing Up and Sterilizing Facilities:
Availability of a wide range of plastic tissue culture reduces the amount of necessary washing
up. However, glassware such as pipettes should be soaked in a suitable detergent, then passed
through a stringent washing procedure with thorough soaking in distilled water prior to drying
and sterilizing.
Pipettes are often plugged with non-absorbent cotton wool before putting into containers for
sterilizing. Glassware, such as pipettes, conical flasks, beakers (covered with aluminum foil)
are sterilized in a hot air oven at 160°C for one hour.
All other equipment, such as automatic pipette tips and bottles (lids loosely attached) are
autoclaved at 121°C for 20 min. Sterilizing indicators such as sterile test strip are necessary for
each sterilizing batch to ensure that the machine is operating effectively. Autoclave bags are
available for loose items. Aluminum foil also makes good packaging material.
g. Liquid Nitrogen Deep Freezer:
Invariably for continuous and finite cell lines, samples of cultures will need to be frozen down
for storage. It is important to maintain continuity in cells to prevent genetic drift and to guard
against loss of the cell line through contamination and other disasters.
The procedure for freezing cells is general for all cells in culture. They should be frozen in
exponential phase of growth with a suitable preservative, usually dimethylsulfoxide (DMSO).
The cells are frozen slowly at 1°C/min to -50°C and then kept either at -196°C immersed in
liquid N2 (in sealed glass ampoules) or above the liquid surface in the gas phase (screw top
ampoules). Deterioration of frozen cells has been observed at -70°C, therefore, -196°C (liquid
N2) seems to be necessary.
To achieve slow freezing rates a programmable freezer or an adjustable neck plug or freezing
tray for use in a narrow-necked liquid nitrogen freezer can be used. Alternatively, ampoules

6. may be frozen in a polystyrene box with 1″ thick walls. This will insulate the ampoules to slow
the freezing process to 1°C/min in a -70°C freezer.
h. Water Still or Reverse Osmosis Apparatus:
A double distilled or reverse osmosis water supply is essential for preparation of media, and
rinsing glassware. The pH of the double distilled water should be regularly checked as in some
cases this can vary. Variations in the quality of water used may account for variation in results.
Therefore, water from one source should be used. Water is sterilized by autoclaving at 121°C
for 20 min. The distilled water must be glass distilled and stored in glass if it is to be used for
the preparation of media. Storage in plastic may result in leaching of toxic substances from the
plastic into the water.
i. Filter Sterilization:
Media that cannot be autoclaved must be sterilized through a 0.22 mm pore size membrane
filter. These are obtainable in various designs to allow a wide range of volumes to be filtered
(e.g., Millipore, Gelman).
They can be purchased as sterile, disposable filters, or they may be sterilized by autoclaving in
suitable filter holders. Culture media, enzymes, hormones, cofactors and bicarbonate buffers
are examples of non-autoclavable substances.
j. Other Requirements:
(a) Temperature:
In most of the mammalian cell cultures, the temperature is maintained at 37°C in the incubators
as the body temperature of Homo sapiens is 37°C.
(b) Culture Media:
The culture media is prepared in such a way that it provides:
a. The optimum conditions of factors like pH, osmotic pressure, etc.
b. It should contain chemical constituents which the cells or tissues are incapable of
synthesizing.
Generally the media is the mixture of in- Organic salts and other nutrients capable of sustaining
cells in culture such as amino acids, fatty acids, sugars, ions, trace elements,vitamins, cofactors,
and ions. Glucose is added as energy source—its concentration varies depending on the
requirement.

7. Phenol Red is added as a pH indicator of the medium. There are two types of media used
for culture of animal cells and tissues:
(a) Natural Media:
The natural media are the natural sources of nutrient sufficient for growth and proliferation of
animal cells and tissues.
The Natural Media used to Promote Cell Growth Fall in Three Categories:
1. Coagulant, such as plasma clots. It is now commercially available in the form of liquid
plasma kept in silicon ampoules or lyophilized plasma. Plasma can also be prepared in the
laboratory taking out blood from male fowl and adding heparin to prevent blood coagulation.
2. Biological fluids such as serum. Serum is one of the very important components of animal
cell culture which is the source of various amino acids, hormones, lipids, vitamins, poly-
amines, and salts containing ions such as calcium, ferrous, ferric, potassium, etc.
It also contains the growth factors which promotes cell proliferation, cell attachment and
adhesion factors. Serum is obtained from human adult blood, placental, cord blood, horse
blood, calf blood. The other forms of biological fluids used are coconut water, amniotic fluid,
pleural fluid, insect haemolymph serum, culture filtrate, aqueous humour, from eyes etc.
3. Tissue extracts for example Embryo extracts- Extracts from tissues such as embryo, liver,
spleen, leukocytes, tumour, bone marrow etc. are also used for culture of animal cells.
(b) Synthetic Media:
Synthetic media are prepared artificially by adding several organic and inorganic nutrients,
vitamins, salts, serum proteins, carbohydrates, co-factors, etc. Different types of synthetic
media can be prepared for a variety of cells and tissues to be cultured.

