Unit III

Animal Cell Culture

 Historical Background
• Ross Harrison made the first attempt to culture animal cells
wherein he cultivated embryonic nerve cells of frog by using hanging
drop method. After supplementing with chick embryo plasma, cells
proliferated better. Animal cell culture gained eminence after the use
of viruses for producing vaccines in late 1940s, and became
indispensable after the production of polio vaccine in cultured cells.

• The cell lines can be from different animal groups (insect cell lines,
human cell lines) and from various tissues. Cell lines are available
with National Centre for Cell Science (NCCS), Pune, India; European
Collection of Animal Cell Cultures (ECACC), UK; and American
Type Cell Culture Collection (ATCC), USA. Although an array of
cell lines exist in these centers.
 Principle of sterile techniques
Wet heat sterilization
• Heat treatment under moist or wet conditions is a commonly used method of
sterilization. Many microorganisms, including bacteria and viruses, are
relatively easily inactivated at temperatures of 60–80 ◦C under these
conditions, and this process is termed pasteurization. In fact, lower
temperatures of 55–60 ◦C are often adequate. However, spore-forming bacteria
and prions are more resistant to heat. Pasteurization at 60 ◦C for 10 h is also
used in the pharmaceutical industry for certain products derived from human
plasma as a method of inactivating any viral contaminants.
• Boiling, or steam treatment, at 100 ◦C for 5–10 min represents a simple
means of sterilizing equipment and fluids. It is very effective and will even
destroy some types of bacterial endospores. By the use of three cycles of
heat treatment with intervening incubation periods at 37 ◦C during which the
spores germinate, even resistant endospores can be inactivated. This type of
procedure can be used in the cell culture laboratory for decontaminating
incubators that have a heat sterilization option.
Autoclaving
• Steam under pressure at 121 ◦C or more is a highly effective sterilization
method which will kill even bacterial endospores. It should be noted that
prions are particularly resistant to even this severe form of heat treatment
and require more extreme conditions for inactivation. The quality of the
steam is critical for effective sterilization. The presence of too much air
(i.e. non-condensable gas) in the steam supply will reduce the effective
steam. pressure in the chamber.
• The presence of air in the chamber, either from non-condensable gas in the
steam supply or because of poor air removal, will lead to a lower
temperature in the chamber and load and thus reduce the efficiency of
sterilization. For pharmaceutical applications filtered ‘clean steam’ derived
from water of conductivity less that 15 µS is used. The types of autoclave
available vary widely in size, complexity and versatility. They range from
small, simple pressure cookers, which generate their own steam, to large
fully automated machines possessing multiple cycle options.
Dry heat sterilization: Heating in the dry state can also be used for
sterilization. However higher temperatures and longer times are required
for effective sterilization.
Incineration
• Because of the risk that microbial contamination may be present in waste
from the cell culture laboratory, it is recommended that such material
should be autoclaved or treated by chemical methods in the worker’s own
laboratory or building prior to being sent for incineration. For an
incinerator to operate efficiently it should reach a temperature of 350◦C
and preferably have effective after-burners fitted to incinerate any
unburned material that may be found in the smoke.
• During routine aseptic technique, the openings of glass bottles and other
containers may be briefly treated with the burning flame from a Bunsen
burner. Metal items and scissors, used for example during the preparation
of primary cell cultures, are re-sterilized between manipulations by being
dipped in ethanol which is then ignited and allowed to burn off.
Hot air ovens
• The main advantage of this method is simpler to maintain and cheaper to
purchase. However, it can only be used with heat-resistant objects such as
those made of glass or metal. Some plasticware can be treated in this way
but requires a lower temperature and extended time, for example 120 ◦C
for 18 h. A hot-air oven comprises an insulated box with electrical heaters.
Heat is distributed by simple convection or more effectively by a fan. For
more critical applications, for example in the pharmaceutical industry, a
filter may be fitted to the air-vent to prevent any possible recontamination
of the load from occurring during cooling. For large-volume sterilization
on an industrial scale, tunnel sterilizers are used in which items are
continuously fed by conveyor through a hot-air tunnel.
Filtration
• Membrane filtration using filters with a porosity of 0.2 µm will remove
bacteria and fungi, and is the method commonly used for sterilizing
solutions that cannot be sterilized by methods such as autoclaving. Uses in
the cell culture laboratory may include sterilizing culture media, sera and
other cell culture supplements, and any biological products made by the
cells.
Types of filter
• A range of filter materials is available that can be used for removing
unicellular microorganisms from liquid by filtration. These include materials
made of cellulose acetate, cellulose nitrate, or a mixture of both, nylon,
polysulphone or polycarbonate. Other materials with particularly low
protein-binding properties, for example polyvinylidene difluoride, can be
used for critical applications.
• Membrane filters come in a range of pore sizes, but 0.2 µm is considered
the standard for removing bacteria and fungi. Larger pore sizes and depth
pre-filters, for example of glass fibre, are useful in serial filtration systems to
increase the filtration capacity where significant levels of particulate material
may exist, such as in serum, complete medium containing serum, or cell
culture harvests. For effectively removing mycoplasma, filters with a pore
size of 0.1 µm are required and these are now used by most commercial
processors of serum products.
• Membrane filters are also used for filtering gases, for example CO2 or O2
supplied to cell cultures growing in mass-culture systems. A membrane filter
is an integral part of the cap of some types of cell culture flasks. Various
types of filter, ranging from membrane filters to simple cotton wool plugs,
are also used to protect liquid-handling equipment.
Irradiation
• Ultraviolet light
Microorganisms and viruses can be inactivated by ultraviolet (UV) light at
a wavelength of about 260 nm. However, because of its poor penetrating
power, its usefulness is very limited. It can be used to sterilize surfaces and
the air in cell culture cabinets and rooms, but only after they have been
cleaned. As the power of UV lamps decreases with use, they should be
checked on a regular basis. As found with heat, bacterial spores and prions
are highly resistant. Also, many organisms possess DNA repair
mechanisms that can overcome limited damage.
• Gamma rays
Gamma irradiation is a commonly used method of sterilization. It is used
with items that cannot be heat sterilized, and has the advantage that,
because of its good penetrating power, items can be completely sealed and
packaged. Various items of disposable plastic ware can be bought pre-
sterilized in this way, for example tissue culture flasks and dishes, filters,
plastic pipettes, syringes, pipette tips and some chemicals such as
antibiotics.
• Chemical sterilization
• Formaldehyde or ethylene oxide can be used for fumigation. Both are
effective against all types of microorganisms including viruses, although
bacterial endospores are somewhat more resistant in the case of ethylene
oxide. Their activity is greatest at higher temperatures, and humidity levels
of 75–100%. However, conditions that reduce the accessibility of the
microorganisms to the gas, for example being dried in organic or inorganic
material, will decrease the effectiveness of fumigation.
• Formaldehyde gas is used to decontaminate or sterilize unidirectional airflow
cabinets and rooms used for handling cell cultures, as well as for small items
of equipment. Microbiological safety cabinets that are used for handling cell
cultures may also be decontaminated with formaldehyde gas but this method
can only be used if the cabinet can be sealed and the extract ducted to the
exterior of the building.
• Hypochlorite is a very widely used disinfectant but is not effective in the
presence of high levels of organic matter. Also, it corrodes metals and only
has a shelf life of 24 h after dilution. An exposure time of at least 30 min,
and preferably overnight, is recommended. For treating spills, sodium
dichloroisocyanurate powder can be used as an alternative to absorb the spill.
• Phenolic disinfectants such as Hycolin are commonly used at a
concentration of 2–5%, or as recommended by the manufacturer. Although
not inactivated by organic matter, they have little activity against bacterial
endospores. They may also leave sticky residues when used for cleaning
surfaces
• Alcohol ethanol or propanol is commonly used for disinfecting surfaces
and gloved hands. Its effect is optimal at a concentration of 70–80%. After
spraying, the ethanol/water mixture is then left to evaporate off naturally.
Although a convenient and easy disinfectant to use, and of low toxicity, it
is not very effective against fungi, bacterial endospores or non-enveloped
viruses. Caution must be exercised in its use because it is flammable. It is
best used as a disinfectant for less critical applications or in combination
with other disinfectants.
 Cell propagation
Primary Cultures
• Primary culture refers to the original culture before passage or subculture.
Theoretically primary cultures can be established from any tissue. For
obtaining best results, the choice of starting material should be judicious.
Choice with regard to species, whether to obtain from adult or embryo, and
whether one wants to work with normal or tumor are extremely important
points to be considered before starting a primary culture. Necessary
permission from the ethical committee must be obtained before embarking
on work with human tissues.
• It is important to choose a particular tumor tissue type to derive maximum
benefits if the experiment requires a particular cell type present abundantly
in tumors as compared to normal tissue. Alternately, normal tissue has
advantage over transformed cell lines as there are rarely any pathological
differences in established cell lines. The disadvantage of normal tissue is that
the cells have a finite life.
 Cell morphologies vary depending on cell type

