Transcript Growing Cells in Culture

Terminology Cells
in
Culture
(PART 1)
Introduction
Cell Culture:
the cultivation or growth of cells outside of the host organism
Advantage
Disadvantages
: Allows direct access a population of cells
: the architecture of the original tissue is lost
Cells change properties over time
Two types of cell culture
Primary culture
Secondary culture
Classification of Cell
Cultures
• Primary Culture
– Cells taken directly from a tissue to a
dish
• Secondary Culture
– Cells taken from a primary culture and
passed or divided in vitro.
– These cells have a limited number of
divisions or passages. After the limit,
they will undergo apoptosis.
Primary cultures
Cells are explanted directly from a donor organism
Capable of one or two divisions in culture
Given the right conditions, survive for some time
Do not continue to grow and eventually senesce and die
Advantages
May represent the best experimental in vitro models
May retain characteristics of normal cells from that organ
Disadvantages
Difficult to obtain
Susceptible to contamination
phase contrast micrograph of a culture of
primary neurons
Growth factors are present in cell media
so that cells will keep dividing
• This is an example of how a cell culture is made from tissue.
Here, PDGF (one type of growth factor) is added--there are many others
Passage number
• The number of times the cells have been
removed (or “split”) from the plate and replated.
• Always write this on your plate or flask as
P#
The “Hayflick Limit”
replicative senescence
Normal human fibroblasts (left) and fibroblasts showing a senescent
morphology (right).
Cells get larger, more diverse morphology
Also, telomeres gradually shorten, % of polyploid cells
increases.
Classic example of a
continuously cultured cell line
HELA
Human cervical carcinoma cells transformed by HPV 18
Cell Lines
• Cell Line
– Cells that have undergone a mutation and
won’t undergo apoptosis after a limited
number of passages. They will grow
indefinitely.
• Transformed cell line
– A cell line that has been transformed by a
tumor inducing virus or chemical. Can cause
tumors if injected into animal.
• Hybrid cell line (hybridoma)
– Two cell types fused together with
characteristics of each
Passaging cells (subculturing cells)
• Process of diluting cell number in
to keep cells actively growing
• For adherent cells, when they cover
tissue culture dish, they need to
passaged
– Otherwise, the cells will become
and stop growing
order
the
be
unhealthy
Cell Culture Enemies
Cells are more susceptible to infection at certain times
• When they have been stressed after recovery from liquid
nitrogen
• Primary cells are often generated by enzymatic disruption and
selection procedures
• Cultures prepared from live animals will often be accompanied
by micro-organisms
• Splitting cells at too high a dilution can allow micro-organisms to
dominate the culture
• Cells release Autocrine growth factors which condition the
medium and favour cell growth
Types of Cell
(PART 2)
Types of cell growth
Attached cultures
-cells require a solid surface on which to grow
-plates are specially coated polystyrene
-without surface cells can’t survive
flasks
Suspension cultures
-Liquid cultures, cells do not adhere to plate surface
-hematopoietic cells
Proliferation Cells:
- high nutrition,
- Growth factor
plates
Differentiation Cells:
-zero/low growth factor
- intermediate nutrition
- usually need cell cycle inhibitor
Environment
for
Cells in Culture
Part 4
Sterilization methods
• Autoclave
– Applies heat under high pressure; this
increases the boiling point of water to 121ºC
(normal boiling point of water is 100ºC)
– 15-20 min. is sufficient to kill most microbes
• Filtration
– Large volumes: suction filter
– Small volumes: syringe filter
• UV radiation
– Causes mutations to form in the DNA of
microbes, causing
genetic damage and
eventual death
– Used to sterilize surfaces (such as the
surface of laminar
flow hoods)
Asceptic technique
-execution of tissue culture procedures without the introduction
of contaminating microorganisms
Work with cells in a cell culture hood
-laminar flow hoods
-prevent airborne organisms from entering your cultures
-always use ETOH to clean hood before and after use
-always use separate sterile pipettes for each manipulation
-never sneeze directly in your culture
-work rapidly but carefully
Asceptic technique
A typical laminar flow hood
Filtered air enters the work space from the back
Do not block vents!
