bcbt 478 introduction to mammalian cell culture 2009
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8/9/2019 BCBT 478 Introduction to Mammalian Cell Culture 2009
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BCBT 478:
Plant and Mammalian Tissue Culture
Introduction to Mammalian Tissue Culture
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Tissue Culture
Tissue Culture:
The general term for the removal of cells,
tissues or organs from an animal or plant
and their subsequent placement into an
artificial environment conducive to growth.
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Organ Culture
Organ Culture
The culture of whole organs or intact organ
fragments with the intent of studying their
continued function or development.
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Cell Culture
Cell Culture
When cells are removed from the organ
fragments prior to, or during cultivation,
thus disrupting their normal relationships
with neighboring cells.
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Mammalian Cell Culture
Mammalian Cell Culture
Cell culture of
mammalian cells.
Eukaryotic cells are
much more difficult to
culture than most
prokaryotes.
They demand complex
media
They are very
susceptible to
contamination and
overgrowth by microbes
such as bacteria, yeastsand fungi.
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Cell Culture
Two types of cell culture
Primary Culture
Cell Line Culture
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Primary Culture
Come from the outgrowth of migrating
cells from a piece of tissue or from tissue
that is disaggregated by enzymatic,chemical, or mechanical methods.
Formed from cells that survive thedisaggregation process, attach to the cell
culture vessel (or survive in suspension),
and proliferate.
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Primary Culture
Primary cells are morphologically similar
to the parent tissue.
These cultures are capable of only a
limited number of cell divisions, after
which they enter a nonproliferative statecalled senescence and eventually die
out.
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Primary Culture
Primary cells are considered by many
researchers to be more physiologically
similar to in vivo cells.
Primary cell culture is generally more
difficult than culture of continuous celllines.
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Primary Culture
Advantages
They are thought to
represent the bestexperimental models
for in vivo situations.
Have the same
karyotype as the
parent tissue normal
or abnormal.
DisadvantagesDifficult to obtain.
Relatively short lifespan in culture.
Very susceptible to
contamination
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Finite Cell Lines
Finite cell cultures are
formed after the first
subculturing (passaging)
of a primary cell culture.
These cultures will
proliferate for a limited
number of cell divisions,after which they will
senesce.
The factors which
control the replication of
such cellsin vitro
arerelated to the degree of
differentiation of the cell
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Finite Cell Lines
The cells will proliferate for an extended time,
but usually the culture will eventually cease
dividing, similar to senescent primary cells.
Use of such cells is sometimes easier than use
of primary cell cultures, especially for
generation of stably transfected clones.
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Finite Cell Lines
MRC5 cells
Human embryoniclung fibroblasts
Undergo between60-70 doublings
before senescence.
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Finite Cell Lines
Advantages
Can obtain a large
population of similar cells.
Most cellular
characteristics are
maintained
Disadvantages
Cells have a
tendency todifferentiate over
time in culture.
Over time the culture
tends to select for
aberrant cell
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Continuous Cell Line
A cell line that hasdemonstrated thepotential to besubculturedindefinitely.
Infinite cell line
Immortal cells line
Immortalized celllines are also knownas transformedcells:
Cells whose growthproperties have beenaltered.
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Continuous Cell Line
Finite cell cultures will eventually either die out
or acquire a stable, heritable mutation that gives
rise to a continuous cell line that is capable of unlimited proliferative potential.
This alteration is commonly known as in vitro
transformation or immortalization and frequentlycorrelates with tumorigenicity.
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Continuous Cell Line
Continuous cell lines are generally
easier to work with than primary or finite
cell cultures.
These cells have undergone genetic
alterations and their behavior in vitromay not represent the in vivo situation.
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HeLa Cells
Classic example of
an immortalized cell
line.
These are human
epithelial cells from a
fatal cervical carcinomatransformed by human
papillomavirus 18
(HPV18).
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Continuous Cell Line
Advantages
Easy to maintain in
culture.
Easy to obtain large
population of cells.
Typically easy to
manipulate gene
expression.
Disadvantages
The more aggressive
the cell line the moreit changes over time
in culture.
Not clear how the
function of these
cells relates to that of
other cells, healthy
or diseased.
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Transformed Cells
Transformed Cells
Changed from normal cells to cells with many of
the properties of cancer cells.
Some of these cell lines have actually been
derived from tumors or are transformed
spontaneously in culture by mutations.
No matter how transformation occurred, the result
is a cell with altered functional, morphological,
and growth characteristics.
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Know Your Cells
The more you know about the cells and
the more finely attuned you are to the
cell¶s quirks, the quicker and more clear the interpretation of results will be.
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Know Your Cells
The more differentiated the cell line, the slower
it will grow.
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Know Your Cells
Adherently cultured transformed cells
are usually highly anchorage-
independent and adhere lightly even totissue culture dishes.
Wash these cells very carefully, as theloose monolayer can be inadvertently
aspirated away.
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Characterization by Cell
Growth
Growth characteristics are functional
descriptions and are dependent upon
cell type.
Suspension and adherent growth are
properties of the cell as well as of theculture conditions
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Characterization by Cell
Growth
Attachment CulturesTo survive and grow,
most cells require asurface to which theycan attach
Without the surface
attachment these
cells cannot survive
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Characterization by Cell
Growth
Anchorage-Dependent and Anchorage-
IndependentGrowth
A subdivision of adherent growth
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Characterization by Cell
Growth
Anchorage-Dependent
Require attachment to the
surface for cell
proliferation
Anchorage-Independent
Do not require
attachment for cell
proliferation
Growth of cells in tissue
culture dishes looks more
haphazzard than thegrowth of anchorage
dependent cells with cells
only loosely attached to
the surface.
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Characterization by Cell
Growth
The advantages of adherent growth is
the ability of the cells to adhere and
spread on surfaces such as coverslips,making microscopy, hydribidizations,
and functional assays more easily
performed.
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Characterization by Cell
Growth
SuspensionCultures
Some cells cansurvive and dividewhile beingsuspended in a fluidmedia and stirred or
shaken.
Flasks
Spinner Cultures
Shaker Cultures
A limited number of cell types can bemaintained and
grown in either format.
Can be switchedbetween formats to
meet experimentalneeds.
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Characterization by Cell
Growth
The advantages of suspension growth
are the large numbers of cells that can
be achieved, and the ease of harvesting.