Download So - Assiut University

Fourth Cycle Funded Project, 2005
Project Title:
Project Manager:
Prof. of Molecular Pathology
Director of Molecular Biology Research Unit
Assiut University, Assiut, Egypt
Some Applications of
Cell Culture Techniques
in Biology
Univ. Prof. Mokhtar Taha
Prof. of Molecular Pathology
Director of the Molecular Biology
Research Unit
Assiut University, Egypt
Research Studies
In Vitro
In Vivo
Cell & Tissue Culture
Exp. Animals
Samples
Cell line
Primary culture
Exam.
Scope of the lecture
1- Stem cell research
2- Tissue engineering
applications
3- Regenerative medicine
4- Oncology
5- Hybridoma technology
6- Cloning
7- Viral diagnosis
8- Toxicity studies
Stem Cell Research
Stem cells are undifferentiated cells
with the capacity for unlimited or
prolonged self-renewal and the ability
to give rise to differentiated cells.
Differentiation can be defined as qualitative
changes in the cellular phenotype that are a
consequence of the onset of synthesis of
new gene products.
Embryonic Stem Cells
 High malleability
 Potential for undesired
development (teratomas)
 Infinite lifespan
 unlimited supply
 High ethical burden
 Uncertain legal status
Adult Stem Cells
 Limited developmental
potential
 Better behaved, easier
to manage
 Lose their ability to
proliferate/differentiate
after a time in culture
 Less moral ambiguity
 Less legal controversy
Procedure to develop ES cell lines
Methods for Isolating Stem Cells
from Adult Tissue
1. Flow cytometry


2.
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3.
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4.
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Hematopoietic stem cells--CD34+, HLA-DRNeural crest stem cells--p75+
Epidermal stem cells--β1 integrin
Microdissection/colony-forming assay
Retinal stem cells
Hair follicle stem cells
Cell adhesion or lack of adhesion
--Human epidermal stem cells adhered most rapidly to Type IV
collagen, fibronectin or keratinocytes ECM.
--Late attachment of human breast epithelial stem cells on
plastic.
Contact insensitive growth on cell mat
Putative human kidney epithelial stem cells.
Location of Adult Stem Cells
Example 1
Mouse Endbuds and Budding Structures
Formed by Two Types of HBEC
Example 2
In Vitro
Clinical Applications of Stem Cells
1.Hematopoiesis as a model of stem and
progenitor cell transplantation .
■ Fanconi Anemia —an autosomal recessive
disorder characterized by progressive bone
marrow failure leading to aplastic anemia.
■ Bone marrow ablative doses of chemotherapy and
radiotherapy to eliminate endogenous cancer〔
leukemia (CML), lymphoma (Hodgkin’s),
multiple myeloma, breast cancer〕.
2. Transplantation therapy for acute and chronic
degenerative disease
*
Epidermal--skin graft for patients with
severe burns
* Neuronal--Parkinson’s disease, multiple
sclerosis and other neurodegenerative
disease
(brain gangliosidosis, demyelinating
disease).
* Liver--For Liver damage by drugs, toxins or viral
infection.
* Islet-for insulin non --producing pancreas
* Skeletal muscle satellite--muscular
dystrophies
or muscle loss; heart disease
* Mesenchymal—tissue engineering in plastic
reconstructive surgery
3. Gene therapy
Tissue Engineering
The design, specification and
fabrication of cells,biomaterials,
or
biomolecules to restore or modify
the
biological function of tissues
Tissue Engineering
Applications
NEEDS:
* Scaffold to support tissue growth
* In vitro models to study such as
Matrigel, Vitrogen
General Approaches to Tissue
Engineering
Regenerative Medicine
 Definition:
It is a technique to replace or
repair defective or disease
tissue or organ by in vitro
design while in vivo usage
Ideal Tissue or Organ Design
Stem Cells
Biofactor Engineering
Cell Selection
Scaffolds Design
& Fabrication
Stem Cells Differentiation
In vitro cell culture on Scaffolds
Transplantation
Scaffolds degradation
New permanent tissue or organ formation
Applications in Oncology
*Although the activity of carcinogens can be
assayed in intact animals, such
experiments are difficult to quantitate and
control.
*The development of in vitro assays to
detect the conversion of normal cells to
tumor in culture, a process called Cell
Transformation, therefore represented a
major advance in cancer research.
* Cell lines differ in important ways from
their normal progenitors in the tissue
from which they were derived.

