Animal Cell Culture

Cell culture
• Animal cell culture refers to the rearing of isolated
animal cells or animal tissues in suitable nutrient
medium under controlled conditions.
• In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with
a medium containing cell nutrients & growth factors is
collectively known as tissue culture.
• Different types of cell grown in culture includes
connective tissue elements such as fibroblasts,
skeletal tissue, cardiac, epithelial tissue (liver, breast,
skin, kidney) and many different types of tumor cells.
 History
• 1885, Roux maintained the cells of medullary
plate of chick embryo in warm saline.
• 1903, Jolly observed cell division in Salamander
leucocytes grown in vitro.
• 1907, Ross Granville and Harrison established a
methodology of animal tissue culture for the first
time.
• 1913, Rous and Jones used penicillin and
streptomycin as antibiotics in animal cell cultures.
 History
• 1916, Rous and Jones used laminar air-flow
hoods for the aseptic transfer of cultures and
media and for cultural operations.
• 1940, Rous and Jones formulated a medium
containing blood serum to culture animal cells
and tissues.
• Katherine prepared the clones of L-cells via
animal cellculture.
 History
• Enders, Weller and Robbin grew animal cell
cultures to manufacture polio vaccine.
• 1949, Alan Parks developed a protocol to
freeze cells and kept them at -196°C
• 1950, Rous and Jones evaluated necessary
conditions such as temperature, pH, media
components, inorganic ions, hormones and
others for culture.
 History
• 1951, famous Hela cell was derived from
cancerous growth of Henrietta Lacks, who
died of cervival cancer.
• 1952, Harry Eagle developed a chemically
defined culture medium.
• 1954, Ender et al., employed human cell lines
for the production of vaccines for humans and
animals.
 History
• 1955, Hayflick and Moorhead discovered the
lifespan of normal human diploid cells in cell
cultures.
• 1960’s and 1970’s, several companies started
developing disposable plastic and glass cell
culture products, improved filtration products
for cell culture works.
• 1966, Alec Issacs demonstrated the presence
of Interferon in animal cell cultures infected
with some viruses.
 History
• 1980, recombinant erythropoietin was
prepared from Chinese hamster ovary (CHO)
cell lines.
• 1982, Thilly et al., established the scaling up
process for the large scale culture of animal
cells.
• 2002, Human Genome society (France) funded
for research leading to cloned human body.
 Why is cell culture used for?
Areas where cell culture technology is currently
playing a major role.
• Model systems for
Studying basic cell biology, interactions between
disease causing agents and cells, effects of drugs on cells,
process and triggering of aging & nutritional studies
• Toxicity testing
Study the effects of new drugs
• Cancer research
Study the function of various chemicals, virus &
radiation to convert normal cultured cells to cancerous cells
 Why is cell culture used for? Contd.,
• Virology
Cultivation of virus for vaccine production, also used to study
there infectious cycle.
• Genetic Engineering
Production of commercial proteins, large scale production
of viruses for use in vaccine production e.g. polio, rabies, chicken
pox, hepatitis B & measles
• Gene therapy
Cells having a functional gene can be replaced to cells which
are having non-functional gene
It has many potential applications in production of vaccines, bioactive
compounds, monoclonal antibodies and cell clones for gene
therapy.
 Three categories
(1) Explant culture
Small pieces of tissues excised from the body of
an animal, for animal tissue culture are called
Explants.

Petriplates, conical flasks, roller bottles etc., are

used to culture the explants.
Explant culture
 Three categories
(2) Organ culture
The culture of intact organ fragments or two or
more cell types that make up the part of an
organ in culture media is called organ culture.
Organ Culture
 Three categories
(3) Cell culture
This is maintenance of individual cells in culture
Media.

Unlike the explants and organ cultures, cell

Cultures are done in large flasks or bioreactors.

