Chapter 27

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Animal cells as host
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27
Animal cells as host
Vijay Kumar
of the production processes. Biopharmaceutical indus-

27.1 Introduction tries are currently facing the challenge of producing
biological recombinant medical products in a quick and
Recombinant DNA technology or genetic engineer- cost-effective manner, while complying with the safety
ing was developed in the 1970s to express mammalian requirements set by the regulatory agencies. Mamma-
genes in bacteria. However, it soon became apparent lian cell lines, particularly the Chinese Hamster Ovary
that large complex proteins, especially those of thera- (CHO) cell line, are the eukaryotic systems of choice for
peutic value, could not be produced in bacteria, as they many biopharmaceutical industries for large-scale pro-
do not have the appropriate cellular machinery to make duction of recombinant proteins and mAbs due to their
appropriate post-translational modifications (PTMs) in ability to grow in high-density suspension cultures and
these proteins. Therefore, genetically engineered ani- to produce proteins with human-likePTMs. However,
mal cells were developed for large-scale commercial the lengthy, expensive and tortuous path for approval
production of such important proteins. Now-a-days, of a new therapeutic candidate has a significant bearing
animal cells are used such asbacteria or yeast to pro- on the timeline and economics of process development.
duce a variety of recombinant proteins of human ori- Now-a-days, animal cell biotechnology has advanced to
gin, including secreted proteins such as monoclonal the stage where it is used in a variety of manufacturing
antibodies (mAb) and growth factors, intracellular pro- processes. In fact, products derived from animal cell
teins such as enzymes, and membrane proteins such cultures are reported to generate nearly half of the rev-
asgrowth factor receptors. The mammalian cells offer enues from the sale of modern biotechnology products.
a distinct advantage of making recombinant proteins This chapter provides a historical perspective of ani-
with fully humanized PTMs which drastically improve mal cell culture and describes the commonly used meth-
their biological activity and minimize their immuno- ods, equipment, supplies and reagents in this process.
genicity. Such proteins can remain in body circulation The key steps required to maintain adherent cell lines
for an extended period to produce desirable pharmaco- and suspension-grown cells, their long-term cryogenic
logical effects. storage and revival from frozen stocks are outlined.
Animal cell culture is a process of growing animal cells More important, different methods of gene transfer in
in vitro in a flask, dish or fermenter under a strictly con- animal cells, their selection and scale-up culture for the
trolled environment. Because maintaining the purity of production of recombinant proteins and methods used
cell cultures and preventing their contamination con- in their quality control are elaborated.
stitute the greatest challenge in animal cell culture, the
principles of aseptic technique followed in cell culture
are of paramount importance. Optimized animal cell
culture has the ability to deliver sufficient quantities
27.2 Some milestones in the
of recombinant products, and thus, it constitutes an development of tissue
important activity of the present-day biopharmaceu- and cell cultures and their
tical industries. The range of commercially available
recombinant pharmaceuticals produced by cell culture use in recombinant DNA
technologies has increased rapidly over the past few technology
years. This is particularly so for therapeutic proteins syn-
thesized from selected or genetically engineered mam- The concept of maintaining live animal cell lines sepa-
malian cells. They are needed in large quantities and rated from their original tissue source was discovered in
hence require careful study of the underlying principles the early 20th century and animal cell culture became a
Chapter 27.indd 1 6/23/2014 2:39:07 PM