Fibroblastic Epithelial

Endothelial Neuronal
 Isolation of cell lines for in vitro culture

Resected Tissue

Cell or tissue culture in vitro

Primary culture
Sub-culture
Secondary culture
Sub-culture
Cell Line
Single cell isolation Successive sub-culture
Immortalization
Loss of control
Clonal cell line of cell growth Senescence
Transformed cell line
Immortalised cell line
Difference between Primary and Secondary
culture
Primary culture
• Derived directly from animal
embryo or adult tissue
• Cultured either as tissue
explants or single cells
• Initially heterogeneous
• Become overpopulated with
fibroblasts
• Finite life span in vitro
• Retain differentiated
phenotype
• Mainly anchorage dependant
• Exhibit contact inhibition
Continuous cultures
• Derived from a primary
culture
• Immortalised – Spontaneous
or Viral transformation
• Homogeneous cell population
• Loss of anchorage dependant
• Loss of contact inhibition
• Infinite life span in vitro
• Differentiated phenotype
• Genetically unstable
Continuous Cell culture
• Continuous cell lines go on dividing for a long time at quite a high rate and
can be passaged easily. Mostly, a primary cell stops proliferating after a
large number of passages. Some occasional cell lines can be passaged
indefinitely in vitro. These are then called established cell lines. The
transition from primary cell line to established cell line is rather smooth
and gradual in some cases, but in others cell transformation or cell
alteration may cause this change. The transformed cell multiplies faster
and may soon outnumber the other cell population, thus becoming the
predominant cell type in the culture. Primary cell lines have normal
number of chromosomes but established cell lines have unusual number of
chromosomes.
• They have short doubling times and are invariably aneuploid. Irrespective
of their origin, their requirements are the same. There is no evidence of
spatial orientation. They grow in higher densities than primary cultures.
They can grow from a single cell or as dilute inocula. If required they can
be established in suspensions, unlike primary cultures.
 Cell lines
WRL-68 Jurkat

HeLa HepG2
Cell Line Morphology Species Medium Applications

MEM+ 2mM Glutamine+ 10% FBS +

Tumourigenicity
HeLa B Epithelial Human 1% Non Essential Amino Acids
and virus studies
(NEAA)
RPMI 1640 + 2mM Glutamine + 10- Differentiation
HL60 Lymphoblast Human
20% FBS studies
3T3 clone DMEM + 2mM Glutamine +5% New Tumourigenicity
Fibroblast Mouse
A31 Born Calf Serum (NBCS) + 5% FBS and virus studies
Gene expression
and virus
COS-7 Fibroblast Monkey DMEM+ 2mM Glutamine + 10% FBS
replication
studies
Nutritional and
Ham′s F12 + 2mM Glutamine + 10%
CHO Epithelial Hamster gene expression
FBS
studies
EMEM (EBSS) + 2mM Glutamine +
Transformation
HEK 293 Epithelial Human 1% Non Essential Amino Acids
studies
(NEAA) + 10% FBS
F-12 K + 10% FBS + 100 µg/ml Angiogenesis
HUVEC Endothelial Human
Heparin studies
Jurkat Lymphoblast Human RPMI-1640 + 10% FBS Signaling studies
Establishment of Continuous Cell Lines
• Spontaneous: A large number of established cell lines have been obtained in
the absence of any known exposure to a transforming agent. Such lines include
the RMP promegakaryocyte line derived from rat bone that retains several
differentiated functions including the ability to synthesize factor VIII, antigen
and fibrinogen. The fibroblast cell line 3T3-L1 is derived from mouse embryo
and is capable of lipid accumulation. However, in general it is unusual to
isolate cell lines spontaneously, and those that have been obtained are
predominantly from fetal rodent tissue.
• Chemical transformation: Methyl cholanthrene has been widely used to
establish a variety of cell lines including the mouse L-cell line and the rat
muscle line L6. Another carcinogen, azoxy methane, has been used to
transform normal human colon mucosal cells to give malignant lines with
altered morphology, culture longevity, growth in soft agar, substrate adherence
and peanut agglutinin binding. Human mammary epithelial cells exposed to
benzopyrene develop an extended lifespan and apparently immortal cell lines
can be isolated. They resemble tumor-derived mammary epithelial cells more
closely than their normal progenitors. The mutagen EMS (ethyl methane
sulphonate) has also been used in conjunction with simian virus 40.
Viral transformation:
• Simian virus 40: The monkey virus SV40 has been used to isolate
transformed mouse and human fibroblast lines. In the case of the human
cells, however, these lines are not truly immortal, but merely display a
delay on the onset of senescence. This approach has led to the isolations of
lines such as TPA30-1 human placental line and the RLA fetal rat
hepatocyte line.
• Epstein-Barr virus (EBV): The EBV has been widely used to isolate
transformed cell lines from human B-lymphocytes. Such lines have
important applications in the development of cell lines from individuals
with chromosome translocations or inherited diseases and in the production
of human monoclonal antibodies.
• Other viruses: Cell lines have been isolated following infection with other
viruses, for example murine lymphoid cell lines with the Abelson murine
leukaemia virus. Infection with the Rous rat sarcoma virus was used to
isolate a cerebellar cell line WC5, which (GFAP) expresses glial fibrillary
acidic protein.
Suspension culture
• Suspension cultures are easier to propagate, since subculture only requires
dilution with medium.
• Clearly, freely suspended cultures do not require trypsinization. They are,
therefore, also easier to harvest.
• In contrast to anchorage-dependent cells, cells cultured from blood, spleen
or bone marrow adhere poorly if at all to the culture dish.
• In the body, these cells are held in suspension or are only loosely adherent.
• It is important to realize this if you are working with this category of cells,
since the methods used to propagate these cells are very different to those
for adherent cells.
 Passaging Cells