Cell cultures are done with a view of producing

tissue culture based products of commercial
importance.
 Cell culture

Hela Cells Fibroblast Cells

Setup
 Requirements
• Requires enough infrastructures such as
equipments and chemicals to grow animal
cells and tissues in a safe and efficient manner.
• Single use facility: isolated work area for cell
culture.
• Work area should be free from contaminants.
• Work surfaces should be thoroughly cleaned
between each activities.
 Requirements
• Materials used should be used after proper sterilization and should
be free from bacteria, fungi and mycoplasma contaminants.
Specific Requirements:
(1) Laminar Flow Hoods.
(2) Centrifuges.
(3) Incubators.
(4) Work area and flooring.
(5) Plasticwares.
(6) Gasswares.
(7) Waterbath.
(8) Inverted microscope.
(9) Vaccum pump.
(10)Liquid Nitrogen Container.
 Specific Requirements:
Laminar Airflow
• Laminar flow hoods are sterile air cabinets designed in
such a way that sterile air passes across the work area
continuously.
• The air is pumped into the cabinet by an electric
blower.
• The air entering the work area is filtered by high
efficiency particulate air supply filter (HEPA filter).
• Contaminated air passes through HEPA filter.
• If air filters are kept in vertical position, are called
vertical laminar flow hoods and when kept in horizontal
position, are called horizontal laminar flow hoods
Vertical Laminar Flow Hood
Horizontal Laminar Flow Hood
 Specific Requirements: Centrifuges

• Routinely used in tissue culture for the

concentration of cells for cryopreservation.
• An ideal centrifuge should have a clear lid so
that the condition of the load can be observed
without opening the lid.
• Care should be taken not to over fill the tubes
and to balance them carefully.
• These simple steps will reduce the risk of
aerosols being generated.
Specific Requirements: Incubators
• They are constant temperature rooms used to grow cells
and tissues.
• Provides controlled temperature environment facilitating
the growth of animal cells and tissues.
• Degree of humidity and CO2 levels can be in a controlled
manner.
• Usually incubators are set to run at 28°C for insect cell lines
and 37°C for mammalian cells.
• Copper coated incubators reduces the risk of microbial
contamination.
• Water bath treatment trays will also reduce the risk of
bacterial and fungal growth in the water trays.
Specific Requirements: Incubators
 Specific Requirements: Work Area and
Flooring
• Work area and flooring must be smooth
enough for easy cleaning.
• They should be waterproof and resistant to a
variety of chemicals such as acids, alkalis,
solvents and disinfectants.
• Cold storage rooms should be resistant to
cracking.
• Walls and windows should be covered with
screens to avoid dusting.
 Specific Requirements: Plasticwares

• Cell culture vessels are commercially available

for single use as sterile packed, plasticwares.
• Not effective when recycling.
• They should be used only once (use and
throw).
• They provide a hydrophilic surface to facilitate
the attachment of binding dependent cells.
Specific Requirements: Plasticwares
Specific Requirements: Glasswares
• Flasks, roller bottle, hollow fibre catridge,
petridishes, glass slides etc., are used.
• Conical flasks are commonly used for
formulation of medium.
• Test tubes are used for trypsinization.
• Pipettes are used for aseptic transfer of cell
cultures and media b/w vessels.
 Water bath
• To warm media, TRED
and PBS before placing
on cells
• Can harbor fungi and
bacteria, spray all items
with 70% ethanol before
placing in the hood.
• Usually takes 10 -15
minutes for media to
warm, 5-10 for TRED to
thaw
 Vacuum pump
• For permanent
aspiration of liquids
(media, PBS and
TRED).
• Use unplugged glass
pasteur pipets, throw
into sharps box when
done.
 Inverted Phase Microscope
• A phase contrast
microscope with objectives
below the specimen.
• A phase plate with an
annulus will aid in exploiting
differences in refractive
indices in different areas of
the cells and surrounding
areas, creating contrast
 Cell Culture
• Pros
• Use of animals reduced
• Cells from one cell line are homogenous
and have same growth requirements,
optimizing growing patterns.
• In vitro models allow for control of the
extracellular environment
• Able to monitor various elements and
secretions without interference from other
biological molecules that occurs in vivo
• Cons
• Removal of cells from their in vivo environment
means removing the cells, hormones, support
structures and various other chemicals that the
cells interact with in vivo.
• It is nearly impossible to recreate the in vivo
environment. The artificial conditions could cause
cells to de-differentiate which will cause them to
behave differently and produce proteins other
than it would in vivo.
– Genotype: the genetic make-up of the cell
– Phenotype: the appearance and behavior of a cell as a
result of their genotype. Most often, scientists are
looking at phenotypic changes in their analysis of cells
in culture