2 CHAPTER 27 Animal cells as host
routine laboratory technique by 1950s. The earliest tis- c ofactors, carbohydrates, and horse serum. By elimi-
sue culture experiment dates back to 19th century when nating one component at a time, he then determined
Sydney Ringer – an English physiologist – developed ‘salt which nutrients were essential for cell growth. His
solutions’ containing chlorides of sodium, potassium, minimum essential medium (MEM) contains 13 amino
calcium and magnesium for maintaining isolated ani- acids (human tissue in vivo requires only eight), eight-
mal hearts outside the body in culture. In 1885, Wilhelm vitamins and cofactors, glucose as an energy source and
Roux – a German embryologist – introduced ‘saline cul- a physiological salt solution that is isotonic to the cell.
ture’ by successfully cultivating the medullary plate of a The pH is maintained at 7.2–7.4 by NaHCO3 in equi-
chick embryo in a warm saline solution for several days librium with CO2. The pH indicator phenol red is usu-
and, thus, opened the roads for tissue culture. Between ally incorporated into the medium; it turns red-purple
1907 and 1910, Ross Harrison from Johns Hopkins Med- if the medium is basic, yellow if the medium is acidic,
ical School published a series of papers describing the and remains red-orange if the pH is in the right range.
methods of tissue culture. He used frog embryonic tis- Serum in concentrations of 1–10% must be added to
sue to grow nerve fibers in the presence of frog lymph the medium to provide the cells with additional poorly
by the hanging-drop method. In 1911, Alexis Carrel and defined factors, without which most cells will not grow.
Montrose Burrows used horse plasma to grow explants Most mammalian cells are incubated at 37oC. However,
of chick heart tissue and introduced strict aseptic tech- the avian, reptilian and arthropod cells may grow opti-
niques so that cells could be cultured for long periods. mally at higher or lower temperatures. CMRL-1066 – a
The development of antibiotics during World War II chemically defined medium – was developed in the late
simplified long-term animal cell culture by minimizing 1950s by Connaught Medical Research Laboratories.
the problems of microbial contamination in cell culture. Later, Richard Ham (1965) introduced a defined, serum-
Peyton Rous and FS Jones (1916) introduced the pro- free medium able to support the clonal growth of certain
teolytic enzyme trypsin for the subculture of adherent mammalian cells. Cherry and Hull (1956) used a mag-
cells. In 1950s, Wilton Earle and his colleagues stand- netic stir bar suspended from a fishing swivel to keep
ardized the technique to dissociate cells from growing cells in suspension in a round bottom flask so that they
chick embryo with the help of trypsin. He observed that could study the effects of varying the medium, speed
when a single-cell suspension was mixed with plasma and seeding densities on cell growth. McLimans and his
and embryo extract and transferred in a sterile glass group (1957) took the magnetic stir bar and attached it
container, the cells adhered to the glass and divided to to a sliding wire so that it could be raised or lowered to
form a monolayer of primary culture that covered the match the medium volume. They added a side port for
entire bottom surface of the vessel and then stopped sampling and called it a ‘spinner flask’, which was used
dividing. The primary culture that contained a variety to grow 400 mL suspension cultures of HeLa and L cells.
of cell types could then be re-dispersed with trypsin and They adapted this approach to work in commercially
planted in new culture vessels containing fresh medium. available 5L microbial fermenter with baffles and sparg-
Often, these secondary cultures were composed entirely ers driven by an overhead impeller and showed that sus-
of spindle-shaped cells called fibroblasts because of pension animal cell culture could be successfully scaled
their resemblance to cultured connective tissue. Earle up to grow products at commercial level. As this method
also isolated mouse L fibroblasts, which formed clones was not suitable for anchorage-dependent cell cultures,
from single cells. In 1952, George Gey and colleagues AL van Wezel (1967) resolved this issue by using small
established a cell line derived from a human cervical porous beads called microcarriers (200–250-µm diam-
carcinoma, which later became the well-known HeLa eter) as substances for cell attachment. These particles
cell line, while the first successful suspension cultures, are made from a variety of materials, including polysty-
lymphoblastic MBIII cells, were grown by Owens, Gey rene, dextran, glass and cross-linked collagen. The use
and Gey(1953). In 1981, the teams led by Martin Evans of these beads in culture required the use of more gentle
and Gail Martin first derived embryonic stem (ES) cells stirring techniques, and by the 1980s, spinner vessels of
from mouse embryos, which laid the foundation of different designs were available to minimize the dam-
‘gene knock-out technology’. Subsequently, the teams age caused by bead–bead collisions and sheer effects.
led by James Thomson and John Gearhart established Today, a number of therapeutic protein drugs and vac-
human ES cells in 1998 that are extremely useful in cines are produced from cells grown in suspension on
regenerative medicine. microcarriers.
In 1955,Harry Eagle made the first systematic inves- Frank Graham and Alex van der Eb (1973) devel-
tigation of the essential nutritional requirements of opedthe famous calcium–phosphate method of DNA
cultured cells and reported that animal cells can prop- transfection into mammalian cells. The viral method of
agate in a defined mixture of small molecules supple- gene delivery was introduced by Paul Berg (1976), who
mented with a small proportion of serum protein. He used a modified SV40 virus containing DNA from the
began by showing that HeLa and mouse L cells would bacteriophage lambda to infect monkey kidney cells in
grow in a mixture of salts, amino acids, vitamins and culture. Manual microinjection of DNA into nuclei of
Chapter 27.indd 2 6/23/2014 2:39:07 PM