8. Synthetic Media are of Two Types:
1. Serum containing media (media containing serum)
2. Serum- free media (media without serum).
Examples of some media are: minimal essential medium (MEM), RPMI 1640 medium, CMRL
1066, F12, etc.
Advantages of Serum in Culture Medium are:
1. Serum binds and neutralizes toxins,
2. Serum contains a complete set of essential growth factors, hormones, attachment and
spreading factors, binding and transport proteins, Contents
3. It contains the protease inhibitors,
4. It increases the buffering capacity,
5. It provides trace elements.
Disadvantages of Serum in Culture Medium are:
a. It is not chemically defined, and therefore, its composition varies a lot,
b. It is sometimes source of contamination by viruses, mycoplasma, prions, etc.,
c. It increases the difficulties and cost of downstream processing,
d. It is the most expensive component of the culture medium.
(c) pH:
Most media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth. The
optimum pH is essential to maintain the proper ion balance, optimal functioning of cellular
enzymes and binding of hormones and growth factors to cell surface receptors in the cell
cultures. The regulation of pH is done using a variety of buffering systems. Most media use a
bicarbonate CO2 system as its major component.
(d) Osmolality:
A change in osmolality can affect cell growth and function. Salt, Glucose and Amino acids in
the growth media determine the osmolality of the medium. All commercial media are
formulated in such a way that their final osmolality is around 300 mOsm.
(e) Buffering:

9. Carbon dioxide in the medium is in a developed state and the concentration depends on the
atmospheric carbon dioxide tension and temperature. The CO2 exists in the medium in the form
of carbonic acid and protons.
CO2 + H2O → H2CO3← H+
+ HCO3
−
It must be noted that there is a direct relationship between the concentration of carbon dioxide,
bicarbonate ions and pH of the media. By increasing the atmospheric CO2, the pH will be
reduced making the medium acidic.
(f) Growth Factors:
In addition to buffering the medium, there are other growth requirements including amino
acids, the requirement for which may vary with cell culture type. Commonly the necessary
amino acids include cysteine and tyrosine, but some non-essential amino acids may be needed.
Glutamine is also required by most cell lines and it has been suggested that cultured cells use
glutamine as an energy and carbon source in preference to glucose, although glucose is present
in most defined media. Glutamine is usually added at a final concentration of 2 mM, however,
once added to the medium the glutamine is only stable for about 3 weeks at 4°C.
(g) Antibiotics and Antimycotics:
Unless good sterile conditions can be maintained (e.g., using laminar flow hoods) it is ne-
cessary to incorporate antibiotics and antimycotics into the media. A wide range of suitable
preparations are available from relatively specific antibiotics, e.g., penicillin/streptomycin
solutions, to broader spectrum antibacterial/antimycotic agents such as kanamycin or
amphotericin B.
The antibiotics chosen should clearly not to be toxic to the cells in culture and may depend on
the type of contamination experienced in the individual laboratory.
What is Animal cell culture?
Animal cell culture is a type of biotechnological technique where animal cells are artificially
grown in a favorable environment.
 The cells used in animal cell culture are usually obtained from multicellular eukaryotes
and their established cell lines.
 Animal cell culture is a common and widely used technique for the isolation of cells and
their culture under artificial conditions.
 This technique was developed as a laboratory technique for particular studies; however, it
has since been developed to maintain live cell lines as a separate entity from the original
source.
 The development of animal cell culture techniques is due to the development of basic tissue
culture media, which enables the working of a wide variety of cells under different
conditions.

