Animal Cell Culture

W.M.M.S Bandara
B.Sc (Hons), MSc (Biotechnology)
MS (USA), M.I.Biol

Introduction to animal cell culture

What is animal Cell Culture?
Removal of cells from an animal
and their subsequent growth in a
favorable artificial environment.

Removal of cells from the tissue

by enzymatic or mechanical means before
cultivation

or they may be derived from a cell line or cell

strain that has already been established

History of animal cell culture

Cell culture was first successfully undertaken
by Ross Harrison in 1907
Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture
1940s: The use of the antibiotics penicillin and
streptomycin in culture medium decreased
the problem of contamination in cell culture.

History of animal cell culture

Figure 1. Early cell culture laboratories (circa 1930) at

Central Cancer Research Labs which would later become
part of the National Cancer Institute. Photos courtesy of NCI
Visuals Online; source: G. Terry Sharrer, Ph.D. National
Museum of American History, unknown photographer.

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.
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.

Historical events in the development of cell culture

Contd..
1916: Rous and Jones introduced proteolytic enzyme
trypsin for the subculture of adherent cells.
1927: Carrel and Rivera produced the first viral vaccine
Vaccinia.
1940s: The use of the antibiotics penicillin and
streptomycin in culture medium decreased the problem
of contamination in cell culture.
1948: Earle isolated mouse fibroblasts which formed
clones from single cells. Fischer developed a chemically
defined medium, CMRL 1066.
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.

Historical events in the development of cell culture

Contd..
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.
1982: Human insulin became the first recombinant
protein to be licensed as a therapeutic agent.
1985: Human growth hormone produced from
recombinant bacteria was accepted for therapeutic use.
1990s: Many recombinant products in clinical trial

1980 to date: Tissue culture becomes less of an

experimental research field, and more of a widely
accepted research tool

Historical events in the development of cell culture

Cell culture laboratory

Safety is very important !
Cell culture laboratory has a number of specific
hazards associated with handling and manipulating
human or animal cells and tissues, as well as toxic,
corrosive or mutagenic solvents and reagents.
Strict adherence to standard microbiological
practices and techniques is essential.

Cell culture laboratory

Safe Laboratory
Always wear appropriate personal protective equipment.
Change gloves when contaminated, and dispose of used
gloves.

Wash your hands after working with potentially

hazardous materials.
Do not eat, drink, smoke, handle contact lenses, apply
cosmetics.
Be careful when handling of sharps (i.e., needles,
scalpels, pipettes).

Safe Laboratory
Decontaminate all work surfaces , glassware
before and after your experiments, and
immediately after any spill with an appropriate
disinfectant.
Decontaminate all potentially infectious materials
before disposal.
Report any incidents that may result in exposure
to infectious materials to appropriate personnel
(e.g., laboratory supervisor, safety officer).

Equipments required for Cell Culture

-Depends mainly on the type of research conducted.

Equipments required for Cell Culture

Basic Equipment
Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)
Incubator (humid CO2 incubator recommended)
Water bath
Centrifuge
Refrigerator and freezer (20C)
Cell counter (e.g., CountessR Automated Cell Counter or
hemacytometer)
Inverted microscope
Liquid nitrogen (N2) freezer or cryostorage container
Sterilizer (i.e., autoclave)

Equipments required for Cell Culture

Expanded Equipment
Aspiration pump (peristaltic or vacuum)
pH meter
Confocal microscope
Flow cytometer
Additional Supplies
Cell culture vessels (e.g., flasks, Petri dishes, roller bottles, multiwell plates)
Pipettes and pipettors
Syringes and needles
Waste containers
Media, sera, and reagents
Cells

Cell Culture Laboratory Facilities

Aseptic Work Area - is the need to maintain an aseptic
work area that is restricted to cell culture work.
(a separate cell culture room)
Cell Culture Hood (biosafety cabinet) - provides an
aseptic work area
Three kinds of cell culture hoods: Class I, II and III.

