Download Slides - Workforce Development in Stem Cell Research

Overview
• Introduction to Cell Culture.
– History and definitions of cultured cells
• General Modern Cell culture techniques
– Aseptic technique
– Media
– Substrates
– Passage methods
• Culturing and Human Pluripotent Stem Cells
– Morphology
– Media
– Passaging
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
History of Cell Culture
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Sydney Ringer (1882): Ca2+ containing salt solution that could keep a
frog heart beating in a dish
– Ringer’s Solution still used to rapidly restore blood volume in trauma and burn victims
• Wilhelm Roux (1885): Successfully maintained chick embryonic neural
tissue in a dish
• Ross Granville Harrison (1907): Grew frog embryonic neural tissues in
hanging-drops of clotted lymph
– Proved that nerve fibers form as extensions from single nerve cells
• Steinhardt, C. Israeli, and R. A. Lambert (1913): grew vaccinia virus in
guinea pig corneal tissue culture
[should we include more history, including use of human fetal cells for vaccine
production - I think there is little awareness of this important fact…]
The “Immortal Cell” Dogma
Lasted 50 years
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Alexis Carrel’s Immortal Cells
– 1906: immortal cells- chick heart fibroblasts,
• Explant of chicken heart placed on glass slide, fed with clotted serum and chicken
embryo extract
• Claimed it continuously cultured for 34 years (long after the lifespan of a chicken)
with no changes in appearance or growth rate
• Hypothesized that all cells were immortal in that they could grow indefinitely without
changing. Therefore the mechanisms of aging at the organism level could not have
root mechanisms inside the cell.
– Other labs couldn’t reproduce Carrel’s results –explained by their poor technique
HeLa as a pioneer
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• HeLa is the first successful human cell culture
• Obtained from Mrs. Henrietta Lacks in 1951
• Advanced stage of cervical adenocarcinoma
Photo source: http://en.wikipedia.org/wiki/File:Henrietta_Lacks_(1920-1951).jpg
http://en.wikipedia.org/wiki/File:Hela_Cells_Image_3709-PH.jpg
Doubting the Dogma
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
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• Hayflick Limit: Normal cells have a limited lifespan
• Human fibroblast had a reproducibly limited number of times that
they will divide before replicative senescence:
–
•
the Hayflick limit ~50 population doublings
Senescence
– Exceptions germ cells, stem cells and transformed cells
– 50 generations –crisis =stop dividing but may be viable for up to 18 months
– Somehow cells have a “replicometer,” a method for keeping track of the number of
times the cell has divided.
Telomeres
References
Lecture
Lecturenotes
notes(hyperlink)
(hyperlink)
Activity
Activitynotes
notes(hyperlink)
(hyperlink)
MoreMore
links…links…
Telomeres
A region of repetitive DNA at the
end of a chromosome protecting
the end of the chromosome from
deterioration.
In normal somatic cells, each time
the cell divides, the length of the
telomere shortens.
When the telomere length gets too
short, the cell dies.
Culturing Stem Cells:
Embedded Assessment 1
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
What’s the Big Deal?
• Discuss with a person next to you:
Why is are immortalized cells so important to advancing scientific
research? What are the advantages? disadvantages?
Culturing Stem Cells:
Embedded Assessment 2
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Compare and Contrast
Carrel’s
“immortal” cells
Genetics
Potency
Lifespan
HeLa Cells
Embryonic
Stem Cells
Aneuploidy
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Aneuploidy refers to any number of
chromosomes other than the normal count
• Gains of chromosomes or translocations
contribute to abnormal cell growth
An aneuploid karyotype in a cell taken
from an individual with advanced stage
leukemia. There are chromosome
losses (Chr. 7 and 11), missing pieces
of chromosomes (Chr. 9 and 16),
completely lost chromosome pairs (Chr.
19), unidentified marker chromosomes
(at bottom), and multiple translocations
among other problems.
Cell lines: Definitions
•
•
A "primary cell line" is a population of cells derived by direct isolation from an animal. It may
not necessarily divide indefinitely.
An “established cell line” refers to a population of cells which has been expanded through
many cell doublings
–
–
•
•
•
•
•
often beyond the Hayflick limit
no apparent changes in growth behavior or phenotype
Cell doubling = the number of times the culture has grown as one cell divides to make two,
two to make four, four to make eight and so on…
Population doubling time = the time it takes for the number of cells in a culture to double
–
•
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
may be different from the time it takes for one cell to complete a cell cycle, e.g. if some of the cells are lost due to
cell death
Cell passaging = expanding or "splitting" a cell culture into a new flask or series of flasks as the
population grows
Immortalization = dividing endlessly
Transformation = alteration in certain growth characteristics. Often associated with cancer.
Aneuploid = an abnormal number of chromosomes
Overview
• Introduction to Cell Culture.
– History and definitions of cultured cells
• General Modern Cell culture techniques
– Aseptic technique
– Media
– Substrates
– Passage methods
• Culturing and Human Pluripotent Stem Cells
– Morphology
– Media
– Passaging
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Aseptic technique
Laminar hood
Airflow =
recycled air = use toxic compounds with care
References
Lecture
Lecturenotes
notes(hyperlink)
(hyperlink)
Activity
Activitynotes
notes(hyperlink)
(hyperlink)
MoreMore
links…links…
Tissue Culture Incubators
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Maintain
– Temperature (37C)
– Humidity (close to 100%)
– Gas concentrations
• CO2 - typically ~5-7% (important for CO32--buffered media)
• O2 - optional (some incubators inject N2 to displace O2 down to O2 concentrations of as low as
2%)
• Vibration-free
• Air inside is not sterile!
• Must be cleaned regularly to reduce risk of mold and bacterial
growth
Media: Food for cells
• Early used crude cell and tissue
extracts.