27.3 Characteristic features of animal cell culture 3
mammalian cells was successfully tried by Graessman Box 27.1 Major discoveries from cell culture
and coworkers (1978) to introduce genes into mam-
• Identification and isolation of almost all animal/human
malian cells and study their expression and functions.
viruses
The method of electroporation to introduce DNA into
• Discovery and study of bacteria associated with cells such
fibroblasts was established by Tai-Kin Wong and Eber- as Chlamydia
hard Neumann (1982), while the cationic liposome-
• Growth and attenuation of viruses in culture for vaccines,
based DNA transfection was initiated by Philip Felgner e.g., rubella, polio, chicken pox, measles, mumps, rabies,
and coworkers (1987). Today, cationic liposome method yellow fever, etc.
is one of the most popular methods of mammalian cell • Studies of the genetic basis of cancer, immunology, neuro-
transfection. science, embryology and in vitro fertilization
In the early days, the vaccine industry used animals • Genomic revolution: Human genomic DNA sequencing
such as artificially infected rabbits for rabies vaccine or made possible
cows for vaccines against smallpox as a source of some • Characterization of expressed genes by DNA microarrays
of the required viruses for producing viral vaccines. (transcriptomics) and pattern of protein expression under
a defined condition (proteomics)
Bacteria or their toxins were also used as the basis for
bacterial vaccines. Between 1920 and 1950, virus and • Production of mAbs from hybridomas
bacterial vaccines against a number of diseases such as • Screening/evaluation of drugs against viruses and cancer
typhoid, diphtheria, tuberculosis, tetanus, cholera, per- • Development of gene therapy/stem cell therapy
tussis, influenza and yellow fever were developed and
marketed. However, the major advancement made in
animal cell culture during 1940s and 1950s due to major
epidemics of polio during these years prompted a lot hGH), haemophilia (coagulation factors VIII and IX),
of effort to develop polio vaccine through cell culture. anaemia (erythropoietin; EPO), cancer and viral infec-
Growing the virus in cell culture paved the way for man- tions (interferons). The list also includes many mAbs
ufacturing effective viral vaccines. The polio vaccine was used in diagnostics. These examples illustrate the tre-
made possible by the incessant efforts of John Enders, mendous impact of animal cell biotechnology on mod-
Thomas Weller and Frederick Robbins, who shared the ern medicine.
Nobel Prize in 1954 for their discovery to grow poliomy-
elitis viruses in tissue culture. In fact, the polio vaccine
developed by Jonas Salk was the first product to be man-
ufactured in mass scale using animal cell culture tech-
27.3 Characteristic features of
niques. The Salk vaccine changed the medical history by animal cell culture
preventing many thousands of cases of crippling illness
and saving thousands of lives. Another important land- Animal cells, just as plant cells, when removed from
mark in the animal cell culture was the development of tissues will continue to grow in a favourable artificial
‘hybridoma technology’ by Ceaser Milstein and George environment having appropriate nutrients and condi-
Kohler in 1975, which allowed the continuous produc- tions. Therefore, cell culture is a technique of growing
tion of a single type of antibody or mAb. The diagnos- and maintaining the cells outside of the multicellular
tic and therapeutic potential of mAbs is evident from organism in specially designed vessels and which are
their production in kilogram quantities from large-scale incubated under conditions to mimic precise environ-
cultures of hybridomas. mental conditions such as temperature, humidity and
In short, the development of science of animal culture nutrition and contamination-free conditions that were
had a tremendous impact on both basic and applied present in that organism. The cells may be removed
research as well as contributed immensely to the area from the tissue directly and disaggregated by mechani-
of biotechnology (Box 27.1). Its application ranges from cal or enzymatic means before culturing, or they may
basic studies such as the cell cycle control mecha- be derived from an established cell line or cell strain.
nisms, molecular biology of cancer, discovery of new The culture process allows single cells to act as inde-
viruses, creation of new disciplines such as genomics pendent units, much like a microorganism. These cells
and transcriptomics, etc. to anticancer drug discovery, are capable of dividing as they increase in size or in a
production of recombinant pharmaceuticals and stem batch culture and can continue to grow until limited
cell therapy. The recombinant DNA and hybridoma by nutrient depletion or other culture variables. Fur-
techniques boosted the use of animal cell technology ther, the cells in culture may be genetically identical
for the production of vaccines and natural therapeu- (homogenous population) or may exhibit some genetic
tic proteins. Today, the list of products resulting from variation (heterogeneous population). A homogenous
these methods is extensive and includes drugs for the population of cells is derived from a single parental cell
treatment of cardiovascular diseases (tissue plasmino- and are therefore genetically identical and referred to
gen activator; tPA), dwarfism (human growth hormone; as ‘clone’.
Chapter 27.indd 3 6/23/2014 2:39:07 PM