Equipments required for Cell Culture

Cell culture hood (laminarflow hood or biosafety

cabinet)
Provides aseptic work area

Basic Layout of a culture hood

Horizontal flow -provides protection to the culture (if the air flowing towards the
user) or to the user (if the air is drawn in through the front)
Vertical flow provides significant protection to the user and the cell culture.

Bio safety Cabinets

Class I
an open-front negative pressure cabinet The exhaust air from
the cabinet is filtered by a high-efficiency particulate air
(HEPA) filter. The Class I biosafety cabinet will provide
personnel and environmental protection, but not product
protection.

Class II
an open-front, ventilated cabinet. This cabinet provides a
HEPA-filtered, recirculated mass airflow within the work
space. The exhaust air from the cabinet is also filtered by
HEPA filters. Thus, the Class II biosafety cabinet will provide
personnel, environment and product protection
Class III
a totally enclosed ventilated cabinet of gas-tight construction.
Operations within the Class III cabinet are conducted through
attached rubber gloves.

Bio safety Cabinets

Class I

Bio safety Cabinets

Class II

Bio safety Cabinets

Class III

Equipments required for Cell Culture

Incubator (humid CO2

incubator recommended)

Inverted microscope
-to observe cells

Incubator
Incubator -provides the appropriate environment
for cell growth.
5% CO2
370C
Appropriate humidity

Equipments required for Cell Culture

Other equipments
Water bath
Centrifuge
Refrigerator and freezer
(20C)
Cell counter (Automated
Cell Counter or
hemacytometer
Liquid nitrogen (N2)
cryostorage container
Sterilizer (i.e., autoclave)

Cell Storage (Cryopreservation)

Cryogenic Storage - Do not store
cells in 20C or 80C freezers,
because their viability quickly
decreases at these temperatures.
Liquid-nitrogen storage systems
are used..

Equipments required for Cell Culture

Expanded Equipment
Flowcytometer To
identify, characterize
and/or sort desired
cell types.

Equipments required for Cell Culture

Expanded Equipment
Phase contrast microscope
a type of light microscopy
that enhances contrasts of
transparent and colorless
objects by influencing the
optical path of light. The
phase contrast microscope is
able to show components in a
cell or bacteria, which would
be very difficult to see in an
ordinary light microscope.

Equipments required for Cell Culture

Confocal Microscope

Additional Supplies
Cell culture vessels (e.g., flasks, Petri dishes, roller
bottles, multi-well plates)
Pipettes and pipettors
Syringes and needles
Waste containers
Media, sera, and reagents
Cells

Cell Counter
Cell Counter -is essential to
measure cell count and
viability (live, dead, and total
cells) accurately and precisely.
Trypan Blue uptake technique.

Counting cells

Total No of Cells: 24
Total No of cells per square = 6
Total volume = 10 4
Dilution factor = D
Cell concentration = 6 X 10 4 X D / mL

Square

# cells

Counting Viable cells

Trypan Blue assay
Mix cells with Trypan blue, fill the haemocytometer and
observe under the microscope.

Dead cells stained blue with trypan blue can be counted

separately for a viability count
Percentage viability =Live cell count X 100
Total cell count

Commonly used glassware for cell culture

Cell culture vessels

Flat-bottom and coated, cell
culture vessels are needed for cell
growth.
Three types of culture vessels are
commonly used : flasks, dishes,
and multiwell plates.
All three types are available in
different sizes with different
surface areas.
The choice of the vessel depends
on the nature of the procedures
and personal preference.

Culture Flasks
PYREX glass flasks and Petri dishes
Disposable plastic culture vessels are
commonly used
Flasks, dishes and 96 well plates
made up of polystyrene, a long
carbon chain polymer with benzene
rings attached to every other carbon
are available commercially
Polystyrene was chosen because it
has excellent optical clarity, easy to
mold and can be sterilized by
irradiation.