Why passage cells?

• To maintain cells in culture (i.e. don’t overgrow)
Check confluency of cells • To increase cell number for experiments/storage

Remove spent medium How?

• 70-80% confluency
• Wash in PBS to remove dead cells and serum
Wash with PBS • Trypsin digests protein-surface interaction to release
cells (collagenase also useful)
• EDTA enhances trypsin activity
Incubate with • Resuspend in serum (inactivates trypsin)
trypsin/EDTA • Transfer dilute cell suspension to new flask (fresh
media)
• Most cell lines will adhere in approx. 3-4 hours

Resuspend in serum
containing media

Transfer to culture flask 70-80% confluence 100% confluence

Difference between Adherent and Non
adherent cells
 CHEMICALLY DEFINED MEDIA FOR CELL
CULTURE
Growth Media
• Cell culture medium is the single most important factor in promoting cell
survival and proliferation. A nutrient is defined as a chemical substance
that enters a cell and is used as a substrate for biosynthesis or energy
metabolism. Any other requirement for cell growth is termed as a
supplement, including serum, undefined media and biological fluids. The
function of media is to provide an environment for survival and also to
provide substances required for the growth of cells. Early tissue culture
was done on biological fluids such as plasma, lymph, and serum or
extracts of embryonic origin.
 Types of Cell Culture Media

Media Type Examples

plasma, serum, lymph, human
Biological Fluids placental cord serum, amniotic
fluid

Natural media Extract of liver, spleen, tumors,

leucocytes and bone marrow,
Tissue Extracts
extract of bovine embryo and
chick embryo

Clots coagulants or plasma clots

Balanced salt solutions PBS, DPBS, HBSS, EBSS

Basal media MEM DMEM

Artificial media

Complex media RPMI-1640, IMDM

 Common Cell Culture Media

• Eagle’s Minimum Essential Medium (EMEM)

• Dulbecco’s Modified Eagle’s Medium (DMEM)
– Low glucose
– High glucose
• RPMI-1640
• Ham’s Nutrient Mixtures
• DMEM/F12
• Iscove’s Modified Dulbecco’s Medium (IMDM)
 Composition of Media
Basal Salt Solution
• Balanced salt solution composed of inorganic salts that maintains the
physiological pH and osmotic pressure. The physiological role played by the
inorganic ions is to maintain the membrane potential. They also work as
cofactors in enzyme reaction and in cell attachment. Na +, K+, Mg2+, Ca2+, Cl− ,
S02 4 −, PO3 4 − and HCO− 3 are the inorganic molecules involved.
• To maintain the osmolality, concentration of NaCl is generally adjusted.
Four main categories of BSS are
• EBSS (Earle’s balanced salt solution)
• DPBS (Dulbecco’s phosphate-buffered saline)
• HBSS (Hank’s balanced salt solution)
• ESSS (Eagle’s spinner salt solution).
• For maintaining the correct pH, HBSS and DPBS are equilibrated with air, while
ESSS and EBSS require equilibration with gas phase containing 5% CO2.
Carbohydrates
• Carbohydrates are a major source of energy in cultured cells. Sugars such
as glucose, maltose, sucrose, mannose, fructose and galactose are
commonly used. Glutamine can also alternatively supply energy required
by some cells.