27.3.1 Purpose of cell culture 27.3.3 Limitations of cell culture
Animal cell culture has proven to be of immense value Cell culture is referred to as an ex vivo study of the
in biomedical research and biotechnology industry cellular milieu. This is a serious limitation because
(Box 27.2). It permits the study of cells under controlled the cell is not in its normal physiological and original
conditions and allows researchers to investigate the nor- environment. Cell culture is simply an attempt to pro-
mal physiology and biochemistry of cells, cell metabo- vide a simulated environment. Thus, the study of cells
lism and the cell cycle. It also allows us to examine the in cell culture is akin to studying animal behaviour in
effects of specific conditions and mutations on cell captivity. With the exception of some tumour-derived
physiology and evaluate the effect such as the toxicity of cells, most primary cell cultures have a limited lifes-
chemical compounds or drugs on a given cell type. Most pan. After a certain number of doubling, cells undergo
important, it allows us to do high-throughput screening the process of senescence and stop dividing, but may
of anticancer agents. retain their viability. This phenomenon is known as the
Hayflick limit (Hayflick and Moorhead, 1961). Also a
27.3.2 Missing features in cell culture problem in cell culture is the usage of cells which are
transformed. For example, HepG2 cells are derived from
Cells are often but not always in contact with other cells human hepatoma or liver cancer and, therefore, do not
because in cultures, cells are never left to complete represent normal human hepatocytes. These cells differ
or 100% confluency. This is a major problem as nor- markedly from normal cells as they have altered or lost
mal cells (primary cells) need cell-to-cell contacts and specific cell functiona due to mutations. Alternatives to
show ‘contact inhibition’. This means that when cells using transformed cells are the cultures of primary cells
grow and reach the walls of the container (i.e. reach or immortalized cells which are untransformed. Immor-
confluency), they stop growing further. Transformed
talized cells can grow continuously in culture, but they
cells, on the other hand, can grow and divide quite well areunique in the sense that they do not form tumours in
in the absence of cell-to-cell contacts and form multi- xenograft models (see Section 27.4).
layers. The following important features are missed in
animal cell culture: 27.3.4 Problems with animal cell culture
•• No original tissue organization and three-dimen- Cell culture has many problems, including cell culture
sional (3D) structure contamination by bacteria, mycoplasma and fungus.
•• Poor or no cell–cell and cell–matrix interaction Cells must be subcultured frequently to prevent over-
•• No original blood circulation crowding of cells, leading to a change in their properties.
•• Altered hormonal and nutritional environment. Fur- Further, the culture media must be changed regularly
ther, hormones are usually added at high concentra- to prevent the build-up of contaminants, metabolites
tions which may not be physiological and toxins and to provide fresh nutrients as the nutri-
tional requirements of fast-growing transformed cells
are higher.
Box 27.2 Importance of animal cell culture

Advantages 27.4 Origin of cell culture, cell
• Has a homogenous population of cells types and cell culture
• Is grown in controlled physicochemical environment
• Has the flexibility to add genes (transfection) or regulate
systems
protein levels (RNAi)
• Generates sufficient number of cells for doing The term ‘tissue culture’ was originally used to denote
biochemistry
the explants of tissue grown in the plasma. The term
• Used in production of biopharmaceuticals subsequently became associated with the culture of
• Is ethical to use and with fewer regulatory controls cells and is now obsolete in its original sense. Cell cul-
• Involves low-cost screening/assays ture refers to tissue dissociated into a single-cell sus-
pension, usually with the help of trypsin. After a brief
Disadvantages wash, cells are counted, diluted in a growth medium
• Requires sensitive techniques to detect changes in tiny
and allowed to settle onto the flat-bottom culture ves-
cells sels. Freshly isolated cultures from mammalian tis-
• Has a challenging scale-up sues are known as primary culture until subcultured
(Fig. 27.1). At this stage, cells are usually heterogeneous
• Does not necessarily represent the original living systems
but still closely represent the parent cell types as well
Chapter 27.indd 4 6/23/2014 2:39:07 PM