• Chemically defined media
based on body fluids and
nutritional biochemistry
• Harry Eagle: Eagle’s Minimal
Essential Media (MEM)
– 13 amino acids, six ionic species,
8 vitamins, and glucose.
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Cell Culture Media
• pH Buffer
– e.g., Bicarbonate, HEPES
• Inorganic salts
– Ca2+, Mg2+, K+, Na+, Cl-, SO42-, PO43-
• Small organic molecules
– Energy substrates (glucose, pyruvate)
– Amino Acids
– Vitamins
• Anti-oxidants
– 2-Mercaptoethanol, ascorbic acid, monothioglycerol, glutathione
• Serum / Serum Replacement
– Proteins (serum albumin)
– Growth factors and hormones (insulin etc)
– Lipids
• Antibiotics
– Penicillin, Streptomycin
– Phenol Red
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Chemically Defined Media
Example: mTeSR hESC Media
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Serum and growth factors
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
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• Most medias still rely on serum.
• Serum: Protein-rich fluid that separates out
when blood coagulates
• Contains
• Commonly used serum
– Fetal bovine serum (FBS)
– Horse serum
NGF
NGF
Y
Y
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Surfaces
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• TC Plates: Gas plasma treated polystyrene
– Modifies the hydrophobic plastic surface to make it
negatively charged and hydrophilic
• Gelatin= hydrolyzed collagen
• Matrigel
– From Engelbreth Holm Swarm (EHS) sarcoma= laminin, fibronectin
and proteoglycans
• Ultra Low Attachment Plates
– Polystyrene treated with a hydrophilic, neutral coating
– Only cell-cell adhesions remain, resulting in formation of cell
aggregates (e.g. embryoid bodies from embryonic stem cells)
– Also useful for culture of non-adherent cell (e.g., hematopoietic cells)
Overview
• Introduction to Cell Culture.
– History and definitions of cultured cells
• General Modern Cell culture techniques
– Aseptic technique
– Media
– Substrates
– Passage methods
• Culturing and Human Pluripotent Stem Cells
– Morphology
– Cell dissociation techniques
– Passaging
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Two Types of ESCs!
mESCs
Inner Cell Mass
(epiblast+hypoblast)
hESCs
mEpiSCs
Epiblast
Amnion
(epithelialized)
posterior
anterior
Trophoblast
Blastocoele
Primary Yolk Sac
(human embryo)
Hypoblast
(primitive endoderm)
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Mouse and Human ES cells are
typically grown on Feeders
mESCs
Phase-Contrast Microscopy
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
hESCs
grown on mouse embryonic fibroblasts (MEFs)
Mouse Embryonic Fibroblasts
(a.k.a. MEFs or feeders)
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Derived from ~13.5 days old mouse embryos
– Culture of adherent cells from whole embryo excluding visceral organs (and head)
• Grown on gelatin-coated dishes in DMEM/FCS media
• Produce Proteins (e.g., ECM and growth factors) that:
– facilitate adhesion,
– promote self-renewal,
– and inhibit differentiation of pluripotent stem cells in culture
• MEFs used as stem cell feeder cells are treated to prevent proliferation
– irradiated or treated with the cell cycle drug mitomycin C
– lack of MEF cell proliferation helps maintain an optimal feeder cell density
• Feeder cell density
– optimal MEF feeder cell density differs between mESCs and mEpiSCs or hESCs
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Stem Cell Microscopy
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Inverted Microscope
Dissecting- or
StereoMicroscope
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Stem Cell Microscopy
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
hESC Colony
Phase Microscopy
Dark-field Microscopy
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Stem Cell Culture Quality
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Watch for changes in morphology, growth and plating rate, as well
as ability to differentiate
• Aneuploidy can occur even in low-passage cultures (particularly
when sub-optimal culture methods are used)
• Regularly check karyotype (~every 10-20 passages, and before/after
experimental manipulations)
• Certain contaminations can be difficult to detect without special
testing (mycoplasma, viruses)
• Take extreme care to prevent cross-contamination of cell lines
(contaminations can be detected by DNA fingerprinting
Stem Cell Homeostasis (1)
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Tissue-restricted adult stem cells reside in their niche as quiescent or
slow-cycling cells
• Homeostasis: adult stem cells proliferate when signaled to do so –
for example, when cells need to be replaced after an injury
• There is no culture system for
keeping ESCs in a stable,
quiescent state (like Alice in
Wonderland’s the “Red Queen”, they
must run to stay in place)
Stem Cell Homeostasis (2)
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
The decision to differentiate occurs during the G1 phase of
the cell cycle (G1 phase is extremely short in fast-cycling ESCs)
• When cultures of ESCs get very dense, metabolic
stresses and their cell cycle time can increase
• ESCs differentiate in an uncontrolled fashion
• IMPORTANT: regularly feed, monitor, and
split (passage) embryonic stem cells in order to
maintain optimal growth conditions and cell
densities!
Cell dissociation techniques
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
• Enzymatic: protease digestion of the ECM, cell-cell-, and
cell-ECM interaction proteins (receptors)
– Trypsin
– Collagenase
– Dispase
• Chemical: many cell-cell interactions are dependent on
bivalent ions (Ca2+)
– EDTA
• Mechanical
– Scraping
– Mechanical dissection
– Triturating
Culturing Stem Cells:
Concept Mapping Terms
References
Lecture notes (hyperlink)
Activity notes (hyperlink)
More links…
Add the key terms/concepts from today’s lecture to your
previous concept map. You should include (but are not
limited to) the following terms/concepts:
• Confluence
• Senescence
• Transformed cells
• Immortalized cell line
• Ploidy
• Aneuploid
• Serum
• Telemere
Due by _________.
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