BCBT 478:

Plant and Mammalian Tissue Culture

Introduction to Mammalian Tissue Culture

 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.
 Organ Culture

 Organ Culture
The culture of whole organs or intact organ
fragments with the intent of studying their
continued function or development.
 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.
 Mammalian Cell Culture

 Mammalian Cell Culture  They demand complex

 Cell culture of media
mammalian cells.
 They are very
 Eukaryotic cells are susceptible to
much more difficult to contamination and
culture than most overgrowth by microbes
prokaryotes. such as bacteria, yeasts
and fungi.
 Cell Culture

 Two types of cell culture

Primary Culture
Cell Line Culture
 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 the

disaggregation process, attach to the cell
culture vessel (or survive in suspension),
and proliferate.
 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 state
called senescence and eventually die
out.
 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 cell
lines.
 Primary Culture

 Advantages  Disadvantages
 They are thought to  Difficult to obtain.
represent the best
experimental models  Relatively short life
for in vivo situations. span in culture.

 Very susceptible to
 Have the same
contamination
karyotype as the
parent tissue normal
or abnormal.
 Finite Cell Lines

 Finite cell cultures are  The factors which

formed after the first control the replication of
subculturing (passaging) such cells in vitro are
of a primary cell culture. related to the degree of
differentiation of the cell
 These cultures will
proliferate for a limited
number of cell divisions,
after which they will
senesce.
 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.
 Finite Cell Lines

 MRC5 cells

 Human embryonic
lung fibroblasts

 Undergo between
60-70 doublings
before senescence.
 Finite Cell Lines

 Advantages  Disadvantages
 Can obtain a large  Cells have a
population of similar tendency to
cells. differentiate over
time in culture.
 Most cellular
characteristics are  Over time the culture
maintained tends to select for
aberrant cell
 Continuous Cell Line

 A cell line that has  Immortalized cell

demonstrated the lines are also known
potential to be as transformed
subcultured cells:
indefinitely.
 Cells whose growth
 Infinite cell line properties have been
altered.
 Immortal cells line
 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 frequently
correlates with tumorigenicity.
 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 vitro
may not represent the in vivo situation.
 HeLa Cells

 Classic example of
an immortalized cell
line.

 These are human

epithelial cells from a
fatal cervical carcinoma
transformed by human
papillomavirus 18
(HPV18).
 Continuous Cell Line

 Advantages  Disadvantages
 Easy to maintain in  The more aggressive
culture. the cell line the more it
changes over time in
culture.
 Easy to obtain large
population of cells.
 Not clear how the
function of these cells
 Typically easy to relates to that of other
manipulate gene cells, healthy or
expression. diseased.
 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.
 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.
 Know Your Cells

 The more differentiated the cell line, the slower

it will grow.
 Know Your Cells

 Adherently cultured transformed cells

are usually highly anchorage-
independent and adhere lightly even to
tissue culture dishes.

 Wash these cells very carefully, as the

loose monolayer can be inadvertently
aspirated away.
 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 the
culture conditions
 Characterization by Cell
Growth

 Attachment Cultures  Without the surface

 To survive and grow, attachment these
most cells require a
surface to which they
cells cannot survive
can attach
 Characterization by Cell
Growth

 Anchorage-Dependent and Anchorage-

Independent Growth
A subdivision of adherent growth
 Characterization by Cell
Growth
Anchorage-Dependent Anchorage-Independent
 Require attachment to the  Do not require attachment
surface for cell for cell proliferation
proliferation
 Growth of cells in tissue
culture dishes looks more
haphazzard than the
growth of anchorage
dependent cells with cells
only loosely attached to
the surface.
 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.
 Characterization by Cell
Growth

 Suspension  A limited number of

Cultures cell types can be
maintained and
 Some cells can grown in either
survive and divide format.
while being
suspended in a fluid  Can be switched
media and stirred or between formats to
shaken. meet experimental
needs.
• Flasks
• Spinner Cultures
• Shaker Cultures
 Characterization by Cell
Growth

 The advantages of suspension growth

are the large numbers of cells that can
be achieved, and the ease of
harvesting.