27.4 Origin of cell culture, cell types and cell culture systems 5
Tissues or slice of Organ
•• Fibroblast like: cells which are attached to a substrate
and appear bipolar and elongated, frequently forming
Collagenase swirls in heavy cultures
•• Lymphoblast like: cells which do not attach normally
to a substrate but remain in suspension and have a
Dissociated cells/Organ explants spherical shape.
Culture medium
27.4.2 Types of cultured cells
Primary culture There are three main types of cultured cells depending
on the number of times the cells can divide:
Subculture/passage
•• Primary cell cultures: When cells are taken freshly
from animal tissue and placed in culture, the culture
Cell line consists of a wide variety of cell types, most of which
are capable of very limited growth in vitro, usually
Single cell Immortalization fewer than 10 divisions. These cells retain their diploid
isolation
Clonal line karyotype, that is, they have the chromosome number
and morphology of their tissues of origin. They also
Continuous Cell line (e.g.NIH3T3, IHH) retain some of the differentiated characteristics that
(Immortalized) they possessed in vivo. For this reason, these cells sup-
port the replication of a wide range of viruses. Primary
Transformation/ cultures derived from monkey kidneys, mouse foe-
Loss of growth co ntrol tuses and chick embryos are commonly used for many
laboratory experiments and for diagnostic purposes.
Transformed cell line •• Diploid cell strains: Some primary cells can be passed
(e.g., COS-1) through secondary and several subsequent subcul-
tures while retaining their original morphological
Tumor cell lines characteristics and karyotype. Subcultures will have
HepG2, MCF7 fewer cell types than primary cultures. After 20–50
passages in vitro, these diploid cell strains usually
Figure 27.1 Scheme showing the possible mode of origin of cell undergo a crisis in which their growth rate slows and
lines [modified from Ian Freshney (2010). they eventually die out. Diploid strains of fibroblasts
derived from human foetal tissue are widely used in
diagnostic virology and vaccine production.
as the expression of tissue-specific properties. When •• Continuous cell lines: Certain cultured cells, notably
these cells are subcultured in new containers with fresh mouse foetal fibroblasts, kidney cells from various
medium, this is called a secondary culture. After several mammalian species and human carcinoma cells, are
subcultures onto fresh media, the cell line will either able to survive the growth crisis and undergo indefinite
die out or ‘transform’ to become a continuous cell line. propagation in vitro. After several passages, the growth
Cells can also be virally transformed Such cell lines rate of the culture slows down; then isolated colonies
show many alterations from primary cultures, includ- of cells begin to grow more rapidly than diploid cells,
ing changes in morphology, chromosomal variations their karyotype becomes abnormal (aneuploid), their
and an increase in the capacity to form colonies in soft morphology changes and other poorly understood
agar (colony formation assay) or give rise to tumours changes take place that make the cells immortal. The
when implanted in immune-deficient hosts (xenograft cells are now ‘dedifferentiated’, having lost the special-
models; Fig. 27.2). ized morphology and biochemical abilities they pos-
sessed as differentiated cells in vivo. Continuous cell
27.4.1 Types of cells lines such as KB and HeLa, both derived from human
carcinomas, support the growth of many viruses.
Cultured cells are usually described based on their mor-
phology or their functional characteristics. Animal cells
exhibit three basic morphologies (Fig. 27.3): 27.4.3 Cell culture systems
•• Epithelial like: cells which are attached to a substrate For growing cells, two culture systems are used. They
and appear flattened and polygonal in shape are mainly based on the ability of the cells to either
Chapter 27.indd 5 6/23/2014 2:39:07 PM

Virus
Monolayer
culture
Stacking of
transformed cells
(a) Viral transformation of cells
Control HBx
c-MYC HBx+c-Myc Control siRNA Specific siRNA
(b) Colony formation assay (c) Xenograft model

