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Cell culture technique and
its implication
341 Zoo
Dr. Gamal Badr
Associate professor
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History of tissue culture
1885: Roux maintained embryonic chick cells in saline
1965:Ham introduced serum free culture medium that supportliving cells
1965: Harris & Watkins successfully fused human and mousecells by virus
1975: Kohler & Milstein produced the first Hybridomas capable
of secreting monoclonal antibodies. 1978: Sato developed a serum free medium with a cocktail of
hormone and growth factors
!982: human insulin is produced
1985: human growth factors were produced
1986: lymphoblastoid gamaIFNlicences
1987:tissue type plasminogen activator(tPA) becamecommercially available
1989:Recombinant erythropoitin in trial
1990:Recombinent products in trial (factorVI.HBsAg, Il2, EGF,mAbs)
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What is cell culture?
Cells, previously growing in a human or animalmodified to grow in plastic or glass In the body = in vivo
On plastic or glass = in vitro
Kept in an incubator to stay at body temperature
We use special media with nutrients so the cellscan grow and divide
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What can we do with cells?
Test pharmaceutical drugs
Watch disease mechanisms
Design potential treatments
Observe the regenerative process
How do cells and tissues repair themselves afterdamage from illness or injury?
Observe the developmental process
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The Dos and Donts of cell culture
The Dos
Use personal protective equipment (PPE)
Wear dedicated PPE for tissue culture facility and keep separate from PPE in the
general lab environment.
Keep all work surfaces free of clutter.
Correctly label reagents including flasks, medium.
Only handle one cell line at a time.
Clean the work surfaces with a suitable disinfectant (e.g. 70% ethanol).
Wherever possible maintain separate bottles of media for each cell line. Examine cultures and media daily for evidence of gross bacterial or fungal
contamination.
Quality control all media and reagents prior to use.
Ensure that incubators, cabinet, centrifuges and microsocpes are cleaned.
Test cells for mycoplasma on a regular basis.
Cell Culture Protocols
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The Donts
Do not continuously use antibiotics in culture medium.
Dont allow waste to accumulate particularly within the incubators
and culture area.
Dont have too many ppl in the lab at any one time.
Dont handle cells from unauthenticated sources in the main cellculture suite.
Avoid keeping cell line continually in culture without returning to
frozen stock.
Avoid cell culture becoming fully confluent. Always sub-culture 70-80% confluency or as advised on ECACCs cell culture data sheet.
Dont allow media to go out of date.
Avoid water baths dirty.
Dont allow essential equipment to become out of ccalibration.
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Technique and instrument
Laminar flow
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Carbon dioxide incubator
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Microscope
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Tissue culture Ware
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Culture Media Sterilization
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Cell counting
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Changing Medium
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Passaging cells (subculturing cells)
Process of diluting cell number in
order to keep cells actively growing
For adherent cells, when they cover the
tissue culture dish, they need to be
passaged
Otherwise, the cells will become
unhealthy and stop growing
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Other miscellaneous Equipment
Fridge Freezer for storing medium
Liquid nitrogen Container for cryopreservation
of cells
Incubator for warming up of the medium
Bench centrifuges to separate out cell pellet.
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Sterile technique
Procedures by which cultures are manipulated
without infecting the worker or contaminatingthe cultures or the laboratory environment
Important for the cell culture
You want to be sure you are growing only the cellsyou want to grow a single unwanted cell can ruin an
experiment or a multimillion $ production run
Important for the lab worker Some cultured cells can pose health threats to
workers if they are inhaled, ingested, or absorbed--
sterile technique prevents exposure of the worker to
cultured cells
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Sterile technique: Bacteria
Media is sterilized in an autoclave
Very high pressure and temperature kills
Minimizing contamination through awareness
Inoculation loop, pipets, pipet tips, etc. should never touch
contaminating surfaces
Containers holding media and other cell additives should be kept
closed until needed, then opened briefly
Tubes and flasks are flamed whenever they are opened
The purpose of flaming is not to sterilize, but to warm the tubeand create warm air convection currents up and away from the
opening. This "umbrella" of warm, rising air will help to prevent
the entrance of dust particles carrying contaminating microbes
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Sterile technique: Tissue culture Media is usually sterilized by filtration
Standard biological filters are 0.22 mm - 0.45 mm; these remove
most microbes by trapping them in the filter This does not remove all microbes (such as mycoplasm), and will not
remove viruses
Unlike bacterial media, animal cell media cannot be autoclaved,because this would destroy many of the growth factors and other
molecules needed for cell growth Working with cells in a laminar flow hood
HEPA filter
Disinfect
70% Ethanol is sprayed in hood, onto bottles entering hood Minimizing contamination through awareness
Inoculation loop, pipets, pipet tips, etc. should never touchcontaminating surfaces
Containers holding media and other cell additives should be keptclosed until needed, then opened briefly
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Sterilization methods Autoclave
Applies heat under high pressure; this increases the boiling point
of water to 121C (normal boiling point of water is 100C) 15-20 min. is sufficient to kill most microbes
Filtration
Large volumes: suction filter
Small volumes: syringe filter
UV radiation
Causes mutations to form in the DNA of microbes, causing
genetic damage and eventual death
Used to sterilize surfaces (such as the surface of laminar flow
hoods)
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What is in the media?
