Transcript Research Focused Undergraduate Education

Plant and Mammalian Tissue Culture
Culture Systems and Aseptic Technique
Culture Vessels
 Mammalian cells can be
grown in a variety of
containers.
 The choice of container
is typically dependent
upon cell growth
characteristics and the
number of cells
required.
Culture Vessels
 Most tissue culture container are disposable,
made of polystyrene, and have been
radiation-sterilized.
 Untreated plastic is usually fine for
suspension cells
 Most adherent cells grow better on treated
plastic.
Culture Vessels
 Treated Plastic
 Permanent modification to the polystyrene surface
 Causes a net charge on the surface of the plastic
 Modifier used include:
• Proteins
• Plasma
• Amino Acids
Culture Vessels
 Some cells types require a specific
attachment substrate be added to the culture
dish.
 Common examples are extracellular matrix
proteins
 Collagen
 Fibronectin
 Laminin
Adherent Cells
 Flasks are commonly
used to carry and
expand cells.
 Either vented or nonvented tops.
Adherent Cells
 Dishes commonly used
for specific experiments
 Scraping cells for SDSPAGE and Western
Blotting
 Fixing and staining cells
for protein localization
and interactions.
Adherent Cells
 Multi-well plates
 6, 12, 24, 96, 384 wells
 Allow for multiple
replicates of
experiments effectively
 Different Growth Areas
for each size
Adherent Cells
Adherent Cells
 Chamber Slides
 Used to prepare
cells for microscope
studies.
Suspension Cells
Suspension cultures are usually grown
either:
In magnetically rotated spinner flasks or
shaken Erlenmeyer flasks
• This actively keeps cells suspended in medium
In stationary culture vessels such at Tflasks and bottles
• Don’t need to agitate because they are unable
to attach firmly to the surface
Suspension Cells
 Spinner Flasks
 Require special
variable speed
magnetic stir plate.
 Erlenmeyer Flasks
 Require platform
shaker
Types of Cells
 Cultured cells are usually described based
upon their morphology.
 Epithelial-like cells
• Attached to substrate and flattened in shape
 Lymphoblast-like cells
• Cells that do not attach to a substrate and have a
spherical shape
 Fibroblast-like cells
• Cells that are attached to a substrate and appear
elongated and bipolar frequently forming swirls in heavy
culture
Handling Cell Cultures
 Adherence to good laboratory practice when
working with cell cultures is essential for two
reasons:
 reduce the risk of exposure of the worker to any
potentially infectious agent(s) in the cell culture
 to prevent contamination of the cell culture with
microbial or other animal cells
Aseptic Technique
Aseptic Technique
Refers to a procedure that is performed
under sterile conditions.
This includes medical and laboratory
techniques, such as with microbiological
cultures.
Aseptic Technique
For Cell and Tissue culture this is the
execution procedures without the
introduction of contaminating
microorganisms
Aseptic Technique
 Work with cells in a
biological safety
cabinet
 laminar flow hood
 prevent airborne
organisms from
entering your
cultures
 always use ETOH to
clean hood before
and after use
Laminar Flow Hood
 A typical laminar flow
hood
 Filtered air enters the
work space from the from
above
 Do not block vents!
 UV lights can be turned
on after the work is
finished to sterilize
surfaces.
Aseptic Technique
 Always use separate
sterile pipettes for each
manipulation
 Never cough, sneeze,
or yawn directly in your
culture
 Work rapidly but
carefully
Incubator
Cell Culture Incubator
Internal temperature is
controlled.
CO2 incubators contain a
continuous flow of carbon
dioxide containing air.
Visualizing Cells
 Inverted Microscope
 Large stage so plates
and flasks can be
used.
 Magnification; 4X,
10X, 20X, 40X
Contamination
The presence of microorganisms can
inhibit cell growth, kill cells, and lead to
inconsistent results.
It is not a question of if, but when, your
cells become contaminated.
Contamination is both observed under
microscope and only by other tests.
Contamination
 Cultures can be infected through:
 Poor handling
 From contaminated media, reagents, and equipment (e.g.,
pipets)
 From microorganisms present in incubators, refrigerators,
and laminar flow hoods
 From skin of the worker and in cultures coming from other
laboratories
Contamination
 Bacteria, yeasts, fungi,
molds, mycoplasmas,
and other cell cultures
are common
contaminants in animal
cell culture.
 Cloudiness (due to
large cells in
suspension) or
filaments from mold are
obvious signs
Microbial Contamination
 The presence of an
infectious agent
sometimes can be
detected by turbidity
and a sharp change in
the pH of the medium
(usually indicated by a
change in the color of
the medium), and/or cell
culture death.
pH 8.0
pH 7.2
pH 6.5
Contamination
Mycoplasma – grow slowly and do not
kill cells but will likely alter their
behavior. Mostly tested by PCR for
specific mycoplasma genes or using kits
based on staining of growth in
cytoplasm of cells
Some labs will test every 6 months for
this kind of contamination
Contamination
Cross-culture contamination: multiple
cells growing together – based on
doubling rate, one cell may take over
the other as the dominant population
Up to 60% of cultured lines are
contaminated (NIH 2009)
Contamination
How to get rid of contamination?
AVOID at all costs: sterile techniques, clean and
properly maintained hood and incubator, clean
room.
Laziness or familiarity are most common causes.
Antibiotics may help reduce contamination but
may also alter cell functions
Clearing contamination – only for novel cell lines,
can be done with some agents.
• Wash cells to reduce contaminant burden
• Use sub-lethal doses of fungacide or antibiotic