Courtsey:Dr.Anil Suri NII, New Delhi
Figure 27.2 Animal cell transformation and its assays. (a) Viral transformation of cells. (b) Colony formation assay. (c) Xenograft model (Court-
sey: Dr Anil Suri, National Institute of Immunology, New Delhi).
attach to a glass or treated plastic substrate, known as flasks, where the cells are keep it in a fully suspended
monolayer culture or ‘anchorage-dependent system’, medium. The advantages and limitations of using
and the floating free in the culture medium is known monolayer and suspension cultures are summarized in
as suspension culture or ‘anchorage-independent sys- Table 27.1.
tem’. Monolayer cultures are usually grown in tissue
culture–treated dishes, T-flasks, roller bottles, petri 27.4.3.1 Monolayer culture
plates or multiple well plates; the choice should be
based on the number of cells needed, the nature of Adherent cell lines will grow in vitro until they have
the culture, personal preference and cost. While the covered the surface area available or the medium is
suspension cultures are usually grown either in mag- depleted of nutrients. Before this point, the cell lines
netically rotated spinners or in shaken Erlenmeyer should be subcultured to prevent the culture from
Chapter 27.indd 6 6/23/2014 2:39:08 PM

(a) Human mammary (b) Human fibroblast cells (c) Human large
epithelial cells lymphoid cells
Figure 27.3 Types of mammalian cell lines. (a) Human mammary epithelial cells. (b) Human fibroblast cells. (c) Human large lymphoid cells.
27.4.3.2 Suspension culture

Table 27.1 Adherent versus suspension cell culture
Adherent cell culture Suspension cell culture Some cells, particularly those derived from haemat-
Appropriate for most cell types, Appropriate for cells adapted to opoietic or certain tumour tissues, are anchorage inde-
including primary cultures suspension culture or cells of pendent and grow in suspension. Propagation of such
hematopoietic origin cells has several advantages over propagation in mon-
Requires periodic passaging Easier to passage olayer as this is just a matter of dilution. Thus, there is
Easy visual inspection under Requires daily cell counts and no growth lag after splitting of cells as there is no trauma
microscope viability determination associated with proteolytic enzyme treatment. Further,
Cells are dissociated Does not require enzymatic or the scale-up of suspension cultures is straightforward
enzymatically (e.g. trypsin) or mechanical dissociation and the cells can be easily grown in bioreactors. The
mechanically
seeding densities range from 2 × 104 to 5 × 105 viable
Growth is limited by surface Growth is limited by cell density cells/mL.
area in the medium
Limits product yield Allows easy scale-up 27.4.4 Procedure for cell culture
Requires cell culture– Can be maintained in any
compatible vessels sterile culture vessels After cultured cells have formed a confluent mon-
No agitation is required Requires agitation for gas olayer on the surface of their culture vessel, they may
exchange be removed from the surface, diluted and seeded into
Good for cytology, many Good for bulk protein new vessels. If the initial culture was primary, the new
research applications, production and batch cultures are called secondary and are likely to con-
harvesting of products harvesting, many research
continuous applications sist of fewer cell types. Removal of cells from glass or
plastic surfaces may be by either physical methods
such as scraping with a sterile rubber policeman or
enzymatic methods using proteolytic enzymes such
as trypsin, dispase or collagenase or chelating agents
dying. For subculture, the cells need to be brought into or a c ombination of the two. After removal, cells are
suspension. The degree of adhesion varies from cell pipetted up and down against the bottom of the flask
line to cell line, but in most cases, proteases, for exam- to break up clumps, diluted and counted. Primary
ple, trypsin, are used to release the cells from the flask. cultures can usually be diluted 1:2 or 1:3 for second-
Note however that trypsin treatment sometimes could ary culturing, and after one becomes familiar with the
be harmful to cells as this may remove membrane growth characteristics of a certain cell type, counting
markers or receptors of interest. In these cases, cells can usually be dispensed with. The same procedure can
should be brought into the suspension into a small vol- be used to transfer both primary cells and a continu-
ume of medium with the help of cell scrapers/rubber ous cell line, removing the cells from flasks with a mix-
policeman. ture of trypsin and EDTA in physiological saline (TES,
Chapter 27.indd 7 6/23/2014 2:39:08 PM

