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Introduction the use of animal cell
culture What is cell culture? ? Cells removed
from animal tissue or whole animals, will
continue to grow if supplied with nutrients and
growth factors ? Cells are selected and
maintained as independent manner ? Cells in
culture may be genetically identical or
genetic variation
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Tissue culture means The ability to survive and
grow tissues outside the body in an artificial
environment.
Embryo
Brain
Dissociate cells
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Brief History In 1902, Leo Loeb placed a
fragments of the skin from the embryos of
guinea pigs in agar and in coagulated serum
and inserted them into adult guinea pigs.
He observed wandering and mitosis of the
epithelial cells.
Pathologist Leo Loeb
1869 - 1959
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In 1907, Ross Harrison discovered a way to
grow cells outside the body. Harrisons first
tissue culture
At that time, "tissue culture" was a
curiosity but in 1998, it was named as one of
"medicines ten greatest discoveries".
Biologist, Ross Harrison 1870 - 1959
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Alexis Carrel and his colleagues are
considered who actually built on Harrisons
idea and laid the main principles for culturing
tissues in an artificial media. They
successfully solved three important problems
that faced others before. These problems
include culture vessels, growing media and
death of cultured tissue.
Surgeon, Alexis carrel 1873 - 1944
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The history of cell culture 1880 Roux
maintain embryonic chick cells in saline
solution 1900 Harrison grow for nerve cells by
hanging drop technique 1940 The antibiotics
penicillin and streptomycin are added to
culture medium Earls isolate mouse L
fibroblast Enders grew poliovirus on
cultured human cells
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1950 Gey culture HeLa Cells Eagle
developed a chemically defined culture
medium 1960 Hayflick and Moorhead showed
that human cells have a finite life span
Ham grew cells in serum free medium Harris
and Watkins fuse human an mice cells 1970
Kohler an Milstein produce an antibody secreting
hybridoma Sato developed serum-free media
from hormones and growth factors 1980 Human
insulin was produced from bacteria
Recombinant tissue plasminogen activator was
produced from animal cells
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Freshney, Animal cell Culture
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Freshney, Animal cell Culture
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Why grow animal cells in culture? ? To
investigate the normal physiology or biochemistry
of cells ? To test the effects of compounds
on specific cell types ? To produce specific
artificial tissue by combining specific cell
types in sequence ? To synthesize valuable
products from large scale cell culture
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Major differences in vitro 1. Specific cell
interactions characteristic of the histology
of the tissue are lost 2. Heterotypic
interactions are lost 3. Loss of homeostatic
regulation e.g. endocrine and nervous system
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Definition of types of tissue culture 1. Organ
culture kept three dimensional structure of
cells uses fresh sample cell structure
should be supported by gel grid or raft 2.
Primary explant culture cells dissociated by
enzyme or mechanical method 3. Cell strains(
cell lines) transformed by primary culture
i) monolayer anchorage dependent ii)
suspension anchorage independent
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Freshney, Animal cell Culture
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Applications of cell culture in modern
biotechnology 1. Production of monoclone
antibody 2. Viral vaccine production 3. DNA
recombination 4. Pharmaceutical industry 5.
Drug activity investigation 6. Tissue
transplant( skin or congenital defect) 7.
Clinical investigation (amniocentesis,chromosome
analysis)
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Studies in cell culture 1. Intracellular flux
e.g. RNA , hormone, metabolites, signal
transduction, membrane traffic
2.Intracellular activity e.g. DNA
transcription, protein synthesis, energy
metabolism, drug metabolism 3.Environmental
interaction e.g. infection, drug action,
ligand receptor interaction,
carcinogenesis.
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4. Cell-cell interaction e.g. embryo induction,
metabolite cooperation, cell
proliferation, contact inhibition/density
limitation of growth,paracrine growth and
differentiation, matrix interaction, invasion
5. Cell products e.g. product
formation, exocytosis 6. Genetics
e.g. genetic analysis, genetic manipulation/
intervention,transformation, immortalization
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Intracellular activity DNA trancription, protein
synthesis, energy metabolism, drug metabolism,
cell cycle, differenciation, apoptosis
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• More recent development
• The three major categories of valuable products
  from animal cells
• ? Viral vaccines
• ? Monoclonal antibody
• Recombinant glycoproteins
• Stem cell studies
• Tissue engineering

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Advantages of Tissue Culture 1. Control of
environment, consistency and
reproducibility e.g. pH, temp, pressure, O2,
CO2 2. Characterization and homogeneity of
sample 3. Economy, scale and
mechanization, less expensive
reduction of animal use 4. In vivo
modeling delivering of specific
compound, regulation of
concentration, duration of exposure time
5. Easier to deal with virus contamination
compared to the animal experiment
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Disadvantages of cell culture 1. Expertise