10.  In vitro culture of isolated cells from different animals has helped in the discovery of
different functions and mechanisms of operations of different cells.
 Some of the areas where animal cell culture has found most applications include cancer
research, vaccine production, and gene therapy.
 The growth of animal cells on artificial media is difficult than growing microorganisms on
artificial media and thus, require more nutrients and growth factors.
 However, advances in the culture media have made it possible to culture both
undifferentiated and differentiated cells on artificial media.
 Animal cell cultures can be performed from different complexities of cells as complex
structures like organs can also be used to initiate organ culture in vitro.
 Depending on the purpose and application of the technique, cells, tissues, or organs can be
used for the culture process.
3. Types of Animal Cell Culture:
i. Primary Culture:
When cells are surgically removed from an organism and placed into a suitable culture
environment, they will attach, divide and grow. This is called a Primary Culture. There are two
basic methods for doing this.
First, for Explant Cultures, small pieces of tissue are attached to a glass or treated in a plastic
culture vessel and bathed in culture medium. After a few days, individual cells will move from
the tissue explant onto the culture vessel surface or substrate where they will begin to divide
and grow.
The second, more widely used method, speeds up this process by adding digesting (proteolytic)
enzymes, such as trypsin or collagenase, to the tissue fragments to dissolve the cement holding
the cells together. This creates a suspension of single cells that are then placed into culture
vessels containing culture medium and allowed to grow and divide. This method is called
Enzymatic Dissociation.
Types of Animal cell culture
 Animal cell cultures can be divided into two distinct groups depending on the number
of cell divisions occurring during the process;
1. Primary cell culture
 Primary cell culture is the first culture obtained directly from animal tissue via mechanical
and chemical disintegration or enzymatic methods.
 The cells of the primary cell culture are slow-growing cells that carry all the characteristics
of the original tissue or cells.
 Since these cultures are obtained directly from the origin, they have the same number of
chromosomes as the original cells.
 Primary cell cultures are performed in order to preserve and maintain the growth of cells
on an artificial growth medium at a particular condition.
 Primary cell cultures can be subcultured to obtained other cultures that either continue to
grow indefinitely or die after a few subcultures.

11.  The subsequent subculture of primary cell culture results in the introduction
of mutations into the cells, which might result in cell lines.
 The morphology of cells in the primary cell cultures might be different and varied, with
the most commonly observed morphological structures being epithelium type, epithelioid
type, fibroblast type, and connective tissue type.
 Primary cell cultures are difficult to obtain and usually have a shorter lifespan. Besides,
these are prone to contamination by bacteria and viruses.
 The increase in cell numbers in the primary cell culture can result in exhaustion of the
substrate and nutrients, which affects cellular activity.
 Usually, primary cell cultures need to be subcultured in order to maintain continuous cell
growth once they reach the confluence stage.
Primary cell cultures can be further divided into two groups depending on the kind of cells
present in the culture;
a. Anchorage-dependent/Adherent cells
 The cells in the culture require a stable biologically inert surface for adherence and growth.
 The surface should be solid and nontoxic as these cells are difficult to grow as cell
suspensions.
 These cells are usually obtained from the tissues of organs where the cells remain
immobilized within the connective tissue.
 Examples of adherent cells include kidney cells and mouse fibroblast STO cells.
b. Anchorage-independent/ Suspension cells
 These cells can grow efficiently as cell suspensions and do not require a solid surface for
attachment.
 These can be grown on liquid media continuously to obtain fresh subcultures.
 The ability of the cells to grow as suspension depends on the source of cells as cells that
remain as suspensions in the body are effective suspension cells.
 Examples of suspension cells include blood cells that are vascular and remain suspended
in the plasma.
Advantages of Primary Cell Culture:
The major advantages of primary cultures are the retention of:
1. The Capacity for Biotransformation:
In many cases, the metabolism of a primary cell culture has greater similarity to in vivo than
that seen with sub-cellular fractions used as an exogenous source for biotransformation.
2. The Tissue-Specific Functions:
The second advantage of primary cultures is the retention of tissue specific functions. For
example, primary cultures of rat myocardial cells consisting of synchronously beating cells can
be prepared. When these cultures were exposed to tricyclic antidepressants that are cardio toxic,
beating were observed.
Limitations of Primary Cell Cultures:
One limitation of primary cultures is the necessity to isolate cells for each experiment.
Procedures to isolate cells require the disruption of the tissue, often with proteolytic enzymes.
This may result in the loss or damage of specific membrane receptors, damage to the integrity
of the membrane, and loss of cellular products.