Dulbecco Modified Eagles Media (DMEM) Contains glucose, some proteins, and essential salts
Contains a pH indicator (phenol red) Media looks
pink/red at pH 7.2
Acidic -yellow or orange (cell growth,
bacterial growth)
Basic -purple (no cell growth, not enough
CO2)
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Media preparation
Process of combining and sterilizing ingredients of a
particular medium Proper media prep is essential for cell culture systems!
If the media is lacking any components, the cells would
either die or be unhealthy
If the media is not properly sterilized, cells will be
contaminated with microorganisms
If glassware, pipettes, etc. used to prepare media are not
properly cleaned, cell cultures can be contaminated with
chemical residues
As a result, the cells would not produce the desired product
effectively
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More media components
Antibiotics (penicillin and streptomycin) Prevent bacterial contamination
Salts and buffers
To simulate in vivo environment Serum
Portion of blood after the cells and fibers have clotted
From cow, horse, sheep
added to media as a nutrient source for growing cells Lipids, proteins
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Phosphate Buffered Saline
Used to wash/remove excess serum thatinhibits the function of TRED.
Must be warmed in the water bath before
use so cells are not shocked by cold liquid.
Trypsin EDTA An enzyme used to detach the cells from a culture
dish.
EDTA binds calcium ions in the media that would
normally inhibit trypsin.
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Bleach
Used to destroy any remaining cells in
dishes and tubes before they are tossed in
the trash can.
Add enough to change media to clear,
wait 5 minutes,
rinse solution down sink
throw away the dish/flask/plate in the trash can.
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Potential sources of contamination in
cell culture
Equipment
Glassware, instruments, incubators.
Solutions
Media or reagents added into media
Room air
Work surfaces Operators
Hands, hair, clothing, breath, etc.
Incoming cells
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Types of contamination in animal cell
culture systems
Biological
Bacteria
Fungi
Cross-contamination by other cell cultures Chemical
Residues left from detergents or disinfectants on
glassware, pipettes, instruments, etc.
Metal ions, other impurities in water
Endotoxin: highly bioreactive part of the cell wall of
some types of bacteria (endotoxin molecules are shed
from bacteria and are left behind even after bacteria die)
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Characteristics of microbial
contamination in cell cultures
pH Sudden change in pH is often a strong
indicator of contamination
Turbidity
Media looks cloudy
Microscopic evaluation Can see individual microorganisms, often
because their motion can be seen easily under
the microscope
F th d t ti f t i ti i ll
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Further detection of contamination in cell
cultures Mycoplasma
Smallest free-living prokaryotes Not killed easily by many antibiotics
Contamination cant be seen by microscopicevaluation
Mycoplasma testing should be doneroutinely (several tests are available)
Long-term effects of mycoplasma contamination includereduced growth rate, changes in cell shape and
metabolism, and chromosome abnormalities Endotoxin
LAL test: an extract from the blood ofhorseshoe crabs is used to test for endotoxin (horseshoecrab blood contains a protein that binds endotoxin & can be
detected)
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Minimizing contamination
Contamination is a fact of life when dealing
with cell cultures
Very difficult to prevent entirely, but good lab
practices can keep contamination incidents to a
minimum
Proper cleaning and sterilization of glassware,
pipettes, and other lab instruments. Practicing sterile technique when working with
cell cultures
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Types of cell culture
Primary Cell culture
Continuous Cell lines
Adherent s S spension cells for tiss e
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Adherent vs Suspension cells for tissue
culture
Adherent cells: cells grow in asingle layer (called a monolayer)attached to the tissue culture dish Cell growth is limited by available
surface area on which cells can grow
To passage adherent cells, the cellsmust be released from the dish (doneeither enzymatically, chemically, ormechanically)
Suspension cells : cells aresuspended in liquid as single cells oras free-floating clumps of a few cells To passage suspension cell cultures, a
proportion of the cells in culture are
diluted into a larger volume of medium
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Secondary or continuous
Cell LinesAttached Cell Lines
Name Species and tissue of origin Morphology
MRC-5 (Prod. No. 84101801) Human lung Fibroblast
