1.
PHARMACEUTICAL MICROBIOLOGY
(BP303T)
Unit-V
Part-4
Animal Cell Culture: Growth of animal cells in
culture..
Name: Ms. Pooja Deepak Bhandare
Assistant Professor
G H RAISONI UNIVERSITY
SCHOOL OF PHARMACY

2.
 Introduction:
• Animal cell culture is a microbiological technique in which animal cells and tissues are
obtained and maintained in a suitable environment.
• It is further divided into,
• Organotypic: Animal Organ Tissue Culture.
• Histotypic: Animal Cell Culture.
• Animal cells can be grown only upto certain generations.
• Their growth requires typical conditions and a physicochemical environment.
• Animal cell culture prepared from collecting tissue or cells from an organism is called a
“Primary culture.”
• The sequence of subcultures prepared from primary culture is called “Cell lines”.

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History:
• It was Ross Harisson from America who first cultured animal cells
successfully in the laboratory.
• Campbell, Wilmut and colleagues at Roslin Institute in Scotland
successfully cloned the first animal cell using nuclear transfer technique
and gave birth to a sheep named “Dolly” in 1996.

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• Growing animal cells in cultures is a difficult task, it requires specially
made culture media.
• The culture media used for animal cell culture are classified as,
• Natural,
• Artificial,
• Synthesized.
• The selection of culture media depends on the objective of experiment
and type of cell to be cultured.

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 NaturalCultureMedia:
• As the name indicates these media are obtained from natural resources like, tissue extracts,
biological fluids, plasma clots or coagulants etc.
a. Blood Plasma:
• It provides good nutrition and support to many types of animal cell culture.
• During subculturing it provides good protection against traumatic damage and better
attachment of cells to glass surfaces.
• Chicken plasma is most favored for its clarity even on dilution.
• The plasma is obtained by centrifugation before coagulation.
• This tissue or cells are placed in such plasma and then coagulation is initiated by addition of
thrombin.

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B. Blood Serum:
• Blood serum is plasma without fibrinogen.
• Blood serum with or without nutritional substances is used as a culture media to grow
animal cells.
• The sera commonly used in animal culture are bovine, horse, and human.
• Calf and fetal bovine serum are most widely used sera in animal cell culture.
• Human serum is used sometimes but is needed to be screened for presence of hepatitis and
HIV.
• The composition of a serum is highly complex as it is a mixture of various plasma proteins,
peptides, lipids, carbs, enzymes and minerals.

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C. Tissue Extracts:
• Commonly used tissue extracts in animal cell culture are, embryo, spleen,
liver, bone marrow etc.
• CHick embryo extract is the most commonly used tissue extract in animal
cell culture, it is obtained from 10-12 days old embryos.

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2. Artificial Media:
• Synthetic or artificial media are prepared by adding organic or inorganic substances, nutrients, vitamins, proteins, carbs,
gaseous phases.
• Different artificial media are prepared for different types of cells as per their requirement.
• Some common examples of artificial media are,
• Minimal Essential Medium (MEM),
• CMRL 1066,
• RPMI 1640.
• Synthetic media re classified as,
• Serum Containing Media.
• Serum Free Media.

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a. Serum Containing Media:
• It is prepared by adding 5 to 20 % serum in serum free medium.
• Serum is one of the best sources of many basic nutrients, contains hormones, growth factors, proteins, and
minerals.
• Serum also acts as a buffer.
• Serum combines and neutralises many toxins.
• Although its a best source of nutrients it has following disadvantages,
• Most expensive of all ingredients of the culture medium.
• It increases difficulties and cost of downstream processing.
• Its content varies from batch to batch.
• Extensive testing is required to check similarity on serum change.
• Supply gets affected in drought or cattle disease spread.
• Source of contamination: May contain viruses, bacteria, prions etc.
• Some growth factors may be inadequate and can not be supplemented.

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b. Serum Free Media:
• They are developed to overcome limitations of serum containing media.
• These media can make the medium more selective for particular cells as
per their requirements e.g. melanocytes can be cultivated in absence of
keratinocytes.
• As serum is not present there is no problem of toxicity, serum change tests.
• Bioassays and other processes become easy due to absence of the serum.

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• However, they also has some disadvantages like,
• Slower growth.
• Useful only for a few generations.
• Purity of ingredients.
• pH control and temperature control required is more.
• Most media are specific to a particular cell type.
• Preparation time is more.

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 Physicochemical Parameters neededfor growthanimal cellculture:
• Control on different physicochemical parameters is necessary during animal cell culture.
• The temperature of the culture has to be the same as that of the body temperature of the
animal from which cells are collected.
• Osmolarity between 260 m Osm/kg and 320 m Osm/kg is good for many cell lines.
• Buffers are incorporated for maintaining the pH as per requirement of the cell culture.
• Most cell cultures grow well at a pH of 7.4, phenol red is commonly used indicator to
detect the pH change in medium.

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• External Carbon dioxide addition is needed for some cell cultures to
replenish CO2 loss.
• Oxygen is a component of the gas phase as most cells require oxygen for
respiration.
• Cell damage in the culture is prevented by increasing viscosity of the
medium by addition of CMC or PVP.
• Balanced Salt Solution (BSS) is used as a diluent for vitamins, amino
acids to make complete media.

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General procedure for cell Culture.