12. ii. Sub-Culturing:
When the cells in the primary culture vessel have grown and filled up all of the available culture
substrate, they must be Sub-cultured to give them room for continued growth. This is usually
done by removing them as gently as possible from the substrate with enzymes. These are
similar to the enzymes used in obtaining the primary culture and are used to break the protein
bonds attaching the cells to the substrate.
Some cell lines can be harvested by gently scraping the cells off the bottom of the culture
vessel. Once released, the cell suspension can then be subdivided and placed into new culture
vessels.
Once a surplus of cells is available, they can be treated with suitable cryoprotective agents,
such as dimethylsulfoxide (DMSO) or glycerol, carefully frozen and then stored at cryogenic
temperatures (below -130°C) until they are needed. The theory and techniques for
cryopreserving cells are covered in the Corning Technical Bulletin: General Guide for
Cryogenically Storing Animal Cell Cultures.
2. Secondary cell culture
 Secondary cell cultures are obtained after the primary cell cultures are subsequently
subcultured over a period of time in fresh culture media.
 The cells of the secondary cell cultures are long-lasting as these have a higher lifespan due
to the availability of appropriate nutrients at regular intervals of time.
 Secondary cell cultures are favored over primary cell cultures as these are more readily
available and are easy to grow and preserve.
 These are formed from the enzymatic treatment of the adherent cells followed by washing
and resuspension of cells in particular volumes of fresh media.
 Secondary cell cultures are prepared when the number of cells in the primary culture
exceeds the capacity of the medium to support growth.
 Secondary cultures help to maintain an optimal cell density necessary for continued
growth.
 The cells of the secondary cell culture might not resemble that on the parental tissue as
mutations, and genetic alterations might be introduced during the subculture process.
 The cells can be transformed as, in some cases, the continuous subculture can lead to
immortal cells.
 The risk of contamination by bacteria and viruses is less as the cells transform and become
less susceptible to infections.
 An important disadvantage associated with secondary cell culture is that the cells might
develop the tendency to differentiate over a long period of time and result in aberrant cells.
Cell Lines
A cell line is a group of cells that are formed from the subculture of primary culture consisting
of a pure culture of cells. Cell lines usually display functional features that are close to the
primary cells, but the genotype and phenotype of the cells can be modified. A cell line consists
of several cell lineages with similar or different phenotypes.

13. Cell lines can be further divided into two groups based on the growth patterns of the cells;
a. Finite cell lines
 Finite cell lines are cell lines where the cells in the culture divide for a limited number of
times, after which they eventually die.
 The cells in the finite cell lines can divide from 20 to 100 times before they eventually die
and cannot divide anymore.
 The number of cell division and lifespan depends on a number of factors like cell lineage
differences, species, culture conditions, and media.
 The cells of the finite cell lines grow as adherent cells on solid surfaces.
b. Continuous cell lines
 Continuous cell lines are cells that exhibit indefinite growth via subsequent subcultures.
 The cells in the continuous cell lines grow faster to form an independent culture. The cells
are immortal and can divide indefinitely.
 The cells in the continuous cell lines can be transformed via genetic alterations and are
also tumorigenic.
 The transformed cells are formed from the normal primary cell cultures after treatment
with chemical carcinogens or by infection with oncogenic viruses.
 The cells are capable of growing to prepare higher cell density and can grow as suspensions
on liquid media.
 These cells can even grow on top of each other to form multilayered structures on the
culture vessels.
Examples of common Cell Lines
The following are some of the common examples of cell lines;
a. HeLa cell line
 HeLa cells are one of the first continuous culture human cell lines with the help of cells of
the cervical carcinoma.
 These cells are used for processes like virus cultivation and preclinical drug evaluation.
b. HL 60 (Leukemia)
c. MCF-7 (breast cancer cells)
Procedure or Protocol of Animal cell culture
1. Growth Conditions
 Animal cell culture requires the use of specific culture media that are more complex and
specific than the basic culture media used for microbial growth.
 Some of the important basic components of the media are inorganic salts, nitrogen source,
energy source, vitamins, fat and fat-soluble vitamins, growth factors, and hormones. In
some cases, pH buffering systems and antibiotics are also added.
 The temperature for the growth depends on the source of the cell as different organisms
require different temperatures for cell growth and division.
 Warm-blooded animal cells can be cultured at 37°C as the optimal temperature, whereas
cold-blooded animals grow between 15°C-25°C.
2. Primary cell culture
 Primary cell cultures are obtained from fresh tissues that are removed from the organs with
the help of an aseptic razor.
 In some cases, the cells are removed by the use of chemical disintegrators or proteolytic
enzymes.
 The cell suspension obtained is washed with buffering liquid in order to remove the
proteolytic enzymes.
 The cell suspension is poured onto a flat surface which can be a culture vessel or a sterile
Petri plate.