HELA (Prod. No. 93021013) Human cervix EpithelialVERO (Prod. No. 84113001) African Green Monkey Kidney Epithelial
NIH 3T3 (Prod. No. 93061524) Mouse embryo Fibroblast
L929 (Prod. No. 85011425) Mouse connective tissue Fibroblast
CHO (Prod. No. 85050302) Chinese Hamster Ovary Fibroblast
BHK-21 (Prod. No. 85011433) Syrian Hamster Kidney Fibroblast
HEK 293 (Prod. No. 85120602) Human Kidney Epithelial
HEPG2 (Prod. No. 85011430) Human Liver Epithelial
BAE-1 (Prod. No. 88031149) Bovine aorta Endothelial
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SUSPENSION Cell lines
Suspension Cell Lines
Name Species and tissue of origin Morphology
NSO (Prod. No. 85110503) Mouse myeloma Lymphoblastoid-like
U937 (Prod. No. 85011440)Human Hystiocytic
Lymphoma Lymphoblastoid
Namalwa (Prod. No. 87060801) Human Lymphoma Lymphoblastoid
HL60 (Prod. No. 98070106) Human Leukaemia Lymphoblastoid-like
WEHI 231 (Prod. No. 85022107) Mouse B-cell Lymphoma Lymphoblastoid
YAC 1 (Prod. No. 86022801) Mouse Lymphoma Lymphoblastoid
U 266B1 (Prod. No. 85051003) Human Myeloma Lymphoblastoid
SH-SY5Y (Prod. No. 94030304) Human neuroblastoma Neuroblast
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Storing and maintaining Continuous
Cell Lines
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Advantages of Cell Culture
Cell types kept Constant and homogenous.
There is direct access to the cells so effect oftoxicity of drug and chemicals studied without
being lost to other tissues and excreted. Replace/ reduce the number of animals. Legal,
moral, ethical issues?(animal rights Group).
Control of the environment (pH, osmoticpressure, temperature, oxygen and Carbondioxide tension.)
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Disadvantages of Cell Culture
Contamination can be Chemical (culturemedium) Or Biological (adding antibiotics).
Finding a Happy Environment.
De-differentiation
Origin of Cells
Major differences from in-vivo
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Implication for Cell Culture
Model system (many specialized functions are restored) Toxicity Test (viability of cells)
Cancer research
Virology
Cell Based manufacturing
Genetic councelling
Genetic engineering
Drug screening & development Gene Therapy
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Production of monoclonal antibodies
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Tissue Engineering
Carbon nanotube
scaffolding
Name of
scaffoldig
Made up of
Nanofiber Like carbon
Textile Polyglycolide
Gas Foam Foam like
structure due
to CO2 gas
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Growing cells in 3D Forming
Tissue in Bioreactor
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Application of Monoclonnal
Antibodies
IMMUNOLOCALIZATION
IMMUNOBlotting
Cancer Treatment
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Immunolocalization
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Immunoblotting
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Cancer Treatment
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Stem cell research
Embryonic Cell Lines
Adult Cell Line
Homeiopoitic Stem Cell
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Different types of stem cells
Early embryonic stem cells
Totipotent: can become any kind of cell in the body
Blastocyst embryonic stem cells
Pluripotent: can become virtually any kind of cell inthe body
Umbilical cord stem cells:
Multipotent: can differentiate into only a limited
number of cell types
Adult stem cells:
Multipotent: can differentiate into only a limited
number of cell types
F t f t ll di t d th
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Future of stem cell-mediated therapy{stem cells to replace damaged organs}
Stem cells are induced todifferentiate into a cell type that is
damaged or missing in the patient
Cells are grown on tissue-promoting matrix or scaffolding
Healthy tissue is transplanted into
patient--unlike other organ
transplants, tissue is an immune
match to patient, so no
immunosuppression drugs would
be necessary
Medical applications of embryonic stem
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Medical applications of embryonic stem
cell research
Diabetes Caused by bodys
destruction of insulin-producing cells in thepancreas.
Researchers in Spain(2001) used mouseembryonic stem cells thatthey differentiated intoglucose-responsive,insulin-producing cells; thecells reversed diabetessymptoms when injectedinto the spleens of mice.
A i l l i
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Animal cloning
Remove nucleusfrom egg cell Add somatic cellfrom adult donor Grow in culture to produce anearly embryo (blastocyst)
Implant blastocyst insurrogate mother
Remove embryonic stemcells from blastocyst andgrow in culture
Induce stem cells toform specialized cells(therapeutic cloning)
Clone of donor is born(reproductive cloning)
Donorcell
Nucleus fromdonor cell
Reproductive cloning
The successful "cloning" of mammals has resulted in a flurry ofresearch, Dolly July 96