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Focus assay: is based on the ability to
recognize a group of transformed cells
as a morphologically distinct “focus”
against background of normal cells on
the surface of a culture dish.
Primary and immortalized cell lines
could be used as a model to compare
efficacy and toxicity of a given
treatment and its mechanism of action.
Properties of Cancer Cells in
Cultures
Not sensitive to densitydependent inhibition.
Reduced growth factor requirements
loss of contact inhibition
less stringent regulation of cell- cell
and cell- matrix interactions
As tumors develop they enter in contact with
cells from nonmalignant tissue
T4-2 phenotype
(tumor)
6
8
10
S1 phenotype
(normal)
days
Contact co-culture: T4-2 cells grow toward S1 glandular structures and surround these structures.
T4-2cells were added to pre-formed S1 acini and cultured for 10 days. T4-2 cells were stained with
DiI (red) prior to being plated, and S1 cells are stably transfected with green fluorescent protein
(GFP). Images show co-cultures of S1 and T4-2 cells at days 6, 8, and 10. At day 6: Tumor cells (red)
proliferate when in contact with S1 structures (green, arrow). Day 8: T4-2 cells expand over several
S1 structures (5 glandular structures can be seen in green). Day 10: A single tumor (red) is in
development; at least four S1 glandular structures (green) have been engulfed.
Monoclonal Antibody Production
Uses for monoclonal antibodies

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Monoclonal antibodies are widely used as
diagnostic and research reagents. Their
introduction into human therapy has been
much slower.
In some in vivo applications, the antibody
itself is sufficient. Once bound to its target,
it triggers the normal effector mechanisms
of the body.
In other cases, the monoclonal antibody is
coupled to another molecule, for example
a
fluorescent
molecule to aid in
imaging the target
a
strongly-radioactive
atom, such as Iodine-131
to aid in killing the target.
Some monoclonal antibodies that have
been introduced into human medicine
To suppress the immune system

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Muromonab-CD3 (OKT3) Used to prevent
acute rejection of organ, e.g., kidney,
transplants.
Infliximab (Remicade®). Binds to tumor
necrosis factor-alpha (TNF-α). Shows promise
against some inflammatory diseases such as
rheumatoid arthritis.
Cloning and Tissue Culture
Benefits:
- Recombinant DNA technology
- Gene therapy
•Knockout technology
inactivating certain
genes and tracing their
effects
•Providing DNA for
the Human Genome
Project and other
species’ genome
projects
Why do Plant Tissue Culture?
• Allows fast commercial propagation of new
cultivars
• Rare and endangered plants can be cloned safely
• Give a continuous supply of young plants
• Provide virus free plants
•Plant ‘tissue banks’ can be frozen, then regenerated
through tissue culture
• Easier to export
Virus Cultivation, Assay and
Diagnosis
Hosts for Virus Cultivation
1- Laboratory animals: play an essential role in
studies of
viral pathogenesis.
2- Embryonated chicken eggs: Several viruses
cause discrete and characteristic foci providing
a method for identification, quantification or
assessing pathogenicity.
3- Cell culture: most widely used and most
powerful hosts for cultivation and assay of
viruses.
Lab Methods for Confirmation of
Orthopoxvirus Diagnosis
. PCR related methods for DNA identification,
(e.g., real-time PCR)
. Electron microscopy
. Histopathology
. Culture
. Serology
− Antigen detection (IFA, EIA ag capture)
− IgM capture
− Neutralization antibodies
− IgG ELISA
Negative Stain Electron
Microscopy
vaccinia
Orthopoxvirus Cell Culture
Isolation
HEK 293
HeLa
BSC-40
Uninfected, 24 hpi
Variola infected, 24hpi
Cell culture isolation can be an important and very sensitive method for
detection of vaccinia since it also amplifies the virus for further
characterization.
Growth of Viruses in Embryonated
Chicken Eggs
Foci formation on the
chorioalantoic membrane by
vaccinia virus
Small Pox Vaccine
Infected Chorioalantoic
Membrane
Development of a Cytopathic Effect (time course):
Mixture of Small Cells and Syncytia
Focus Formation
Cells pile up rather than grow as a monolayer
Hemadsorption
Adsorption of erythrocytes to the surface of
infected cells
Foamy Virus CPE
ECHO Virus: Round Cells
Syncytium Formation
Syncytium Formation
Foamy Virus Vacuoles
Toxicity Studies
Liver cell culture can be used in co-culture
with brain cell aggregates, providing a
potential model for complementary
toxicological and metabolic studies.
Tissue culture has been used for analysis of
feed stuffs and fungal extracts for the
presence of mycotoxins .
Toxicity Studies
In vitro methods provide a necessary and
useful adjunct to the in vivo studies in
testing potential biomaterials by using cell
lines.
Primary and immortalized cell lines could
be used as a model in order to compare
efficacy and toxicity of a given
experimental treatment and to exploit the
differences in the mechanism of action of
a drug.