14.  The cells that can adhere to the base of the vessel are overlaid with an appropriate culture
medium and incubated at room temperature.
3. Cell thawing
 In the case of subsequent subcultures, the preserved cell culture might have to be used.
 The water bath is heated to a temperature of 37°C, and the growth media where the cells
are to be plated is warmed.
 The warm medium is added to the culture vessel. The vial with the frozen cells is then
placed in the water bath until thawed.
 After thawing, the via is washed with 70% alcohol on the outside. The cell suspension is
pipetted into the cell culture vessel and swirled gently to mix everything.
 The medium is then incubated overnight under the usual growth conditions. The growth
medium is replaced the next day.
4. Trypsinizing Cells
 Trypsinization is the method of separating adherent cells from the surface of the culture
vessel with the help of proteolytic enzymes. It is done when the cells are to be used for
passaging, counting, or other purposes.
 The medium is removed, and the cells are recovered. The cells are then washed with
phosphate buffer.
 Warm trypsin-EDTA is added to the vessel so as to cover the monolayer. The vessel can
be rocked to ensure that the monolayer is coated.
 The vessel is incubated in a CO2 incubator at 37°C for 1-3 minutes.
 The vessel is removed from the incubator, and the flask is firmly tapped on the side with
the palm of the hand to assist detachment.
 Once the cells are dislodged, they are resuspended in an appropriate growth medium
containing some amount of serum.
 The cells are then separated with the help of syringe needles by disrupting the cell clumps
and used accordingly.
Applications of Animal cell culture
The following are some of the applications of animal cell culture;
a. Production of vaccines
 Animal cell culture is an important technique used for the development of viral vaccine
production.
 The technique has been used for the development of a recombinant vaccine against
hepatitis B and poliovirus.
 Immortalized cell lines are used for the large-scale or industrial production of viral
vaccines.
b. Recombinant proteins
 Animal cell cultures can also be used for the production of recombinant therapeutic
proteins like cytokines, hematopoietic growth factors, growth factors, hormones, blood
products, and enzymes.
 Some of the common animal cell lines used for the production of these proteins are baby
hamster kidney and CHO cells.
c. Gene Therapy
 The development of animal cell culture is critical for the advances in gene therapy.
 Cells with faulty genes can be replaced by a functional gene in order to remove such
defects and diseases.

15. d. Model systems
 Cells obtained from cell culture can be studied as a model system for studies related to cell
biology, host-pathogen interactions, effects of drugs, and effects due to changes in the cell
composition.
e. Cancer Research
 Animal cell culture can be used to study the differences in cancer cells and normal cells as
cancer cells can also be cultured.
 The differences allow more detailed studies on the potential causes and effects of different
carcinogenic substances.
 Normal cells can be culture to form cancer cells by the use of certain chemicals, viruses,
and radiation.
 Cancer cells can also be used as test systems for studies related to the efficiencies of drugs
and techniques used in cancer treatment.
f. Production of Biopesticides
 Animal cell lines like Sf21 and Sf9 can be used for the production of biopesticides due to
their faster growth rate and higher cell density.
 Organisms like baculovirus can be produced through animal cell culture as well.
Advantages of Animal cell culture
The following are some of the advantages of animal cell culture;
1. Cell culture is superior to other similar biotechnological approaches as it allows the
alteration of different physiological and physiobiological conditions like temperature, pH,
and osmotic pressure.
2. Animal cell culture enables studies related to cell metabolism and understand the
biochemistry of cells.
3. It also allows observation of the effects of various compounds like proteins and drugs on
different cell types.
4. The results from animal cell cultures are consistent if a single cell type is used.
5. The technique also enables the identification of different cell types on the basis of the
presence of markers like molecules or by karyotyping.
6. The use of animal cell culture for testing and other processes prevents the use of animals
in experiments.
7. Animal cell culture can be used for the production of large quantities of proteins and
antibodies, which would otherwise require a large investment.
Disadvantages of Animal cell culture
Even though animal cell culture has been used as a technologically advanced method, there are
some disadvantages associated with this approach.
1. It is a specialized technique that requires trained personnel and aseptic conditions. The
technique is an expensive process as it requires costly equipment.
2. The subsequent subculture of the cell culture might results in differentiated properties as
compared to the original strain.
3. The method produces a minuscule amount of recombinant proteins, which further
increases the expenses of the process.
4. Contamination with mycoplasma and viral infection occur frequently and are difficult to
detect and treat.
5. The cells produced by this technique lead to instability due to the occurrence of aneuploidy
chromosomal constitution.

