Académique Documents
Professionnel Documents
Culture Documents
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Outline
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Cell Culture
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Maintaining cell culture
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Animal cell culture media is
complex
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Growth factors are present in cell media
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Culture Medium
Culture medium should provide;
– basic nutrition requirements;
– Amino acids, vitamins, minerals and
carbohydrates which cells used to build new
proteins and other components and provide
the energy necessary for metabolism
– Necessary growth factors and hormones
– 5-20% of animal blood such as calf serum
– Help to regulate and control the cell’s growth
rate and functional characteristics.
– Regulate the pH and osmolarity
– The medium control the pH range and buffers
the cells from abrupt changes in pH.
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Cell Culture
Removal of cells, tissue or organ from an animal
or plant and their subsequent placement into
an artificial environment conducive to growth.
• Organ Culture/tissue culture
A three dimensional culture of undisaggregated
tissue retaining some or all of the features of
the tissue in vivo.
• Cell Culture
Single cells, no longer organised as tissues.
Derived from dispersed cells taken from the
original tissue.
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Isolation of cells
There are 2 basic methods;
• Explant Cultures : small pieces of tissues are
attached to a glass or treated plastic culture vessel
and bathed in culture medium. After a few days,
individual cells will move from the tissue explant
out onto the culture vessel surface or substrate
where they will begin to divide and grow.
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Explant Enzymatic Dissociation
1 week old
Poeciliopsis lucida
cells from embryo
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Cell culture
(Primary cell VS Continuous cell)
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Continuous Cell Lines
An established or immortalized cell line has
acquired the ability to proliferate indefinitely
through;
– Spontaneously modification such as
random mutation by drug, radiation or
viruses.
– Deliberate modification such as artificial
expression of the telomerase gene.
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Continuous cell lines
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Cell Culture Systems
• Monolayer Culture Systems
• Adherent cells or Anchorage-
Dependent cells
• cells attach to a glass or
treated plastic substrate
• Cell growth is limited by
available surface area on which
cells can grow
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Monolayer Culture Systems
Dishes T-flaskes
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Suspension Culture Systems
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Types of Cells
• Epithelial-like :
• Cells that are attached to a
substrate and appear flattened
and polygonal in shape.
• Lymphoblast-like :
• Cells that do not attach
normally to a substrate but
remain in suspension with a
spherical shape.
• Fibroblast-like
• Cells that are attached to a
substrate and appear elongated
and bipolar, frequently forming
swirls in heavy cultures.
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Cell Culture and Vaccine
Manufacturing
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Cell cultures can be scaled-up for
biomanufacturing using bioreactors
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Cell Culture and Vaccine Production
•Viral vaccines
Cultivation of virus for vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles
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Viral Vaccine Production
cells
Infection
with seed virus Splitting, inactivation and purification
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Cells for virus production
Propagation of cells
Virus-
Infection
Seed Preculture
Virus Propagation
cells
Production
fermenter
Fermenter 4
Virus
Production
Fermenter 2 Fermenter 1
Fermenter 3 Cell propagation
Cell propagation Cell propagation
High Cell Density
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Purification
Virus-
Infection Virus Propagation
Seed Preculture
cells Harvest
Centrifuge
Ultrapurification (Cells/Debris)
Ultracentrifugation;
BPL-Inactivation/ Chromatography +
+ Virus splitting Ultra-/Diafiltration
Filter
(small particles)
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Japanese Encephalitis
Vaccines
• JE virus (JEV), a mosquito-borne flavivirus,
is the most common vaccine-preventable
cause of encephalitis in Asia
• Two JE vaccines are licensed in the United
States.
¾ An inactivated mouse brain–derived JE
vaccine (JE-VAX [JE-MB])
¾ An inactivated Vero cell culture-derived
vaccine (IXIARO [JE-VC])
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Japanese Encephalitis
Vaccines
• An inactivated mouse brain–derived JE vaccine [JE-MB])
• It has been licensed since 1992 to prevent JE in persons aged
≥1 year traveling to JE-endemic countries.
• Use of mouse brains as the substrate for virus growth has
raised concerns about the possibility of neurologic side effects
associated with the JE vaccine.
• Moderate to severe neurologic symptoms including
encephalitis, seizures, gait disturbances, and parkinsonism,
have been reported at a rate of 0.1-2 cases per 100,000
vaccinees with variation by country.
• Supplies of this vaccine are limited because production has
ceased.