i.e.Tris-EDTA-Saline with 10 mM Tris chloride, pH 7.4, determined by the media formulation. Salt and glu-
150 mM NaCl, 1 mM EDTA). cose are the major contributors to the osmolality of
the medium, but amino acids are equally important.
27.4.5 Culture conditions Most commercial media are formulated to have a final
osmolality of ~300 mOsm, which can be checked with
Culture conditions vary widely for each cell type, but the help of an osmometer.
the artificial environment in which the cells are cul- •• Culture medium: Apart from temperature and CO2,
tured invariably consists of a suitable vessel containing the most common variable in culture systems is the
the following: growth medium. Recipes for growth media can vary
in pH, glucose concentration, growth factors and the
•• A substrate or medium that supplies the essential presence of other nutrients. Glucose (0.8g to >5g/L)
nutrients (amino acids, carbohydrates, vitamins, min- is commonly used as an energy source, which helps
erals) in maintaining osmolarity of the medium. Many
•• Growth factors of the media contain phenol red as a pH indicator,
•• Hormones which is very helpful in monitoring the pH of the
•• Gases (O2, CO2) culture medium in a CO2 incubator. Highly acidic
•• A regulated physicochemical environment (pH, conditions turn the phenol red into yellow, while
osmotic pressure, temperature) highly alkaline conditions turns the phenol red into
pink. The growth factors used to supplement media
Cells can also be adapted to different culture environ- are often derived from animal blood, such as foe-
ments (e.g., different nutrients, temperatures, salt tal bovine serum (FBS). One complication of these
concentrations, etc.) by varying the activities of their blood-derived ingredients is the potential for con-
enzymes. Most cells are anchorage dependent and must tamination of the culture with viruses, mycoplasmas
be cultured while attached to a solid or semi-solid sub- or prions and shows variation in growth factor and
strate (adherent or monolayer culture), while others can hormone contents. Therefore, current practice is to
be grown floating in the culture medium (suspension minimize or eliminate the use of these ingredients
culture): wherever possible and use chemically defined media.
Commercially available medium are highly sterile
•• Incubation conditions: Most mammalian cells are and ready-to-use liquids in a concentrated liquid or
grown and maintained at an appropriate tempera- powdered form. Instead of providing nutrients for
ture and gas mixture (typically at 37°C with 5% CO2 growing cells, the medium is generally supplemented
for mammalian cells) in a CO2 incubator. This tem- with fungicides and antibiotics, or both, to inhibit
perature is selected because it is the core temperature contamination.
of the human body. Besides, most cells derived from •• Dulbecco’s Modified Eagle Medium (DMEM) is a basal
other warm-blooded animals grow well at 37oC. medium consisting of amino acids, vitamins, glucose,
•• pH: The extracellular and intracellular pHs are criti- salts and a pH indicator which contains no proteins
cal for the survival of mammalian cells. They help in or growth-promoting agents. Therefore, it needs sup-
maintaining the appropriate ion balance and opti- plementation to be a ‘complete’ medium. It is com-
mal function of cellular enzymes, hormones and monly supplemented with 5–10% FBS. DMEM used
growth factors. Fluctuations in pH adversely affect cell a sodium bicarbonate buffer system (3.7 g/L) and
metabolism, which can lead to cell death. Most media therefore requires artificial levels of CO2 to maintain
strive to achieve and maintain the pH between 7 and the required pH 7 and 10% CO2 is optimal, but many
7.4. Most media use the bicarbonate–CO2 buffering researchers successfully use it in 5% CO2.
system. The interaction of CO2 derived from cells or •• Serum and antibiotics: Serum is one of the most
the atmosphere with water leads to a drop in the pH, important components of animal cell culture as it
while the bicarbonate content of the medium neu- supports cell proliferation. It is the source of peptide
tralizes the effect of increased CO2 until equilibrium hormones and hormone-like growth factors that pro-
is reached at pH 7.4. This kind of system is called an mote a healthy growth of cells. Serum is also a source
open system and can be described by the following of various amino acids, hormones, lipids, vitamins,
equation: polyamines and salts of calcium, potassium and iron,
etc. However, one of the key limitations of FBS usage is
H2O + CO2    H2CO3    H+ + HCO3– the variation in its growth factor contents and chances
of its contamination with viruses or prions that may
•• Osmolality: The osmolality of the culture medium adversely affect the culture. Therefore, current prac-
also plays a crucial role in cell growth and function tice is to minimize the use of FBS in cell culture and
by maintaining the outside and inside osmotic pres- use serum-free medium instead. Although not essen-
sure of the cell. Osmolality of the medium used is tial for cell growth, antibiotics such as penicillin and
Chapter 27.indd 8 6/23/2014 2:39:08 PM