16. 4. Characteristics of Cultured Animal Cells:
i. Cell Culture Systems:
Two basic culture systems are used for growing cells. These are based primarily upon the
ability of the cells to either grow attached to a glass or treated plastic substrate, called as mono-
layer culture systems, or floating free in the culture medium called as Suspension Culture
Systems.
Monolayer cultures are usually grown in tissue culture treated dishes, T-flasks, roller bottles,
Cell-STACK® Culture Chambers, or multiple well plates, the choice being based on the
number of cells needed, the nature of the culture environment, cost and personal preference.
Suspension Cultures are usually Grown Either:
1. In magnetically rotated spinner flasks or shaken Erlenmeyer flasks where the cells are kept
actively suspended in the medium;
2. In stationary culture vessels such as T-flasks and bottles where, although the cells are not
kept agitated, they are unable to attach firmly to the substrate. Many cell lines, especially those
derived from normal tissues, are considered to be Anchorage-Dependent, that is, they can only
grow when attached to a suitable substrate.
Some cell lines that are no longer considered normal (frequently designated as Transformed
Cells) are frequently able to grow either attached to a substrate or floating free in suspension;
they are Anchorage-Independent. In addition, some normal cells, such as those found in the
blood, do not normally attach to substrates and always grow in suspension.
ii. Types of Cells:
Cultured cells are usually described based on their morphology (shape and appearance) or their
functional characteristics.
There are three basic morphologies:
1. Epithelial:
Like: cells that are attached to a substrate and appear flattened and polygonal in shape.
2. Lymphoblast-Like:
Cells that do not attach normally to a substrate but remain in suspension with a spherical shape.
3. Fibroblast-Like:
Cells that are attached to a substrate and appear elongated and bipolar, frequently forming
swirls in heavy cultures. It is important to remember that the culture conditions play an

17. important role in determining shape and that many cell cultures are capable of exhibiting
multiple morphologies.
Using cell fusion techniques, it is also possible to obtain hybrid cells by fusing cells from two
different parents. These may exhibit characteristics of either parent or both parents. This tech-
nique was used in 1975 to create cells capable of producing custom tailored monoclonal
antibodies.
These hybrid cells (called Hybridomas) are formed by fusing two different but related cells.
The first is a spleen-derived lymphocyte that is capable of producing the desired antibody. The
second is a rapidly dividing myeloma cell (a type of cancer cell) that has the machinery for
making antibodies but is not programmed to produce any antibody.
The resulting hybridomas can produce large quantities of the desired antibody. These
antibodies, called Monoclonal Antibodies due to their purity, have many important clinical,
diagnostic, and industrial applications with a yearly value of well over a billion dollars.
iii. Functional Characteristics:
The characteristics of cultured cells result from both their origin (liver, heart, etc.) and how
well they adapt to the culture conditions. Biochemical markers can be used to determine if cells
are still carrying on specialized functions that they performed in vivo (e.g., liver cells secreting
albumin).
Morphological or ultra-structural markers can also be examined (e.g., beating heart cells). Fre-
quently, these characteristics are either lost or changed as a result of being placed in an artificial
environment. Some cell lines will eventually stop dividing and show signs of aging.
These lines are called Finite. Other lines which become immortal can continue to divide
indefinitely and are called Continuous cell lines. When a “normal” finite cell line becomes
immortal, it undergoes a fundamental irreversible change or “transformation”. This can occur
spontaneously or be brought about intentionally using drugs, radiation or viruses.
Transformed Cells are usually easier and faster growing, may often have extra or abnormal
chromosomes and frequently can be grown in suspension.Cells that have the normal number
of chromosomes are called Diploid cells; those that have other than the normal number are
Aneuploid. If the cells form tumours when they are injected into animals, they are considered
to be Neo-plastically Transformed.