Amino Acids
• Animal cell culture requires essential amino acids as they cannot be
synthesized from raw materials by heterotrophic organisms. The
concentration of amino acids influences cell yield, survival and growth
rates. Amino acid deficiency inhibits cell division, induces chromosomal
damage and increases lysosomal activity and cell death. Imbalance of
amino acid concentrations can also produce karyotype changes. During the
lag phase of the growth cycle, there is an enhanced requirement of
cysteine, glutamine, isoleucine and serine. Glutamine serves both as an
energy source and carbon source in the synthesis of nucleic acids. It is the
most unstable (labile) of the amino acids and needs to be replenished
regularly.
Vitamins
• Vitamins are required as cofactors in metabolism [e.g. Niacin, NAD(P),
riboflavin, FMN, FAD]. The vitamins added to the basal media are para-
amino benzoic acid, biotin, choline, folic acid, nicotinic acid, pantothenic
acid, pyridoxal, riboflavin, thiamine and inositol. Retenoids help cells to
adhere to the substrate. A sufficient supply of choline is necessary for
incorporation into membrane phospholipids. Vitamins A and E are added
in Medium199.
Protein supplements
• Common examples of protein supplements are fetuin, globulin, fibronectin,
albumin and transferrin. Low molecular weight factors are carried into
cells by binding onto transport proteins. Albumin is a transport protein,
which carries vitamins, lipids and hormones into cells. Transferrin is
involved in binding and transport of iron.
• Prostaglandins E and F2, are involved in cell growth. Their action may
possibly be in conjunction with epidermal growth factor (EGF) and other
growth factors. Insulin is essential for all media. It is very sensitive to
inactivation by cysteine and hence large quantities need to be added.
• Glucocorticoid hormones such as hydrocortisone and dexamethasone can either
stimulate cell growth or inhibit cell growth depending on cell type. Growth
factors like nerve growth factor (NGF), EGF have been used to promote cell
growth. Certain interleukins, colony stimulating factors and fibroblast growth
factors (FGF) are also used.
• Copper, zinc, cobalt, manganese, molybdenum and selenium are considered to
be activating enzymes and protecting against free radicals, which cause damage
to DNA. Nontransformed cells have to adhere to solid substrate in order to
multiply. Only haemopoetic and transformed cells can multiply without
attachment. Chondronectin is required for the adhesion of chondrocytes and
laminin for adhesion of epithelial cells. Fibronectin is also important for cell
adhesion.
Antibiotics
• Antibiotics are routinely used in laboratories although their use in
biopharmaceutical production is not acceptable. Careful selection of the
antibiotics is a necessity. Ideally, antibiotics should have no toxicity, preferably a
broad spectrum type and be economical in cost. Commonly used antibiotics are
penicillin (100 IU ml−1) and streptomycin (50 g ml−1). Gentamycin (50 g ml−1) is
expensive but is widely used as an antifungal agent. Nystatin (25 g ml−1) also is
an effective antifungal agent.
Serum as a Complex Supplement
• Blood serum may be used either as the entire culture media or as a
supplement in basal medium. Serum provides a nutritive substrate and a
supporting structure of many types of cell culture. It provides a cocktail of
growth factors necessary for maintenance and proliferation of cells.
• Proteins of molecular weight 5–30 kDa are the peptide growth factors
which act via cell surface receptors and stimulate cells into proliferation or
differentiation. Serum requirement varies depending on the cell type and
the concentration of the cells.
• Albumin, major protein component of serum, acts as a carrier for small
molecules across cell membrane. Transport of fatty acids is an important
function of albumin. Steroids and fat-soluble vitamins also bind to albumin.
Lipids such as cholesterol, triglycerides and phospholipids are transported
in serum in micellar form complexed with specific lipoproteins.
• Transferrin is a major iron transport protein in vertebrates, representing 3–
6% of total serum protein. The transferrin–Fe3+ complex is taken by cell
surface receptors after the release of iron; apotransferrin is liberated from
the cell and recycled.
 Components of Serum
• Specific growth factors - EGF, PDGF, IGF, FGF, IL-1, IL-6, insulin
• Trace elements – Iron, Zinc, Se, Co, Cu, I, Mn, Mo, Cr, Ni, As, Si, Sn, F
• Lipids - Cholesterol and Linoleic acid
• Steroids - Polyamines
• Attachment Factors – Fibronectin, Laminin and fetuin
• Mechanical protection - Albumin
• Buffering - Albumin
• Neutralization of toxic factors - Albumin
• Transport of metals - Transferrin
• Protease inhibitors – Antitrypsin and Macroglobin
Freshney.(1992) Animal Cell Culture.
Constituents of serum with their respective functions
• Albumin - Transports lipids, hormones, minerals and provides osmotic
pressure
• Fibronectin - Promotes cell attachment
• Fetuin - Enhances cell attachment
• Transferrin - Binds iron
• Macroglobulin - Inhibits trypsin
• Insulin - Uptake of glucose and amino acids
• Platelet derived growth factor (PDGF) - Mitogen for fibroblasts, smooth
muscle cell
• Fibroblast growth factor (FGF) - Mitogen growth factor
• Endothelial growth factor (ECGF) - Mitogen growth factor
• Epidermal growth factor (EGF) - Mitogen growth factor
• Hydrocortisone - Promotes cell attachment
• Steroid hormones - Mitogen growth factor
• Thyroid hormones (T3, T4) - Oxygen consumption, metabolic rate control,
growth and differentiation of various cells.
 Serum free Media
• The mixture of supplements required for the serum-free culture of neural
cells, defined by Bottenstein and Sato in 1979, still forms the basis of many
serum-free supplements for a wide variety of cell types.
• Serum-free media has no growth factors, viruses or growth inhibitors. A
great deal of effort with very limited success has gone into developing serum-
free media.
• A great deal of effort with very limited success has gone into developing
serum-free media.
• A great deal of effort with very limited success has gone into developing
serum-free media.
• With the identification of growth factors and nutrients required by different
cells, several effective serum-free media have been formulated.
• In a defined serum-free media the components are formulated together to
optimize performance of a single cell type. Each component is of known
concentration and purity. The origin of the cell line, that is species and tissue;
the compatibility of media components and their interactions; and the specific
application for which the cell line is being cultured.
Advantages of serum-free media:
• It has the ability to make a medium selective for a particular cell type,
since each cell type appears to require a different recipe.
• It has high degree of purity of reagents and water.
• It needs high degree of cleanliness of all apparatus
Disadvantages of Serum-free Media
• The quality of serum varies from batch to batch and deteriorates within one
year. Therefore, every batch of serum needs fresh testing.
• If more than one cell type is used, each may require different serum batch,
therefore, many batches are to be maintained and co-culturing may be
difficult.
• The demand of serum usually exceeds the supply for a variety of reasons.
• When cell culture is used for downstream processing to cover cell
products, the presence of serum is an obstacle for purification.
• Serum increases the cost of medium.
• Serum may stimulate undesirable growth and may even inhibit growth in
some cases.
 Scale up of Mammalian cell culture
• In the biotechnological and bioprocessing industries, scale-up and scaling-
out are two widely employed strategies to generate large numbers of cells.
• Scale-up systems progressively increase the surface area/culture volume
as the cell number raises .
• Scale-out systems are based on the use of multiple culture
vessels/bioreactors working in parallel.
• There are advantages and disadvantages associated with each approach.
• For instance, compared to scale-up processes, scaling-out can better deal
