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BASIC ANIMAL CELLCULTURE HANDLINGHISYAM ABDUL HAMID IPoPS2013
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Ice-breaking
Transfer cells from subjects/models into artificialenvironment finite/continuous cell
Convenient tool to study animal cell biology
Fussy discipline requires a lot of considerations
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Getting started - environment
Safety
Certain human-derived cancer cell harbouring pathogen(potentially harmful yet to be known)
Equipment and chemical hazardeg: trypan blue, EtBr carcinogenic, UV irradiation, thin glassslide
NO mouth pipetting!
NO smoking, eating or drinking!
DO label!
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Getting started - environment
Contamination
Sources of contamination: bacteria, yeast, fungi, mycoplasma,cross-contamination etc..
Sterile
Air circulation/Ventilation
Aseptic technique
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Aseptic technique
Sterile media and reagents autoclave/filter
Avoid aerial contamination of solutions
Avoid repeated opening of bottles
70% ethanol swab
UV irradiation before and after
Disposable items one time usage only
Wear proper and clean cloth, glove/mask
Pipetting technique and cell handling
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Getting started materials andinstrumentation
1. Equipments
Biosafety cabinet
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Getting started materials andinstrumentation
1. Equipments
Centrifuge Refrigerator
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Getting started materials andinstrumentation
1. Equipments
IncubatorInverted microscope
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Getting started materials andinstrumentation
1. Equipments
Liquid nitrogen tankAutoclave
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Getting started materials andinstrumentation
2. Accessories
Pipettor
Haemocytometer
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Getting started materials andinstrumentation
2. Accessories
Consumable items
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Getting started materials andinstrumentation
3. Materials
Reagents
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Choosing the Cell
What type of study?
Cytotoxic preliminary study
Mechanism of reaction
Detection, production and function of hormones, growthfactors etc..
The study of interactions: cell-cell and cell-matrix
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Choosing the Cell
What type of cell?
Cell line?
- AKA immortalized/ transformed cells More morphologically
and physiologically resemble the parent cell
- Acquired stable and heritable mutation giving the cell ability toproliferate infinitely
Disadvantages
Prone to mutation
behavioral and physiological changes whichmay interfere the result of the experiment
Adapted from ATCC animal cell culture guide; retrieved from
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Maintaining the Cell Growth Media
Sodium bicarbonate stabilize pH by regulating the level of CO2
Phenol red monitor media pH (can mimic certain steroid
hormone)
L-glutamine protein production, energy source and nucleic acidmetabolism.
Amino acid
Vitamins
INGREDIENTS
Adapted from ATCC animal cell culture guide; retrieved from
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
+ MEDIA SUPPLEMENTS such as antibiotic/antimycotic, animal sera
Adapted from ATCC animal cell culture guide; retrieved from
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Maintaining the Cell Culture Vesseland Surfaces
Glass vessel were originally used heavy, expensive, labor-intensive cleaning and poor microscopic viewing
replaced by plastic vessel surface treatment techniques were
developed for polystyrene
Provide additional protection from contamination and simplerincubator requirements
Plastic walls is slightly permeable to CO2 and O2 might interferewith anoxia study and long-term storage media
Loose cap and vented cap
Adapted from ATCC animal cell culture guide; retrieved from
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
Adapted from ATCC animal cell culture guide; retrieved from
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Maintaining the Cell Culture Vesseland Surfaces
3 types of cell culture:
Anchorage
Suspension
Anchorage/suspension
4 basic culture systems:
Stationary monolayer - flask, petri dish, well plate
Moving monolayer roller bottles
Stationary suspension flask, petri dish, well plate
Moving suspension spinner flask (stirred), bioreactors
SELECTING YOUR VESSEL
Adapted from ATCC animal cell culture guide; retrieved from
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
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Maintaining the Cell Cell Growth
Growth cycle of culture
Adapted from ATCC animal cell culture guide; retrieved from
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Maintaining the Cell - Subculture
Defrost/stabilize the
temperature of trypsin,
growth media
appropriate with the
temparature of the cell
(370C)
Discard cellculture medium
from the flask
Rinse the cell
with PBS
Add 2-3mL
trypin and
incubate for 5-
15 minutes
Observe the cell
detachment under
microscope gentle tap
if necessary
Add 6-8mL
complete growth
media to inactivate
the trypsin
Count/simply divide the
cell into new flask with
new complete growth
medium and incubate.
Examine the culture on
the next day to monitor
reattachment of the cell
Adapted from ATCC animal cell culture guide; retrieved from
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
Adapted from ATCC animal cell culture guide; retrieved from
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Maintaining the Cell Cell counting
1. Assemble a cleaned and dry
haemacytometer with the cover slip
2. Transfer a small amount of cell suspension
to each counting chambers
3. View under inverted microscope at 100Xmag.
4. Focus on quadrant labeled 1,2,3 and 4.
5. Record number of cell in each section.
Number of
cells insample
Average
number of
cell in
1,2,3,4
Dilution
factor104
p g ;
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
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Hayflicks Phenomenon
Normal human embryonic cells can divide up to 50 times before enteringsenescence phase
The more cell divides, the shorter the telomeres
Rubin H. (2002)
It is advisable to use cell lines with range passages
from 40-60 at most
Adapted from ATCC animal cell culture guide; retrieved from
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Advantages of cryopreservation:
Generation of safety stocks
Elimination of time, energy and materials required for
maintaining cultures
Preservation of finite cells
Insurance against phenotypic drift in culture
Creating standard reagent to be used for a series of
experiments
Cryopreservation
p g
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
Modified from ATCC animal cell culture guide; retrieved from
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Cryopreservation - procedure
Identify cell needs to be
stored
Resuspend the cell
with complete
growth medium
and transfer into
cryovials
Collect 1x106 to
5x106 viable
cells/ml
Label cryovials
properly (name,
cell line, date)
SealPut on
ice
Transfer into
-200
C freezer
Put into -800C
freezer
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
30
+ 5% DMSO
60
24 hours
Liquid nitrogen tank
Remove one vial,restore to determine
viability and sterility
24 hours
Adapted from ATCC animal cell culture guide; retrieved from
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Thawing
Decontaminatethe vial using 70%
ethanol
Remove vial
from liquid
nitrogen tank
Thaw until icemelted (gentle
agitation in
waterbath)
Centrifuge
tube
Discardsupernatant
Resuspend the cell
gently using complete
growth medium
https://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_
Guide.pdf
+ 9ml complete
growth medium
Culture vessel (should
contain at least 10ml
culture medium)
Examine and subculture
if necessary
24 hours
125 x g, 10
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References
ATCC Animal Cell Culture Guide; retrieved fromhttps://www.atcc.org/~/media/PDFs/Culture%20Guides/AnimCellCulture_Guide.pdf at 27 August 2013
Celis, J.E. Cell Biology (Third Edition) A LaboratoryHandbook. Elsevier Inc., 2006. Imprint.
Mehra A., McDonald I., Pillay T.S. (2007). Variability in3T3-L1 adipocyte differentiation depending on cellculture dish.Analytical Biochemistry362; 281-283
Rubin H. (2002). Promise and problems in relatingcellular senescence in vitro to aging in vivo.Archives ofGerontology and Geriatrics 34; 275-286
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