18. Advantages of Animal Cell Culture:
a. Controlled physiochemical environment (pH, temperature, osmotic pressure, O2, etc.)
b. Controlled and defined physiological conditions
c. Homogeneity of cell types (achieved through serial passages)
d. Economical, since smaller quantities of reagents are needed than in vivo.
e. Legal, moral and ethical questions of animal experimentation are avoided.
Disadvantages of Animal Cell Culture:
a. Expertise is needed, so that behaviour of cells in culture can be interpreted and regulated.
b. Ten times more expensive for same quantity of animal tissue; therefore, reasons for its use
should be compelling.
c. Unstable aneuploid chromosome constitution.
Applications of Animal Cell Culture:
A. Model Systems:
Cell cultures provide a good model system for studying;
a. Basic cell biology and biochemistry.
b. The interactions between disease-causing agents and cells.
c. The effects of drugs on cells.
d. The process and triggers for aging.
f. Nutritional studies.
B. Toxicity Testing:
Cultured cells are widely used alone or in conjunction with animal tests to study the effects of
new drugs, cosmetics and chemicals on survival and growth in a wide variety of cell types.
Especially important are liver and kidney derived cell cultures.

19. C. Cancer Research:
Since both normal cells and cancer cells can be grown in culture, the basic differences between
them can be closely studied. In addition, it is possible, by the use of chemicals, viruses and
radiation, to convert normal cultured cells to cancer causing cells.
Thus, the mechanisms that cause the change can be studied. Cultured cancer cells also serve as
a test system to determine suitable drugs and methods for selectively destroying types of
cancer.
D. Virology:
One of the earliest and major uses of cell culture is the replication of viruses in cell cultures (in
place of animals) for use in vaccine production. Cell cultures are also widely used in the clinical
detection and isolation of viruses, as well as basic research into how they grow and infect
organisms.
E. Cell-Based Manufacturing:
While cultured cells can be used to produce many important products, three areas are gen-
erating the most interest.
The first is the large-scale production of viruses for use in vaccine production. These include
vaccines for polio, rabies, chicken pox, hepatitis B and measles.
The second is the large-scale production of cells that have been genetically engineered to
produce proteins that have medicinal or commercial value. These include monoclonal
antibodies, insulin, hormones, etc. The third is the use of cells as replacement tissues and
organs. Artificial skin for use in treating burns and ulcers is the first commercially available
product.
However, testing is underway on artificial organs such as pancreas, liver and kidney. A po-
tential supply of replacement cells and tissues may come out of work currently being done with
both embryonic and adult stem cells.
These are cells that have the potential to differentiate into a variety of different cell types. It is
hoped that learning how to control the development of these cells may offer new treatment
approaches for a wide variety of medical conditions.
F. Genetic Counselling:
Amniocentesis, a diagnostic technique that enables doctors to remove and culture fetal cells
from pregnant women, has given doctors an important tool for the early diagnosis of fetal

20. disorders. These cells can then be examined for abnormalities in their chromosomes and genes
using karyotyping, chromosome painting and other molecular techniques.
G. Genetic Engineering:
The ability to transfect or reprogram cultured cells with new genetic material (DNA and genes)
has provided a major tool to molecular biologists wishing to study the cellular effects of the
expression of these genes (new proteins).
These techniques can also be used to produce these new proteins in large quantity in cultured
cells for further study. Insect cells are widely used as miniature cells factories to express
substantial quantities of proteins that they manufacture after being infected with genetically
engineered baculoviruses.
H. Drug Screening and Development:
Cell-based assays have become increasingly important for the pharmaceutical industry, not just
for cytotoxicity testing but also for high throughput screening of compounds that may have
potential use as drugs. Originally, these cell culture tests were done in 96 well plates, but
increasing use is now being made of 384 and 1536 well plates.
I. Gene Therapy:
In modern molecular biology, Gene Therapy is an experimental technique that involves
insertion of cloned/altered genes into cells using r-DNA technology to replace defective genes
causing genetic abnormalities or to prevent potential disorders.
Followings are the purpose of Gene Therapy:
a. Swapping harmful mutant alleles with functional ones by selective reverse mutation.
b. Deactivating improperly functioning mutated gene.
c. Inserting a new gene into the body to help battle a disease.
d. Interchanging non-functional gene with normal gene through homologous recombination.
References
1. Kaur, Gurvinder, and Jannette M Dufour. “Cell lines: Valuable tools or useless
artifacts.” Spermatogenesis vol. 2,1 (2012): 1-5. doi:10.4161/spmg.19885
2. Yao, Tatsuma, and Yuta Asayama. “Animal-cell culture media: History, characteristics,
and current issues.” Reproductive medicine and biology vol. 16,2 99-117. 21 Mar. 2017,
doi:10.1002/rmb2.12024
3. Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W.
H. Freeman; 2000. Section 6.2, Growth of Animal Cells in Culture. Available
from: https://www.ncbi.nlm.nih.gov/books/NBK21682/