with changes in product demand and improves process performance,
however reproducibility can be difficult to achieve.
• Instead, scaling-up processes are more difficult to handle and control due
to the high working volumes involved, but it can lower the costs of goods
in the long term.
T-Flasks
T-flasks are the most commonly used plastic consumables for early stage cell
expansion, usually when growing cells starting from a cryovial. T-flasks vary in
size, ranging from a culture area of 12.5–225 cm2 and are made of disposable
plasma treated polystyrene, or tissue culture plastic. While conveniently
economical, these flasks are labor-intensive and become cost-inefficient when
expanding cells beyond bench scale, mainly because of their high footprint.
Multi-Layered Flasks
They are large T-flasks composed of stacked flat surfaces. The aim is to
increase the available surface by incorporating a multi-tray unit reaching a total
area that depends on the number of layers. This type of flask must be treated as
an individual unit with the cells from each layer to be seeded, cultured and
detached at the same time. Although being a useful device for scaling-up at
bench scale, there are concerns regarding the cell quality and the associated
labor intensity. For instance, there might be a heterogeneous availability and
distribution of nutrients and gasses between the different layers of the flask.
Moreover, simple operations like cell seeding, media change and cell harvest
become challenging due to their size and weight. In this respect, system
automation would greatly improve these day-to-day operations.
Roller Bottles
• These bottles consist of cylindrical vessels to grow cells in a dynamic
system. They are usually placed in a heated environment on a rack that
slowly revolves (ranging between 5 and 240 revolutions/hour).
• They are inexpensive and are a common method used for the initial scale-
up of adherent cells. The cells attach and cover the inner surface of the
bottle; hence the cells are cyclically bathing in culture medium and
exposed to gases.
• In addition, the rotation provides a level of mixing, preventing gradients
from forming within the medium that may affect cell growth.
• In this system, the cells are most of the time covered by a thin layer of
medium, thus facilitating superior gas exchange. The surface available for
cell expansion is between 500 and 1,700 cm2 , in a total volume ranging 1
to 1.5 L, suitable for culture volumes of 0.1–0.3 L.
• Like static flasks, rotating flasks are also labor intensive. For high cell
numbers, a further constraint of a roller bottle process through scaling-out
is the limitation in the control of O2 and CO2 in both the gas and the liquid
phase of culture.
Spinner Flasks
• These devices are flat-bottom flasks commonly used at a benchscale for
stirred suspension cultures that can be used to initially validate
microcarriers and media composition.
• The culture is maintained in suspension and the stirring is achieved by a
magnetic stir bar, also called magnetic driven impeller.
• The media is inoculated with cells to fill the flask with a volume of 100–
200 ml at a stirring speed of 50 rpm.
• The spinner flasks can generate high cell numbers, provide a better
aeration system, a more homogeneous nutrient supply, longer culture
period and reduced costs.
• Microcarriers can be added to spinner flasks mainly to do preliminary tests
before moving to larger bioreactors. Microcarriers are small spheres with a
diameter ranging between 90 and 300µm and available in different sizes,
materials, coatings, and surface charges.
• As the choice of the microcarrier depends on the cell type, product, and
operational set-up, it is highly recommended to run preliminary tests with
different microcarriers
Rocking Motion Bioreactors
• This type of reactor utilizes the wave motion of culture medium generated
by a rocking platform to provide a cell-beads (microcarrier) suspension. The
beads are placed inside a disposable bag with ports allowing for air
circulation and bag inflation.
• The disposable bag system has advantages for clinical applications in
terms of safety providing the ultimate ease in operation and protection
against cross-contaminations.
• The chamber is placed on a special rocking platform causing low shear stress
to the cells. The agitation provides proper mixing and mass transfer while the
circulating air provides the necessary oxygen exchange.
• It is possible to connect culture medium bags for perfusion via additional
ports and it can operate via batch, fed-batch, repeated fed-batch, and
perfusion mode.
• This setting facilitates scale-up and automation, which has been
demonstrated for culture volumes up to 500 L
• Such a system is widely used for the expansion of mammalian cells, for
example embryonic feline lung fibroblasts, neutrophils from HSCs, and
T cells.
Stirred Tank Bioreactors
• The stirred tank reactors are possibly the most used system for large-scale
culture of mammalian cells.
• They apply the same operational principles as the spinner flask, just in much
larger volumes which can reach up to 2,000 L in a single vessel.
• The impeller keeps the solution in agitation to maintain the particles (i.e.,
organoids, suspension cells, or microcarriers) in suspension whilst
homogenizing the distribution of oxygen, nutrients, and heat.
• The tank provides a closed and automated platform and can operate in
different modes, such as batch, fed-batch, and perfusion.
• When it is used to grow cells attached to microcarriers, it can provide in situ
assistance in dissociating the cells from the carriers when cells reach
confluency.
• The strategy is based on coupling the addition of trypsin with intense agitation.
The generated shear stress improves the cell detachment efficiency, thus
increasing the final yield.
• Industrial stirred tank bioreactors are available as single-use, however they are
traditionally made of stainless steel, considering it is easy to clean, well-
compatible with biologics and highly resistant to pressure and erosion.
Packed Bed Bioreactors
• Also called fixed bed, they consist of a hollow tube packed at the bottom
with immobilized surfaces such as scaffolds, microcarriers or porous fibers.
• The cells are seeded on the fixed bed while fresh media is continuously
circulating within the system transferring oxygen and supplying nutrients,
whilst providing a large surface to volume ratio for cell attachment and
expansion.
• They are commercially available bench scale (up to 4 m2 ) and for
industrial scale manufacturing (up to 500 m2 ). High cell densities of 5.1 ×
108 cells/mL have been reported with packed bed bioreactors.
• Very early progenitor cells (CFU-GEMM) were expanded up to 4.2-fold
while later progenitor cells (CFU-GM and BFU-E) exhibited up to seven-
fold and 1.8-fold expansion, respectively.
• Moreover, an average seven-fold expansion of MSC was reported with a
starting cell density of 6.0 × 107 cells, after 7 days of culture.
• Cell harvesting can also be problematic due to the presence of high cell
densities and the difficulty of effectively introducing the detachment
supplements into the culture.
Hollow Fiber Bioreactors
• They consist of a cylindrical chamber stacked with semipermeable hollow
fibers. Cells can be inoculated both within the fibers and on the
extracapillary surfaces, permitting high cell densities in the order 1.0 × 109
cells/mL.
• The fibers mimic blood vessels in coordinating nutrient supply and removal
of waste while oxygen exchange is managed by diffusion between intra-
capillary and extra-capillary spaces.
• The culture medium can flow through the fiber or chamber or both using
proper channels and ports. Depending on the inoculation method, pore size
for the semi-permeable membrane can be chosen to determine which
particles shall pass through or retained by the membrane.
• For instance, if the cells are inoculated in the intracapillary surface, then the
media is perfused from outside or extra-capillary space. This flow operation
is known as intra-capillary inoculation with extracapillary perfusion.
