Main Points
Learn how to culture cells
Cell culture as a research tool
DEFINITION
Cell culture: Culture of cell from chemical or
enzymatic desegregation of tissue slice or from
suspension of liquid body.
Explants culture : tissue slice (1-3 mm) place in
substrate solid to form 3 dimension structure
called Histolytic culture.
Tissue culture: non desegregated tissue, 3
dimension structure still intact.
DEFINITION
1. Primary culture: Directly remove from organ,
and place in culture dish, never sub-culture.
2. Cell line: Primary culture which has been sub
cultured
- Finite cell line 20-80 generations, dies after
several sub culture
- Continuous cell line >100 generation.
transformed immortal
Primary cultures
Remove from organ, trypsin
digest and place in culture dish
Advantages
Similar chromosome
number as parent tissue
- Perform specialized
biochemical properties a
parent tissue
Mouse Embryonic Fibroblasts (MEF)
Disadvantages
Limited lifespan
- Low survival rate
Meaning
Organism
HEK-293
HeLa
CHO
Sf-9
human
kidney (embryonic)
human
Cervical cancer
hamster
Ovary
Insect -Spodoptera frugiperda (Moth) Ovary
NIH-3T3
bEnd.5
MCF-10A
HMEC
MDCK II
COS-7
aethiops
HL-60
Jurkat
Origin tissue
mouse
embryo
brain
mammary gland
mammary gland
kidney
Ape -Cercopithecus
Myeloblast
T-Cell-Leukemia
Untreated Polystyrene
--------------------
-----------------TC-treated Polystyrene
~ uncharged / neutral
~ hydrophobic
~ hydrophilic
Embryoid
Bodies (EB)
muscles, blood,
blood
vessels,
connective
tissues,
heart.
pancreas,
stomach,
liver, lungs,
bladder
Petri dish
Estimated
<5%
50 - 60%
Mechanical or enzymatic
methods are used
Plate in serum-free
media+ EGF in
ULA vessels
After 6 -8 days,
mechanically harvest
neurospheres
Adapted from:
Reynolds,
Nature Methods 5:333,
2005
Polycarbonate (PC)
Membrane
Transwell Insert
Upper
Compartment
Porous Membrane
Lower Compartment
Collagen-coated
Polytetrafluoroethylene (PTFE)
Membrane
Polyester
(PET)
Membrane
CellBIND
Surface Review
attachment
of
cells
over
Pre-Wash 22
Pre-Wash 43
Post Wash 24
Post Wash 58
CellBIND
Standard TCT
CellBIND
Surface Review
Amino acids 2%
Inorganic salts
11%
Vitamins
1%
Sugars 5%
Other 3%
Serum 10%
Water 68%
pH Control
Physiological pH 7
pH can affect
Cell metabolism
Growth rate
Protein synthesis
Availability of nutrients
CO2 acts as a buffering agent in
combination with sodium bicarbonate
in the media
Essential equipment
Additional equipment
Sterilization equipment
Balance(s)
Vortex
Water purification
pH meter
Magnetic stirrer
Micropipettor(s)
Cell counter
Video camera and monitor
Hoods
No hood necessary if you have an
isolated clean room
Horizontal
Vertical or biological safety cabinets
Usually equipped with UV light for
sterilization of the work surface use it
BEFORE and AFTER not during work
Hood is not a storage area!
Incubator
For most of mammalian cells
Temp = 36oC - 37oC
Humidity 95%
CO2 5%
All mammalian cells require CO2
incubator
Non mammalian cells might require
different culture conditions
Inverted microscope
It is vital to look at cultures regularly
A morphological change is often the
first sign of deterioration in culture
Inverted because it is not good to open
tissue culture dishes
It should have phase contrast
Most cells are not dense enough to be
visible in a regular light without staining
Aseptic technique
Sterile Hood - All manipulations must be carried out in a
sterile cabinet
10-20 minutes prior to experiment turn on UV light and
blower
Turn the UV light off (you dont want to kill the cells or
sunburn your hands)
Open the cabinet
Wipe down with 70% ethanol
Bring materials into the hood
Wash all items inserted into the hood with 70% ethanol
when appropriate
Begin your work
Tightly close all bottles and caps
Remove materials from the hood
Clean after yourself !!!
Sterilization Techniques
Metal -A
Glass - A, R
Plastics A, R
Medium -R, F
Serum -R, F
Salt solutions - A, R, F
Filtration
Always filter through 0.2 m filter to remove
bacteria (bacteria are 1-2 m)
0.45 m filters remove only particulates
Mycoplasma and viruses will still filter through
Filters come in many sizes, all are
Cellulose, polycarbonate, PTFE, best are low
protein binding
Contamination
Happens to even the best
Fungal - yeast
Bacterial
Mycoplasma filterable
bacteria which will pass
across 0.2 m filter. Big
problem. It is treated with
cephalosporin antibiotics.
Can take over laboratories.
Passaging or sub-culture
Disadvatages
Need skill
Relative expensive
PRINCIPLE:
DIFERENTIAL CENTRIFUGATION
ON A DENSITY GRADIENT GIVES
HIGH-PURITY LYMPHOCYTE
PERIPHERAL BLOOD
FICOLL-HYPAQUE DENSITY
GRADIENT CENTRIFUGATION
PROCEDURE:
CULTURE FOR MITOGEN-INDUCED
PROLIFERATION OF PERPHERAL BLOOD
MONONUCLEAR CELLS
[3H] Thymidine
MTT-Assay
Gamma counter
ELISA Reader
CELL PROLIFERATION
Radioisotope-Assays
Estimating
Incorporating
Of [3H] Thymidine
Into DNA
Colorimetric-Assay
Estimating
Metabolism of the TetraZolium Salt (MTT) to The
Colored Formazan Product
By Mitochondrial Succine
Dehydrogenase Activity in
Viable Cells
Measures Energy
Metabolism of Living Cells
PRINCIPLE
MACROPHAGES CAN BE REMOVED FROM
A CELL SUSPENSION USING EITHER
THEIR ADHERENCE OR
PHAGOCYTIC PROPERTIES
ANALYSIS
I. MORPHOLOGICAL ALTERATION
II. FUNCTIONAL TEST
- Phagocytosis Assay (Latex Beads)
- Reactive Oxygen Intemediate Assay
- Cytokine Production Assay