21. 4. Hudu, Shuaibu Abdullahi et al. “Cell Culture, Technology: Enhancing the Culture of
Diagnosing Human Diseases.” Journal of clinical and diagnostic research : JCDR vol.
10,3 (2016): DE01-5. doi:10.7860/JCDR/2016/15837.7460
5. Verma, Anju et al. “Animal tissue culture principles and applications.” Animal
Biotechnology (2020): 269–293. doi:10.1016/B978-0-12-811710-1.00012-4
6. Gilgenkrantz S. Regard sur soixante années de culture de cellules HeLa [Sixty years of
HeLa cell cultures]. Hist Sci Med. 2014 Jan-Mar;48(1):139-44. French. PMID: 24908793.
7. Lucey BP, Nelson-Rees WA, Hutchins GM. Henrietta Lacks, HeLa cells, and cell culture
contamination. Arch Pathol Lab Med. 2009 Sep;133(9):1463-7. doi: 10.1043/1543-2165-
133.9.1463. PMID: 19722756.
8. https://www.biologydiscussion.com/cell/cell-lines/cell-lines-types-nomenclature-
selection-and-maintenance-with-statistics/10517
9. https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/animal-
cell-culture/essential-protocols-for-animal-cell-culture/essential-protocols-for-animal-
cell-culture#Maintaining-cell-cultures.

22. B.Sc.III, Sem-VI: Unit-I
Gene Therapy: Types, Demonstration and Drawback
Authored By: Dr. Rajendra Chavhan, Department of Zoology, RMG College Nagbhid
Gene Therapy: Types, Demonstration and Drawback
Types of Gene Therapy:
1. In Vivo Therapy:
The gene is inserted into an individual with the desire that it may find the appropriate target
cell (thereby making it inefficient).
2. Ex Vivo Therapy:
The cell from patient is manipulated externally in cell culture and reintroduced into patient.
3. Somatic Cell Therapy:
Fixing of genetic malfunctions in somatic cells is restricted to patient only.
4. Germ Line Therapy:
Germ cells (sex cells) are modified by introduction of functional genes and passed on to future
progeny. Genes directly introduced into a cell become non-functional; therefore, carriers
known as vectors are genetically engineered to deliver the gene.
Genes are inserted via retroviruses, adenoviruses, adenoassociated viruses and herpes simplex
viruses by removal of virulent gene or inserted via non-viral methods by naked DNA,
oligonucleotides, lipoplexes and polyp lexes.
Use of genetic therapy is mainly directed towards oncology for eradication of tumour cells. In
spite of its low success rate, it is hopeful that gene therapy may be used as an adjuvant therapy
to cure diseases like cystic fibrosis, ADD, phenylketonuria and immunodeficiency syndromes.
Demonstration of Gene Therapy:
1. Adenosine deaminase is needed for maturation of white blood cells, and people without this
enzyme have severe immune system deficiencies. A functional gene for adenosine deaminase
was introduced via a viral vector into the white blood cells of a girl with a genetic deficiency
of this enzyme.
Unfortunately, mature white blood- cells were used, and although they survived for a time in
the girl and provided some therapeutic benefit, they eventually died, as is the normal fate of
such cells. Further clinical trials have used stem cells, the bone marrow cells that constantly
divide to produce white blood cells.

23. 2. Hemophilia is a disease in which patients do not make enough of a blood clotting protein.
Some cells from the skin of patients’ arms were removed and transfected with a plasmid
containing a normal allele of the clotting protein gene. The cells were then reintroduced into
the patients’ body fat, where they produced adequate protein for normal clotting.
Drawbacks of Gene Therapy:
Followings are the factors posing as obstacles for effective gene therapy treatment:
a. Short-lived nature of gene therapy.
b. Occurrence of severe immune responses.
c. Problems associated with viral vectors.
d. Ineffective treatment of multi-gene disorders.