• The use of oxygen carriers that increase the flow rates and/or rotate the
hollow-fiber bioreactor in timed cycles to reduce oxygen gradients.
• Hollow fiber bioreactors have been employed to expand MSCs and human
umbilical cord derived HSCs.
 Cell Culture Contamination
Contamination of cell cultures
It is easily the most common problem encountered in cell culture
laboratories, sometimes with very serious consequences. Cell culture
contaminants can be divided into two main categories, chemical
contaminants such as impurities in media, sera, and water, endotoxins,
plasticizers, and detergents, and biological contaminants such as bacteria,
molds, yeasts, viruses, mycoplasma, as well as cross contamination by other
cell lines. While it is impossible to eliminate contamination entirely, it is
possible to reduce its frequency and seriousness by gaining a thorough
understanding of their sources.
Bacterial Contamination
• Bacteria are a large and ubiquitous group of unicellular microorganisms.
They are typically a few micrometers in diameters, and can have a variety of
shapes, ranging from spheres to rods and spirals. Because of their ubiquity,
size, and fast growth rates, bacteria, along with yeasts and molds, are the
most commonly encountered biological contaminants in cell culture.
• Bacterial contamination is easily detected by visual inspection of the
culture within a few days of it becoming infected; Infected cultures usually
appear cloudy (i.e., turbid), sometimes with a thin film on the surface. A
sudden drop in the pH of the culture medium is also frequently
encountered.
• Under a low-power microscope, the bacteria appear as tiny, moving
granules between the cells, and observation under a high-power
microscope can resolve the shapes of individual bacteria.
Mycoplasma Contamination
• Mycoplasmas are simple bacteria that lack a cell wall, and they are
considered the smallest self-replicating organism. Because of their
extremely small size (typically less than one micrometer).
• Mycoplasma are very difficult to detect until they achieve extremely high
densities and cause the cell culture to deteriorate; until then, there are often
no visible signs of infection.
Detection of Mycoplasma contamination
• Some slow growing mycoplasma may persist in culture without causing
cell death, but they can alter the behavior and metabolism of the host cells
in the culture. Chronic mycoplasma infections might manifest themselves
with decreased rate of cell proliferation, reduced saturation density, and
agglutination in suspension cultures; however, the only assured way of
detecting mycoplasma contamination is by testing the cultures periodically
using fluorescent staining (e.g., Hoechst 33258), ELISA, PCR,
immunostaining, autoradiography, or microbiological assays.
Mold Contamination
• Molds are eukaryotic microorganisms in the kingdom of Fungi that grow
as multicellular filaments called hyphae. A connected network of these
multicellular filaments contains genetically identical nuclei, and is referred
to as a colony or mycelium. Similar to yeast contamination, the pH of the
culture remains stable in the initial stages of contamination, then rapidly
increases as the culture become more heavily infected and becomes turbid.
• Under microscopy, the mycelia usually appear as thin, wisp-like filaments,
and sometimes as denser clumps of spores.
Yeast Contamination
Yeasts are unicellular eukaryotic microorganisms in the kingdom of Fungi,
ranging in size from a few micrometers (typically) up to 40 micrometers
(rarely). Like bacterial contamination, cultures contaminated with yeasts
become turbid, especially if the contamination is in an advanced stage.
There is very little change in the pH of the culture contaminated by yeasts
until the contamination becomes heavy, at which stage the pH usually
increases. Under microscopy, yeast appears as individual ovoid or
spherical particles that may bud off smaller particles.
Cross contamination
While not as common as microbial contamination, extensive cross-
contamination of many cell lines with HeLa and other fast growing cell
lines is a clearly-established problem with serious onsequences. Obtaining
cell lines from reputable cell banks, periodically checking the
characteristics of the cell lines, and practicing good aseptic technique are
practices that will help you avoid cross-contamination. DNA
fingerprinting, karyotype analysis, and isotype analysis can confirm the
presence or absence of cross-contamination in your cell cultures.
Chemical contaminants
• Chemical contaminants may be the Media from reagent and water, sera:
batch to batch variation in hormone and growth factors, water borne
endotoxins, and some other culture additives.
• Storage vessels may further add to the problems if not cleaned thoroughly.
Excessive use of fluorescent lights and addition of sodium azide in the
CO2 incubators are generally a common cause of chemical contaminants.
• Occasionally there may be white deposits on glassware, pipettes and on
instruments that may have been left by disinfection or detergents.
• Metal ions, endotoxins, and other components of media, generally the sera
form a thin coat on the plastic ware, in plastic tubing and storage bottles.
• Free radicals may be generated in the media, possibly by photoactivation
of riboflavin or tryptophan. HEPES (4-(2-hydroxyethyl)-1-piperazine
ethane sulfonic acid), commonly used for buffering the media also gets
reduced when exposed to fluorescent light.
• Residues from germicides used to disinfect equipment and incubators
including the laboratories, also cause white deposits. The impurities in
gases used in CO2 incubators may also create unwanted deposits.
 Eradication of Contamination
• Firstly, accidents can be reduced by keeping the laboratory area clean and free
from dust. There should be a standard protocol for checking the contaminants
routinely. The frozen cell repository should be used strategically.
• Antibiotics are best avoided but if necessary, should be used cautiously.
• Routine cleaning of laboratories-work surfaces, floors and the equipment
should be done diligently.
• Biosafety cabinets should be tested for the quality of purified air coming from
the HEPA(High Efficiency Particulate Air) filters. The velocity of air coupled
with the particulate count also determines the contaminant periodicity.
• The CO2 incubator is a very good source of fungus as 90% humidity is
maintained. The air jacketed incubators are safer but many a times they are
supplemented with water trays that are good breeding grounds for fungus as
well as bacteria. So it should be sterilized for every to weeks.
• The microscope is important for regularly examining all the tissue culture
flasks for any contamination.
• The contaminated flask should be topped with an equal amount of 10%
sodium hypochlorite and left for two hours before spilling down the sink
with running tap water.
• Wipe the outside of all the non-infected flasks with 2.5% sodium
hypochlorite and 70% isopropanol/alcohol/ethanol.
• Further, remember to discard all the aliquots of media,
penicillin/streptomycin, fetal calf serum and any open bottles of water that
could have been a source of contamination.
• It is always advisable to fumigate the Class I/II cabinet by formaldehyde
and if possible the whole laboratory.
• Also, the incubator should be decontaminated by the usual cleaning
procedure i.e. by spraying 70% ethanol and wiping it dry.
 Detection of contaminants
• In general: turbid culture media, change in growth rates, abnormally high
pH, poor attachment, multi-nucleated cells, graining cellular appearance,
vacuolization, inclusion bodies and cell lysis
• Yeast, bacteria & fungi usually shows visible effect on the culture
(changes in medium turbidity or pH)
• Mycoplasma detected by direct DNA staining with intercalating
fluorescent substances e.g. Hoechst 33258
• Mycoplasma also detected by enzyme immunoassay by specific antisera
or monoclonal abs or by PCR amplification of mycoplasmal RNA
• The best and the oldest way to eliminate contamination is to discard the
infected cell lines directly
 Preservation of Animal Cells