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Japanese Encephalitis
Vaccines
• An inactivated Vero cell culture-derived vaccine (IXIARO
[JE-VC])
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Japanese Encephalitis
Vaccines
MMWR Recommendations and Reports March 12, 2010 / Vol. 59 / No. RR-1 KMUTT
Influenza Vaccines
• Influenza Vaccine Manufacturing Today
• Vast majority (90%) of licensed capacity is in egg-based
products
• Reliable process for seasonal production but the egg-
based process has significant limitations and lacks
flexibility (embryonated eggs require~6 months from
order to delivery)
No Chickens 1x
No Eggs ~ 1x
No Vaccine
Influenza Vaccines
• Cell Culture: An alternative to Egg-based
production
No egg lead time involved, production can start
as soon as seed virus is available
Cell culture is a practical alternative
• Industrializable – can be run routinely and cost
effectively
• Established processes can be run at practical scale
to provide sufficient vaccine for interpandemic and
pandemic needs
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Cell-based vaccine production:
critical points
• Cell line
Virus production
Regulatory path
Industrialization capacity
• Growth in suspension or on microcarriers
• Synthetic or complex medium
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Influenza Vaccines
• Cell lines
• PerC.6 (Crucell)
• EBx™: a stem cell line derived from chicken embryos
(Sigma-Aldrich Group)
• VERO: a kidney cell from the African green monkey
(GSK)
• MDCK : Madin-Darby canine kidney cells used by
Chiron
• Requirements
• Good cell-virus interaction
• Broadly and highly permissive for a wide variety of flu strains
• Restricted growth of non-flu human pathogens that may be
present in the viral seed
• Scalable, industrializable high yield, high volume production
• Cell growth in chemically defined medium (no animal-derived
components)
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Cell-based vaccine production:
regulations
• There must be documentation to support the complete
removal of the cells from the final product and
documentation to show that the cell line does not bring
any transforming agent (oncogenic transformation) into
the final product
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Influenza Vaccines
• EU approves Novartis's cell-based
flu vaccine
• Jun 13, 2007 (CIDRAP News) – The European
Union (EU) has approved Novartis's seasonal
influenza vaccine, Optaflu, the first flu vaccine
grown in cell culture rather than eggs.
• The vaccine has been approved for use in all 27
EU member states plus Iceland and Norway.
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Rabies Vaccine
• Rabies is a zoonotic disease caused by RNA viruses in the
Family Rhabdoviridae, Genus Lyssavirus.
• Virus is typically present in the saliva of clinically ill
mammals and is transmitted through a bite.
• After entering the central nervous system of the next host,
the virus causes an acute, progressive encephalomyelitis that
is almost always fatal.
• Cell-based rabies vaccines which are licensed in the United
States:
• human diploid cell vaccine, MRC-5 (HDCV, Imovax®
Rabies, sanofi pasteur),
• purified chick embryo cell vaccine, primary cultures of
chicken fibroblasts (PCECV, RabAvert®, Novartis
Vaccines and Diagnostics)
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Cell Culture and Vaccine Production
•Viral vaccines
Cultivation of virus for vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles
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Established & Novel Expression
Established Systems
Systems
Slow SPEED Fast
yeast E. coli
transgenic plant mammalian cells baculovirus
$$$$ COST $
Cell Nucleic
free acid
duckweed crustacean
Animal Cell Expression System
A. Transient transfection
B. Stable transfection
C. Viral vector-mediated protein expression (e.g. adenovirus, baculovirus)
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Influenza Virus Vaccine
based on the Baculovirus-Insect Cell Expression
System
• FluBlok®
• First recombinant influenza vaccine
• First cell-based influenza vaccine in U.S.
• FDA licensure in 2010
– No additional safety or efficacy studies required
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HA (Hemagglutinin)
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Recombinant HA protein
from Baculovirus-Insect Cell Expression System
Baculovirus with
influenza gene Days post infection
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Safety & Immunogenicity of FluBlok
Potential Benefits
• rHA antigens are produced in insect cells – protein based
vaccine with low endotoxin content
• highly purified and does not contain egg protein or other
contaminants from eggs
• Selection or adaptation of influenza virus strains that
produce at high levels in eggs is not required
• FluBlok can be prepared within 2 months
• FluBlok does not require large amounts of embryonated
chicken eggs
• Manufacturing of FluBlok does not require biocontainment
facilities
• Manufacture of rHA does not include formalin inactivation or
organic extraction procedures
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Pandemic Preparedness
Development Timeline recombinant HA1 vaccine
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Virus-Like Particle
(VLP)-based Influenza Vaccines
• What are VLPs?
• Made of recombinantly-expressed viral proteins that
• spontaneously assemble into 3-D structures similar to
parent virus
• By electron microscopy, VLPs mimic the parent virus
• Immune response is similar to that which would be
seen if exposed to the parent virus
• Do not contain genetic material
• Cannot replicate
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How are Influenza VLPs Constructed?
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Potential Immunologic Advantages
of Influenza VLPs
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Possible Limitations of Vaccine
Production using cell culture
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Personalized Cancer Vaccine
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Antigen-Presenting Cells and cancer
DC-based cancer vaccine
• Dendritic cells (DC), a leukocyte population, represent unique
antigen-producing cells capable of sensitizing T cells to both
new and recall antigens.
• DCs are the most potent antigen-presenting cells (APS).
• Tumor antigens in different forms (DNA, RNA, proteins,
peptides, viruses, cell lysates) become immunogenic when
presented to T-lymphocytes by DCs.
• Immunization with ex vivo generated DC has proven feasible
and permits the enhancement as well as the dampening of
antigen-specific immune responses in man.
• Several DC-based clinical trials have demonstrated potent
immunological and some clinical responses.
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DC loading and activation
Ex vivo generated dendritic cells(DC) can be loaded with antigens and re-infuse to
the patients, or they can be used for ex vivo expansion of anti-tumor lymphocytes.
DC loading and activation
• Ex vivo generated dendritic cells(DC) can be loaded with
antigens and re-infuse to the patients, or they can be used
for ex vivo expansion of anti-tumor lymphocytes.
scale up
of patient
Derived cells
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Ex vivo expansion of loaded APCs
AutovaxID
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Thank you
คณะทรัพยากรชีวภาพและเทคโนโลยี
School of Bioresources and Technology
Bangkhuntien Campus
Established in 1993
www.bioresources.kmutt.ac.th