UV lights can be turned on after the work is finished
to sterilize surfaces.
Incubators are required for
mammalian cell
A typical incubator for cell culture.
-internal temperature is controlled.
-CO2 incubators contain a continuous flow of
carbon dioxide-containing air.
The tanks to the right of the incubator are carbon dioxide tanks
used to provide the carbon dioxide for cell culture.
Growing cells in culture
Place in culture dish in proper media at appropriate density
Passage cells using a dilution appropriate for the cell type
-often 1:5, 1:10 or 1:20
Remove attached cells using trypsin to break attachments
Resuspend cells in new media and put into fresh dish
Put back in incubator
Confluency
• How “covered” the growing
surface appears
• This is usually a guess
• Optimal confluency for
moving cells to a new dish
is 70-80%
– too low, cells will be in lag
phase and won’t proliferate
– Too high and cells may
undergo unfavorable
changes and will be difficult
to remove from plate.
YEAST & BACTERIAL
CULTURE
Part 6
Typical ingredients list in media
to grow:
E. coli bacteria
Yeast
• Tryptone
– Peptide; source of
amino acids
• Yeast extract
– Source of vitamins,
minerals, and
nucleic acids
• Glucose
– Energy source
• Salts
• Peptone
– Peptide; source of
amino acids
• Yeast extract
– Source of vitamins,
minerals, and
nucleic acids
• Glucose
– Energy source
Working with bacterial cells
• Bacterial cells are grown as either
liquid or solid cultures
– Solid cultures (nutrient agar plates) are used to
isolate single bacterial cells with specific
properties (more on this later)
– Liquid cultures are used to scale up cell cultures
(grow larger volumes of cells)
• Starting liquid cultures
– Involves seeding liquid media with cells from
either another liquid culture or a colony from a
solid culture plate
• Starting solid cultures
– “Streaking plates”
Nutrient agar plates
for solid cultures of bacteria
• Agar: an
unbranched
polysaccharide
obtained from
the cell walls of
some species
of seaweed
– Nutrients are
added to allow
bacteria to
grow
– Liquefies with
heat, solidifies
as it cools
(~60ºC)
Streaking a plate
(to start a solid bacterial culture)
• Use sterile loop to add cells to plate; then resterilize
loop to repeatedly spread and dilute cells on plate in
such a way as to obtain single bacterial colonies
– Each colony arose from one cell Obtaining single colonies is
the goal of this procedure
Monitoring cell growth in liquid
bacterial cultures
• OD: stands for optical density
• The number of cells in a bacterial culture
can be estimated by reading the
absorbance at 600 nm (OD600).
• Want to maximize cell
density while keeping
cell cultures in growth
phase
Yeast
• Yeast are small, unicellular eukaryotic cells
• Grow at 30ºC (sometimes lower when expressing
certain proteins)
• Most common yeast
species in BT/BM is
Saccharomyces
cerevisiae (S. cerevisiae)
• S. cerevisiae is also
known as brewer’s
yeast or baker’s yeast
• S. cerevisiae are
budding yeast--they
grow by budding off
a daughter cell from
the mother cell
Working with yeast cell cultures
• Similar in many ways to working
with bacterial cultures
• Cells can be grown as liquid or
solid cultures, and lab technicians
frequently go from one type of
culture to the other
– Seed a liquid culture with a colony from a plate
– Streak a plate from a liquid culture
Example of a plasmid used to produce
recombinant proteins in E. coli
Bacterium
Cell containing
gene of interest
1 Plasmid
isolated
Gene
Inserted
3
into plasmid
Bacterial Plasmid
chromosome
Recombinant DNA
(plasmid)
2
DNA
isolated
Gene of
Plasmid
put
interest
4
into cell
DNA
Recombinant
bacterium
5
Cell multiplies with
gene of interest
Copies of gene
Gene for pest
resistance
inserted into
plants
Copies of protein
Clone of cells
Gene used to alter bacteria
for cleaning up toxic waste
Protein used
to make snow
form at higher
temperature
Protein used to dissolve blood
clots in heart attack therapy