Passage cells Why cryopreserve cells?

• Reduced risk of microbial contamination.
• Reduced risk of cross contamination with other cell
Resuspend cells in serum
containing media lines.
• Reduced risk of genetic drift and morphological
changes.
Centrifuge & • Research conducted using cells at consistent low
Aspirate supernatant
passage.

Resuspend cells in How?

10% DMSO in FCS • Log phase of growth and >90% viability
• Passage cells & pellet for media exchange
Transfer to cryovial • Cryopreservant (DMSO) – precise mechanism
Freeze at -80oC unknown but prevents ice crystal formation
• Freeze at -80oC – rapid yet ‘slow’ freezing
• Liquid nitrogen -196oC
Transfer to liquid
nitrogen storage tank
 Characterization of Mammalian cells
Cytogenetics
• There is need to verify that the cells are derived from the particular
species. Chromosome content and analysis is species specific and is a
reliable method of species characterization. The correlation of
chromosomal structure with heredity and variation is termed
cytogenetics.
Karyotype
• Karyotype is the collective term used to describe the chromosome
number, size and shape. Primary cell lines retain diploid chromosomes
and also the chromosomes of the donor. Lines from individuals with
cytogenetic abnormalities have been extensively used for mapping genes,
not merely to individual chromosomes but also to specific regions of
chromosomes.
Isoenzymology
• Enzymes that exist in multiple forms in animal tissue and in different
molecular forms, catalyzing the same reaction, are called isoenzymes.
Immunological Test
• This test depends on the specificity of antigen–antibody reaction. Species-specific
antigens can be detected by immunofluorescence.
Recombinant DNA methods
• For many genes there are slight variations between individuals of a species, and
such molecular differences in the DNA of a particular gene are called
polymorphism. The presence of dissimilar arrangements of DNA bases means that
each individual’s DNA is cleaved at slightly different sites by restriction enzymes,
generating distinct lengths of restriction fragments. DNA probes can be used to
detect polymorphisms in a technique known as restriction fragment length
polymorphism (RFLP).
Allozyme analysis
• Every individual belonging to the same species expresses different alleles for a
given enzyme locus. Allelic isoenzymes are referred to as allozymes. This
analysis is like a genetic signature for that cell line.
Blood group antigens and HL-A
• This involves the determination of the antigens present on the surface of the cell.
Blood group antigens are present on normal human epithelium in primary culture
and on some continuous epithelial lines. The use of anti-human A, B or AB typing
antiserum can be used to type these cell lines.
 Organotypic culture
Organ Culture
• Organs cannot be propagated, but pieces of organs can be cultured on artificial
medium. Care should be taken to handle organ culture in such a way that tissue
dose not get damaged.
• Organ culture technique demands more tactful manipulation than tissue culture.
The culture media on which organ is cultured are the same as described for cell
and tissue culture.
• It is easier to culture embryonic organs than the adult organs from animals.
Methods of culturing embryonic organ and adult organs differ.
• A major deficiency in tissue architecture in organ culture is the absence of a
vascular system, limiting the size (by diffusion) and potentially also the polarity
of the cells within the organ culture.
• When cells are cultured as a solid mass of tissue, gaseous diffusion and the
exchange of nutrients and metabolites become limiting.
• To alleviate this problem, organ cultures are usually placed at the interface
between the liquid and gaseous phases to facilitate gas exchange while retaining
access to nutrients.
Organ Culture on Plasma Clots
• A plasma clot is prepared by mixing five drops of embryo extract with 15
drops of plasma in a watch glass placed on a cotton wool pad. The cotton
wool pad is put in a petridish. Time to time cotton is moistened so that
excessive evaporation should not occur. Thereafter, a small piece of organ
tissue is placed on the top of plasma clot present in the watch glass. In the
modified technique the organ tissue is placed into raft of lens paper or
ryon. The raft makes it easy to transfer the tissue; excess fluid can also be
removed.
Organ Culture on Agar
• Solidified culture medium with agar is also used for organ culture. The
nutrient agar media may or may not contain serum. When agar is used in
medium, no extra mechanical support is required. Agar does not allow the
support to liquefy. The tumors obtained from adults fail to survive on agar
media, whereas embryonic organs grow well. The ingredients of the
media are agar (1% in basal salt solution), chick embryo extracts and
horse serum in the ratio of 7:3:3.
Organ Culture in Liquid Media
• The liquid media consist of all the ingredients except agar. When liquid
media are used for organ culture, generally perforated metal gauze or
cellulose acetate or a raft of lens paper is used.
Whole Embryo Culture
• For the culture of chick embryo, eggs are incubated at 38 ◦C for 40–42 h
so that a dozen of embryos could be produced. The egg shell sterilized
with 70% ethanol is broken into pieces and transferred into 50 ml of
balanced salt solution (BSS). The vitelline membrane covering the
blastoderm is removed and kept in petridish containing BSS. With the help
of a forceps the adherent vitelline membrane is removed.
• The embryo is observed by using a microscope so that the developmental
stage of blastoderm could be found out. The blastoderm is placed on the
medium in the watchglass placed on sterile absorbent cotton wool pad in
petridishes. Excess BSS is removed from medium and embryo culture of
chick is incubated at 37.5 ◦C for further development.
Histotypic Culture
• When one characterized cell line is propagated at high density in the presence
of appropriate extracellular matrix and soluble factors then such a culture is
called histotypic. For example vascular endothelial cells will form capillary
tubules in the presence of appropriate soluble factors when grown in a collagen
matrix. Homologous cell interactions occur because the cells are cultured at
high density. An alternative approach is the use of cellulose sponges coated
with extracellular matrix components such as collagen. Cells may penetrate the
sponge and form glandular structures.
Organotypic Culture
• It is not possible to study heterologous cell interactions in a histotypic culture.
When cells of different lineages are recombined to create a tissue-like structure
then we call it organotypic culture. To begin with the simplest is to maintain a
co-culture of two cell types. For example co-culture of epithelial cells and
fibroblast clones derived from mammary gland allow the epithelial cells to
differentiate functionally. This has been demonstrated by their ability to
produce milk proteins in optimal hormonal environment. This functional
differentiation is preceded by the formation of characteristic structures, such as
threedimensional cords in which fibroblast cells organize themselves into
bundles that are then enveloped by epithelial cells.
• Artificial skin:
• The Skin was the first organ to be artificially grown.
• In early 1970s, irradiated 3T3 fibroblast cell line, was grown with
keratinocytes which constitute 90% skin cells.
• 3T3 was found to stimulate the growth of skin cells and helped them to
differentiate into epidermis.
• Three biotechnology companies are developing artificial skin, mainly for
treating burn patients.
• The supporting materials for these skin equivalents are natural or synthetic.
Bovine collagen type I (acid extracted) as well as polygalactic acid (PGA)
have been used for growing skin from neonatal foreskin.
• Synthetic polymers allow cells to grow into skin without scars; hence are a
potential organ developer. Many industries are involved in creating
allogenic grafts.
• The company ‘Organogenesis’ seems to have created a ‘living skin
equivalent (LSE)’ comprising of dermis and epithelial layer. The support
for growth is provided by collagen matrix. These LSE grafts elicit no
immunogenic response in host and are eventually replaced by host cells.
• Artificial cartilage
• Cartilage tissue replacement holds promise in tissue engineering owing to
lack of vascularization.
• The chondrocyte cells which are the precursors of cartilage are combined
with PGA or collagen and are grown in culture. Given a suitable
microenvironment, artificial cartilage can grow.
• Tissue engineering is a new field based upon a relatively simple concept:
Start with some building material (e.g. extracellular matrix or
biodegradable polymer), shape it as needed, seed it with living cells and
bathe it with growth factors.
• When the cells multiply, they fill up the scaffold and grow into three-
dimensional tissue, and once implanted in the body, the cells recreate their
intended tissue functions.
• Blood vessels attach themselves to the new tissue, the scaffold dissolves
and the newly grown tissue eventually blends in with its surroundings.
 Application of Animal Cell Culture
Cell Culture Based Vaccines
• A vaccine is an important immunizing agent. Vaccines are prepared either by
dead organisms or attenuating live microorganisms.
• These are chemical substances prepared from the proteins (antigen) of other
animals, which confer immunity to a particular virus.
• Cell culture based vaccines are purer and safer than existing vaccines. The
existing vaccines contain antigens obtained after treatment of animals with
particular bacteria or virus causing the disease, are used. This causes
‘overloading’.
• In cell culture based vaccines, cells are cultured with the specific bacteria or
virus and only those antigens or antibodies that are specifically required
against that disease are injected into the human; hence they are considerably
purer.
• Cell culture gained eminence after the large-scale production of vaccine. By
using animal cell technology we are able to produce very potent antigens. The
antigen vaccine derived from cell culture system stimulates both humoral and
cell-mediated immunity.
Model Systems
• Animal cell culture provides a great model system for studying basic cell
biology and metabolism. Animal cell culture has been used as 2D and 3D
culture models in research for various experiments related to drug or
infectious agent studies.
Toxicity Testing
• Animal cell cultures are widely used to prevent animal testing for toxicity
testing of new drugs, chemicals, and cosmetics. Major animal cell cultures
used in this domain are Kidney- and liver-derived cell cultures.
Cell-based Manufacturing
• Animal cell culture can be used for the production of genetically modified
products for medical and commercial value. The types of products can be
monoclonal Abs, insulin, hormones, etc.
Drug Screening and Development
• In the pharmaceutical industry, animal cell culture-based assays have
become increasingly im­portant. They are used not only for toxicity but also
for high throughput drug screening.
Cancer Research
• Animal cell cultures have been used for biomarker and molecular studies
in oncology research. Moreover, cancer cells in culture can also serve as
drug testing models for several anticancer compounds. Recent studies in
cancer look forward to selectively kill cancer cells in a population mixed
with normal primary cells.
Genetic Engineering
• Genetic engineering revolves around the idea of reprogramming genes to
produce new proteins. Transfection is the ability to induce new genetic
material in cells. Animal cell cultures can be used for transfection to
produce new proteins in large quantities for clinical study or therapeutic
purposes.
Gene Therapy
• As we know animal cell cultures can be used for genetic engineering, these
engineered cells can be also be used for therapy. Cells can be removed
from a patient and can be replaced by engineered cells having the desired
functional gene. An alternative approach is to add the missing gene in the
patient’s cells using a viral vector.
 Cell culture as source of therapeutic products