2. Quantity
3. Differentiation and selection
characteristic change, adaptation
to nutrient change
4. Origin of cells 5.
Instability
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Biology of Cultured Cells I. The
culture environment the influence of
environment on culture cells is expressed in
four ways 1. The nature of substance or phase in
which the cell grow e.g. plastics,
semisolid( gel, or agar), or liquid 2.The
physiochemical and physiological institution
of medium 3. The constitution of gas phase 4. The
incubation temperature
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Major differences of cultured environment
and animal model In Vivo
In Vitro 1. some
cell type proliferate cells does
proliferate 2. cell/cell interacts
loss of cell interaction 3. hormones and
nutrient no effects of hormone
affect
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1.Cell adhesion Cell adhesion Proteins Three
classes of transmembrane protein I. .
Mediate interactions between homologous
cells Cell-cell
adhesion molecule CAMs( calcium
dependent)-calmodulin cadherins II. Mediate
cell-substrate interaction Integrin
matrix protein receptor III. Interact with
matrix Proteoglycan
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CAM
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2. Cell Proliferation cell cycle is divided
by four stages
Receptor kinase EGFR, erbB
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Cell Cycle Arrest
P53 mutation
M
Mitosis
G2
G1
Gap1
Gap2
Restriction point
Rb/E2F
S
DNA repair or Apoptosis?
DNA synthesis
Check point
P53 mutation
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Cell Cycle Progression
M
Mitosis
p53
G1
G2
Gap2
Gap1
Restriction point
Rb-P
S
DNA synthesis
Check point
p53
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?Control of proliferation of cell culture by
a. signals from the environment e.g.
growth factors, EGF, FGF, PDGF b.
Intracellular control cyclins, Rb gene
products c. cell membrane receptors
link intra and extra cellular pathway
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3. Differentiation Dedifferentiation caused
the inability of cell lines to express in vivo
phenotype ?dedifferentiation may occur due to
1) undifferentiated cell of the same lineage
over grow terminally differentiated cell or
reduce proliferative capacity 2) the absence of
the appropriate inducers ( hormones cell or
matrix interaction) cause deadaptation
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Differences between dedifferentiation,
deadaptation and selection 1)
Dedifferentiation specialized properties of
cells are lost irreversibly e.g. Hepatocyte
loss of enzyme activity of arginase,
aminotransferase, could not store
glycogen or secret serum proteins
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2) Deadaptation products re-induced by
certain culture environment hormones,
cell/cell interaction, cell/matrix
interactions.) e.g. induction of tyrosine
aminotransferase in hepatocyte by
floating collagen raft)
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3) Selection isolation of cultured cell
type by specific methods e.g. confluent
feeder layer or selection media for
epidermal cells e.g. use of D-valine
containing medium for growth of
epithelium
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Initiation of culture ? Cells form primary
culture if 1. Survive the disaggregation
process 2. Adhere to the substrate or survive
in suspension ? Cell lines
may be established if cells are
capable of proliferation
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Freshney, Animal cell Culture
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Elements of selection in the evolution of cell
lines
factors influencing selection stage
primary explant
enzymatic disaggregation isolation
mechanical damage enzymatic
damage primary culture adhesion of
explant cell adhesion and
outgrowth spreading
migration first
subculture trypsin
sensitivitynutrient,hormone,and substrate
limitation
propagation as a cell relative growth rates
of different cellsselective line
overgrowth of one
lineagenutrienthormone,and
substrate limitationseffect of
cell density on
predominance of normal and transformed
phenotype senescence normal
cells die outtransformed cells
overgrow transformation
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Characteristics of continuous cell lines 1.
Chromosomes are usually aneuploid 2. Chromosome
numbers are always between diploid and
tetraploid 3. Cells forming continuous cell
lines are tansformed or preexisted 4. A
number of properties of continuous cell lines
are associated with malignant transformation
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• A cell culture contain
• ? multiple stem cell
• undifferentiated but committed cell
• mature differentiated

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CHO cell Chinese hamster Ovary
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PC12 cell
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HeLa cell
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Breast Cancer MCF
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Melanoma cell B16 F10