Download Introduction to tissue culture cell line

Introduction to tissue culture
cell line
Dr Ghada Abou El-Ella
Definition of Tissue Culture
Tissue culture is the branch of biology in which
tissues or cells of higher animals and plants are
grown artificially in a controlled environment.
Such studies were undertaken in the hope that
the behavior of various body components could
be studied and their potentialities more readily
analyzed under the simpler and more readily
manipulated conditions possible in the test tube.
Definition of Tissue Culture
Tissue culture is the term used for “the process
of growing cells artificially in the laboratory”
Tissue culture involves both plant and animal
cells
Tissue culture produces clones, in which all
product cells have the same genotype (unless
affected by mutation during culture)
History of tissue culture
The 1st culture of animal tissue was done in 1907 by
Ross Harrison where he cultured nerve tissue of frog in
blood clot also cultured at room Temp cold blooded.
Tissue culture had its origins at the beginning of the 20th
century with the work of Gottleib Haberlandt (plants)
and Alexis Carrel (animals)
The first commercial use of plant clonal propagation on
artificial media was in the germination and growth of
orchid plants, in the 1920’s
History of tissue culture
In the 1950’s and 60’s there was a great deal of
research, but it was only after the development of a
reliable artificial medium
A more recent advance is the use of plant and
animal tissue culture along with genetic
modification using viral and bacterial vectors and
gene guns to create genetically engineered
organisms
Basic requirements for plant and
animal tissue culture
Both plant and animal tissue culture have
several critical requirements:
Appropriate tissue (some tissues culture better
than others)
A suitable growth medium containing energy
sources and inorganic salts to supply cell
growth needs. This can be liquid or semisolid
Basic requirements for plant and
animal tissue culture
Aseptic (sterile) conditions, as microorganisms
grow much more quickly than plant and animal
tissue and can over run a culture
Growth regulators as auxins & cytokinins in
plants or it will be provided in serum as in animals
Frequent sub culturing to ensure adequate
nutrition and to avoid the build up of waste
metabolites
Animal tissue /cell culture
differences from plant tissue culture
Animal cell lines have limited numbers of cell cycles
before they begin to degrade
Animal cells need frequent sub culturing to remain
viable
Animal Tissue culture media need in addition to
inorganic salts, energy sources, amino acids, vitamins,
etc., they require the addition of serum (bovine serum is
very common, but others are used)
Animal tissue cultures can pose biohazard concerns, and
cultures require special inactivation with hypochlorite
(e.g. Janola, Chlorox, etc.) and then incineration
Uses of Animal Tissue Culture
Growing viruses - these require living host cells
Making monoclonal antibodies, used for
diagnosis and research
Studying basic cell processes
Genetic modification & analysis
‘Knockout’ technology - inactivating certain
genes and tracing their effects
Providing DNA for the Human Genome Project
(and other species’ genome projects)
Advantages of Tissue Culture
Need fewer animals
Can look at direct effect of a substance on a cell type
without interaction with any other substance.
Homogeneity of cell type
Economy
a- where we used fewer reagents
b- less costly than maintaining animals
N.B
(A) Cancer cells divide forever, i.e are immortal.
(B) Cancer cells don't show contact inhibition.
Primary culture
How to get cells from the animal
A) Explant method:
-Cut block of tissue from animal
-Let cells migrate out, remove tissue
B) Disaggregation method:
-Cut small tissues from animal
-Incubate in trypsin
-Agitate to disaggregate tissue to cell suspension
-Plate in culture medium
C) Can count cells in hemacytomer before plating
-Count # cells in 0.l µl , multiply by 10,000 to get cells / ml of medium
viable cells / total cells plated = plating efficiency
TYPES OF CELLS GROWN IN
CULTURE
Tissue culture is often a generic term that
refers to both organ culture and cell culture.
Cell cultures are derived from either primary
tissue explants or cell suspensions.
Primary cell cultures typically will have a
finite life span in culture .