CELL LINE PRODUCT

Human leucocytes Interferons
Mouse fibroblasts Interferons
Human Kidney Urokinase
Dog Kidney Canine distemper vaccine
Chick embryo fluid Vaccines for influenza, measles and mumps

Duck embryo fluid Vaccines for rabies and rubella

Cell lines of mouse, rat or human origin Monoclonal antibodies
Chinese hamster ovary cells (CHO) Tissue-type plasminogen activator (tPA)
 What is Tissue Plasminogen Activator (t-PA)?

• t-PA is an enzyme that serves in the cascade of events leading to

dissolution of blood clots

Damaged Blood Clot Dissolution

Tissues t-PA Clot

Fibrin
Breakdown
t-PA
Plasminogen Activation
Plasmin
Urokinase Streptokinase
From the Kidneys From Bacteria
 t-PA Has Been Developed As A Drug By Genentech

• The biotechnology company Genentech has cloned human t-PA for

use in treating unwanted or life threatening blood clots

• Activase (Alterplase recombinant) is the trade name of

Genentech’s t-PA

• Activase is useful in treating heart attacks and strokes when

administered within 5 hours of thrombosis formation or embolism
lodging in the heart or brain
 Production of t-PA

• Human mRNA coding for t-PA from a human

myloma cell line was isolated

• The mRNA was converted into cDNA

• Human t-PA coding cDNA was inserted into Chinese

hamster ovary cell lines

• When grown in culture, the CHO cells excrete

human t-PA into their growth medium

• Activase is produced by isolating t-PA from the

growth medium
 Growing t-PA In CHO Cells

Collect Cells RNA Reverse

From Cell Extraction Transcription
Culture

mRNA

cDNA
Human Myoloma
Cell Culture Insertion Into
Grow CHO Chinese Hamster Ovary Cells
cells in
culture
Collect Extract
culture T-PA
medium
Chinese Hamster Ovary
Cell Culture
t-PA