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• A piece of tissue of an organism is quite a complex substance as it is associated
with cells of other tissues also like, cells from connective tissue, blood cells etc.
• In the beginning the cell suspension is isolated and inoculated in a new culture
vessel with fresh culture medium, such culture is called “Primary Cell Culture or
Primary Cell Line”.
• Following three stages are performed while isolating a primary culture,
• Isolation of the tissue.
• Disaggregation of the tissue.
• Seeding of culture into the culture vessel.
• All these stages are performed under a Laminar AirFlow hood in order to avoid any
chance of contamination.

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1.Isolationof thetissue:
• A piece from a surgically removed portion of the body of an animal is used for the culture of animal cells in
a suitable nutrient medium.
• The major explant tissues for animal cell culture are collected from lab animals like rabbits, mice, guinea
pigs etc.
• The human cells like muscle cells, erythrocytes, leukocytes are also collected and cultured in suitable
media.
• Organs from which cells to be collected are surface sterilized with 70% alcohol and then aseptically
removed.
• The collected tissues are immediately transferred to a sterile nutrient medium or balanced salt solution
(BSS) containing antibiotics.
• The tissue is usually used immediately or stored in the freezer.

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2. Disaggregationof theTissue:
• A tissue collected is disaggregated (Separated) from surrounding
tissues by mechanical, enzymatic means or using chelating agents.

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a. Mechanical disaggregation:
• Also called “Physical Disaggregation”.
• The mechanical approach involves slicing or harvesting tissue and subsequent
harvesting of spill out cells.
• This can be achieved by sieving, syringing and pipetting.
• This procedure is inexpensive, rapid and simple, however, all these approaches
involve the risk of cell damage, thus mechanical disaggregation is only used
when the viability of the cells in the final yield is not very important.

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b. Enzymatic Disaggregation:
• This approach involves efficient disaggregation of cells with high yield
by using enzymes such as trypsin, collagenase and others.
• Enzyme based disaggregation allows hydrolysis of fibrous connective
tissue and the extracellular matrix.
• Currently, the enzymatic method is extensively used as it offers high
recovery of cells without affecting the viability of cells.

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A. Trypsin based disaggregation or trypsinization:
• The process of enzymatic disaggregation of tissues using trypsin is called trypsinization.
• Trypsinization is of two types,
• Cold trypsinization.
• Warm trypsinization.
• This allows disaggregation of tissue using trypsin, usually crude trypsin because this trypsin
contains other proteases.
• In addition, cells can tolerate crude trypsin well and the ultimate effect of crude trypsin can
easily be neutralized by serum or trypsin inhibitor (supplementation of trypsin inhibitor is
required in the case of serum-free media).

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•Warm trypsinization:
• This approach is extensively utilized for the disaggregation of cells.
• During the initial step, sliced tissue is washed with dissection basal salt solution
and is subsequently transferred to a container of warm trypsin (37 °C).
• At regular intervals of 30 min the contents are stirred properly.
• Then, the supernatant having dissociated, the cells are separated to disperse in a
suitable medium.
• Efficient dispersion of cells can be achieved by placing the container over ice.

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•Cold trypsinization:
• This method is also called trypsinization with cold pre-exposure.
• In this process the chance of cellular damage due to constant exposure
to trypsin is reduced, which results in a high yield of viable cells with
an improved survival rate for the cells (after 24 h of incubation).
• Since this method does not involve frequent stirring or centrifugation,
it can be conveniently adopted in the research laboratory.

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• During this process, after washing and chopping, tissue pieces are kept over ice
in a vial and then subjected to treatment with cold trypsin for 6–24 h.
• Then, after the cold trypsin treatment the trypsin is removed and discarded.
• However, the tissue fragments still contain residual trypsin. These fragments are
incubated at 37 °C (for 20–30 min) followed by repeated pipetting.
• This will encourage the dispersion of cells. The fully dispersed cells can be
counted using a cell counter and properly diluted, and then further utilized.

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• Drawbacks of trypsin disaggregation:
• Trypsinization of cells can damage some cells, such as epithelial cells, and
sometimes it is not effective for certain tissues, such as fibrous connective tissue,
thus other enzymes are also recommended for dissociation of cells.

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B. Collagenase based disaggregation:
• Collagenase is an enzyme which is responsible for the cleavage of peptide
bonds in collagen.
• Collagen is a structural protein which is abundantly found in higher
animals, mainly in the extracellular matrix of connective tissue and
muscle.

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• Collagenase, mainly crude collagenase, can be successfully used for the
disaggregation of several tissues that may or may not be sensitive to trypsin.
• This process involves an initial transfer of the desired tissue into a basal salt
solution which contains antibiotics.
• This is followed by washing with settling and then transfer into a medium
containing collagenase.
• The solution is incubated for 1–5 days, followed by repeated pipetting for
uniform dispersal of cells.
• Separation of these dispersed cells is encouraged by keeping the solution in a
stationary phase to further encourage the settling of cells.

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C. Chelating Agents:
• These are mainly used for preparation of cell suspensions.
• These agents form chelats along with ions like Calcium,
Magnesium etc.which are essential for maintaining integrity of
epithelial cells.

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3. Seedingof Culture:
• The primary culture cell grows well when seeded into culture plates at high
density.
• Most Of the cell require support of substrate for growth, these cells are called
“Anchorage Dependent Cells” e.g. liver, kidney etc.
• Some cells don't require support, these cells are called “Anchorage independent
Cells”.
• The support materials used for Anchorage Dependent Cell are glass, plastic metals
like stainless steel, titanium etc.
• The Anchorage independent cells are cultivated in liquid medium.