Continuous cell lines are, by definition,
abnormal and are often transformed cell lines.
II. WORK AREA AND EQUIPMENT
A. Laminar flow hoods.
There are two types of laminar flow hoods,
vertical and horizontal.
The vertical hood( biology safety cabinet): is
best for working with hazardous organisms since
the aerosols that are generated in the hood are
filtered out before they are released into the
surrounding environment.
Horizontal hoods are designed such that the air
flows directly at the operator hence they are not
useful for working with hazardous organisms but
are the best protection for your cultures.
A. Laminar flow hoods
Both types of hoods have continuous
displacement of air that passes through a HEPA
(high efficiency particle) filter that removes
particulates from the air.
In a vertical hood, the filtered air blows down
from the top of the cabinet; in a horizontal hood,
the filtered air blows out at the operator in a
horizontal fashion.
NOTE: these are not fume hoods and should not
be used for volatile or explosive chemicals.
They should also never be used for bacterial or
fungal work.
A. Laminar flow hoods
The hoods are equipped with a short-wave UV light
that can be turned on for a few minutes to sterilize the
surfaces of the hood, but be aware that only exposed
surfaces will be accessible to the UV light.
Do not put your hands or face near the hood when the
UV light is on as the short wave light can cause skin
and eye damage.
The hoods should be turned on about 10-20 minutes
before being used.
Wipe down all surfaces with ethanol before and after
each use.
Keep the hood as free of clutter as possible because this
will interfere with the laminar flow air pattern.
B. CO2 Incubators
The cells are grown in an atmosphere of 5-10%
CO2 because the medium used is buffered with
sodium bicarbonate/carbonic acid and the pH must
be strictly maintained.
Culture flasks should have loosened caps to allow
for sufficient gas exchange. Cells should be left out
of the incubator for as little time as possible and the
incubator doors should not be opened for very long.
The humidity must also be maintained for those
cells growing in tissue culture dishes so a pan of
water is kept filled at all times.
C. Microscopes
Inverted phase contrast microscopes are used
for visualizing the cells. Microscopes should
be kept covered and the lights turned down
when not in use. Before using the microscope
or whenever an objective is changed, check
that the phase rings are aligned.
D. Vessels
Cells require a nontoxic, biologically inert, and
optically transparent surface that will allow cells
to attach and allow movement for growth.
The most convenient vessels are specially-treated
polystyrene plastic that are supplied sterile and are
disposable.
Suspension cells are shaken, stirred, or grown in
vessels identical to those used for anchoragedependent cells.
III. Media and growth requirements
Physiological parameters
Temperature - 37ºC for cells from homeotherm
pH - 7.2-7.5
humidity is required
gas phase - bicarbonate conc. and CO2 tension in
equilibrium
visible light - can have an adverse effect on cells;
light induced production of toxic compounds can
occur in some media; cells should be cultured in the
dark and exposed to room light as little as possible;
Medium requirements
z
z
z
z
z
z
Bulk ions - Na, K, Ca, Mg, Cl, P, Bicarb or CO2
Trace elements - iron, zinc, selenium
sugars - glucose is the most common
amino acids - 13 essential
vitamins - B, etc.
choline, inositol.
Growth and morphology
Visually inspect cells frequently. Cell culture is sometimes
more an art than a science.
Get to know what makes your cells happy!!! Frequent
feeding is important for maintaining the pH balance of the
medium and for eliminating waste products.
Cells do not typically like to be too confluent so they
should be subcultured when they are in a semi-confluent
state.
In general, mammalian cells should be handled gently.
They should not be vortexed, vigorously pipetted or
centrifuged at greater than 1500 g.
Cell feeding
prewarmed media and have cells out of the
incubator for as little time as possible.
a. Suspension cultures: Feeding and subculturing
suspension cultures are done simultaneously.
About every 2-3 days, dilute the cells into fresh
media.
b. Adherent cells. About every 2-3 days, pour off
old media from culture flasks and replace with
fresh media.