Note #4: Cell Biology Methods
1
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Culture of cells
 http://www.studiodaily.com/main/searchlist/6850.html
 The
Inner Life of a Cell
 http://www.youtube.com/watch?v=Id2rZS59xSE
 Medical animator David Bolinsky’s interpretation
 Text
Reading: From Ch. 8 pp566-573 and Ch. 9
pp585-596
How to obtain cells to study
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Tissue culture: A terminology for procedures used for maintenance
and growth of cells or organs in an artificial and aseptic
environment.
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Cell culture: Cells can be isolated from intact tissues and cultured as
isolated cells
Organ culture (explant cultures): The culture of the whole or fragments
of an organ to study the development.
Started early 1900; an explant of spinal cord tissue
Late 1940’s and early 1950’s, tissue culture became easier to do by:
1.
2.
3.
The use of antibiotics to control contamination;
Development of chemically defined, nutrient-rich media to grow cell lines;
The use of trypsin (protease) and collagenase to remove cells from the
tissue and the plastic vessel.
Culture of primary cells
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Removal of tissue or organs from animals and
plants, and growing in artificial, aseptic
environment.
Cut and mince with scissors
Dissociate the tissue with trypsin and collagenase
enzymes to prepare cell suspension.
Artificial environment can be glass or polystyrene
plastic vessels containing liquid or semi-solid
medium that supplies nutrient.
Adhering Cells can be grown on plastic, usually
grown in humid environment, in atmosphere of 510% CO2 pH 7.2-7.5
Some cells require attachment factors - plastic can
be coated with gelatin, collagen, fibronectin,
laminen (extracellular matrix).
Cell Properties
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Many of the cell properties are maintained in culture, including
cell shape, indicative of the cell origin, and function.
Figure 8-4 Molecular Biology of the Cell (© Garland Science 2008)
Primary Cells
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Primary cells have a finite lifespan.
Replicative cell senescence: process by which primary cells slow proliferation and
then eventually die (95%).
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“Immortalized” Cell line can grow continuously.
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Cells can be immortalized by introducing oncogenes or telomerase gene.
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Normal cells are anchorage dependent
and contact inhibited
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Monolayer
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Reach confluency after a certain
time.
The surface of the plate is covered
with one layer of cells (monolayer)
Transformed cell lines (cancer cells)
are anchorage-independent; not
contact inhibited
Exception: Blood cells (lymphoblast
cells), which are normal cells, do not
normally attach to substrates and
always grow in suspension.
Multilayer
Spinner bottles
Summary
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Primary Cells
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From living organism - normal
cells
Have to isolate from living
organism.
Have a finite lifespan.
Undergo replicative cell
senescence.
Normal cells
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Contact inhibited
Grow as mono-layer on a
plastic dish
Reach confluence
Anchorage-dependent
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Cell lines
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Easier to obtain (access)
“Immortalized” cell line can
grow continuously.
Cell line can have abnormal
chromosome number.
Transformed cells
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Not contact inhibited
Can grow as multi-layer on a
plastic dish.
Form foci
Achorage-independent - can
grow in suspension or in soft
agar
Monoclonal antibody production in
hybridoma
Figure 8-8 (part 1 of 2) Molecular Biology of the Cell (© Garland Science 2008)
Figure 8-8 (part 2of 2) Molecular Biology of the Cell (© Garland Science 2008)
Stem cells:
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Multiopotent: can differentiate into several
other cell types. Typical of adult stem cells.
Pluripotent: can differentiate into all other
cell types. Typical of embryonic stem cells.
Embryonic stem cells
Embryonic Stem Cells
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Embryonic stem cells (ES) harvested from the inner mass of the early
embryo can proliferate indefinitely; have ability to give rise to different
differentiated cells after treatment with retinoic acid, hormones, growth
factors, etc.
Pluripotent
Figure 23-68 Molecular Biology of the Cell (© Garland Science 2008)
Cell Isolation and Cell sorting
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1.
Prepare cell suspension by enzymatic digestion
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2.
Treat tissue with Trypsin, Collagenase
Cell Sorting
by cell size: Gravity sedimentation through a density
gradient
 by Fluorescence activated cell sorter
 by Magnetic beads
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3.
4.
Laser Capture Microdissection
Use an unusual characteristic of specific cells
Preferential adherence to the surface of culture dishes
 Sensitivity to pH
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Cell Sorting by cell size: Gravity
Sedimentation
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Using Magnetic beads
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Cell sorting by Fluorescence Activated
Cell Sorter
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Cell surface protein is
marked by fluorescence.
Fluorescent cells are
sorted from nonfluorescent cells
DNA can be marked by
fluorescence. 2N, N, 4N
cells are sorted by the
amount of fluorescence.
Laser Capture Microdissection
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Differential Adhesion
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Barkhatov I.M.1, Roumiantsev S.A.2, Vladimirskaya E.B.2, Afanasyev B.V.1 “Composition and functional
properties of monolayer cell culture from human umbilical cord blood “ Cellular Therapy and
Transplantation (CTT), Vol. 1, No. 2, 26 December 2008