Tissue Culture Treated Flasks

Drawback of polystyrene: it is a very
hydrophobic - cells have difficulty attaching.
Hydrophobic polystyrene surface are modified
to a more hydrophilic surface.
Corona discharge , extracellular matrix,
collagen, laminin and fibronectin, and heparin
sulfate, hyaluronidate and chondroitin sulfate,
poly-D-lysine (PDL), as coatings

Vented and Plug sealed caps

Flasks with vented caps

are preferred, since nonvented caps need to be
loose when the flasks are
in the incubator to allow
for exchange of gases

-Commercially available disposable

culture vessels are sterile
-No need to sterilize
-Transfer of media must be done using a
sterile pipettes

Applications of Cell Culture

Excellent model systems
for studying the normal
physiology and
biochemistry of cells (e.g.,
metabolic studies, aging)
Study the effects of
drugs and toxic
compounds on the cells
Drug screening and
development
Large scale manufacturing
of biological compounds
(e.g., vaccines, therapeutic
proteins)

Advantages and disadvantages of cell culture

The major advantage of using cell culture is the
consistency and reproducibility of results that can be
obtained from using a batch of clonal cells.
Represent the best experimental models for in vivo
situations.
Can have the same karyotype as the parent tissue
normal or abnormal.
Can maintain cells in undedifferentiated states.

Advantages and disadvantages of cell culture

Control of the environment

Characterization and homogeneity of the samples
In vitro modeling of in vivo conditions
Economy, scale and mechanization of culture
Avoid animal experiments
Mimic of in vivo cell behavior (e.g cancer cells)

Advantages and disadvantages of cell culture

However the cells can acquire undesirable
characteristics and mutations in culture.
Considerable variation in population and between
preparations
Difficult to obtain the cells from tissues.
Some cell types have relatively short life span in
culture.
Contaminations are common.
The cells may not fully act like in vivo (tissue/organ)

Cell Culture associated problems

Two main categories
1. Chemical contaminants such as impurities in
media, sera, and water, endotoxins, etc.
2. Biological contaminants such as bacteria, molds,
yeasts, viruses, mycoplasma, as well as cross
contamination by other cell lines.

It is essential to have understanding of the sources of

contaminants and follow good aseptic technique.

Cell culture contaminations - Bacteria

Bacteria
- unicellular microorganisms
- few micrometers in diameters
- variety of shapes (spheres, rods
and spirals)
- fast growth rates.
Bacterial contamination is easily detected by visual within a few days of
infection; infected cultures usually appear cloudy (turbid)
Sudden drops in the pH of the culture medium occurs.
Under a low power microscope, the bacteria appear as tiny granules
between the cells, and under a high-power microscope can identify the
shapes of individual bacteria.

Cell culture contaminations - Yeast

Yeasts - unicellular eukaryotic microorganisms (Fungi)
cultures become turbid, especially if the contamination is
in an advanced stage.
little change in the pH of the culture at the initiail stages.
Under microscopy, yeast appear as individual ovoid or
spherical particles, that may bud off smaller particles.

Cell culture contaminations - Yeast

Cell culture contaminations - Molds

Molds -eukaryotic microorganisms (Fungi)
-grow as multicellular filaments called hyphae
-the pH of the culture remains stable in the initial stages
of contamination
Under microscopy, the mycelia usually appear as thin,
wisp-like filaments, and sometimes as denser clumps of
spores.
Spores of many
mold species can
survive extremely
harsh
environments

Cell culture contaminations -Mycoplasma

Mycoplasma -are simple bacteria that lack a cell wall, and they
are considered the smallest self-replicating organism.
Mycoplasma are very difficult to detect until they achieve
extremely high densities and cause the cell culture to deteriorate;
until then, there are often no visible signs of infection.
Detection: by testing the cultures periodically using fluorescent
staining, ELISA, PCR, immunostaining, autoradiography, or
microbiological assays.

Cell culture contaminations - Viruses

Viruses -microscopic infectious agents that take over the
host cells machinery to reproduce. T
heir extremely small size makes them very difficult to detect
in culture, and to remove them from reagents used in cell
culture laboratories. can be a serious health hazard to the
laboratory
Viral infection of cell cultures can be detected by electron
microscopy, immunostaining with a panel of antibodies, ELISA
assays, or PCR with appropriate viral primers.