streptomycin are often used in the culture medium to 5. Seed an appropriate volume of cell suspension for the size
control microbial growth. of the flask to get the desired cell density.
•• Seeding density: Plating density (the number of cells
per volume of the culture medium) plays a critical role 27.4.7 Cell counting
for some cell types. For example, HepG2 cells when
seeded at lower density do not grow optimally in cul- Cell counting is done in the following steps with the
ture vessels. help of a haemacytometer (Fig. 27.4):
•• Mode of culturing: Cells can be grown either in sus-
pension or adherent cultures. Some cells naturally live 1. Clean the chamber and cover slip with alcohol. Dry and fix
in suspension, without being attached to a surface, the coverslip in position.
such as cells that exist in the bloodstream. There are 2. Harvest the cells. Add 10 μL of the cells to the haemacy-
also cell lines that have been modified to be able to tometer.
survive in suspension cultures so they can be grown 3. Place the chamber under the inverted microscope under a
to a higher density than adherent conditions would 10× objective. May use phase contrast to distinguish the cells.
allow. Adherent cells require a surface, such as tissue 4. Count the cells in four 16-square grids (marked 1 to 4) and
culture plastic or microcarrier, which may be coated take their average.
with extracellular matrix components to increase
adhesion properties and provide other signals needed The following example illustrates how to convert your
for growth and differentiation. Most cells derived from cell count to the number of cells in the original suspen-
solid tissues are adherent. Another type of adherent sion. Assume a 1:10 dilution is being used to count cell
culture is organotypic culture, which involves grow- numbers; if you count an average of 12 cells in the four
ing cells in a 3D environment as opposed to two- 16-square grid (1 mm2), then the cell numbers in a given
dimensional culture dishes. This 3D culture system suspension can be derived as follows:
is biochemically and physiologically more similar to
in vivo tissue, but is technically challenging to main- 12 × 104× 10 = 1.2 × 106 cells/mL
tain because of many factors (e.g. diffusion). Cultures
should be examined daily for their morphology, col-
our of the medium and density of cells. Loading groove
Mirrored surface
27.4.6 Typical experimental protocol

for cell culture
A typical experimental protocol for cell culture is as

follows:
Grid
1. Examine the cells growing in a 25-cm2 flask with an
inverted microscope to see if they have formed a confluent
(~80%) cell monolayer. If there are sufficient cells, pour off
the medium.
2. Wash the monolayer with 2 mL of phosphate-buffered
saline. Rinse well without shaking the flask (shaking pro-
duces bubbles) and pour off. Repeat. 1 2
3. Add 1.0 mL of TES to the flask and incubate at 37oC for
2–10 minutes with TES covering the cells. Observe periodi-
cally to determine when cells are loosened from the plas-
tic. (Note: TES should contain a pH indicator. Below pH 7,
trypsin is inactive. However, above pH 8, trypsin is damag- 5
ing to cells.)
4. When cells are seen to detach from the surface upon shak-
ing or jarring against the heel of one’s hand (this can be
checked under the microscope), add 4 mL of fresh growth
medium with 10% serum and suspend cells by pipetting 3 4
up and down a few times. Count cells in a haemacytom-
eter, calculate the volume of additional medium needed to
bring the cell concentration to ~5 × 105 cells/mL and add Figure 27.4 Haemacytometer along with an enlarged view of the
this volume to the cell suspension. counting chamber or grids.
Chapter 27.indd 9 6/23/2014 2:39:09 PM

where 12 is the average number of cells in four grids, × should be characterized and checked for contamina-
104 converts to cells per millilitre, × 10 is the dilution fac- tion. There are several common media used to freeze
tor and 1.2 × 106 cells/mL is the number of cells in the cells.
original suspension. For serum-containing medium, the constituents may
be as follows:
27.4.8 Cell viability
•• Complete medium containing 10% glycerol
Viability as says measure the number of viable cells in •• Complete medium containing 10% DMSO (dimethyl-
a population and provide an accurate indication of the sulphoxide)
health of cells in culture. These as says rely upon the •• 50% cell-conditioned medium with 50% fresh
integrity of the cell membrane as an indicator of cell via- medium containing10% glycerol or 10% DMSO.
bility. Stains such as trypan blue are actively excluded
by viable cells, while they are taken up and retained by Cells are stored in sterile cryogenic storage vials or
dead cells that lack an intact membrane. cryovials that have a screw-top and a rubber seal to
keep the nitrogen out. Cryoprotective agents such
27.4.9 Cryopreservation (freezing, asDMSO help in reducing the freezing point and facili-
storage and revival) of cells tate a slow cooling rate. Gradual freezing decreases the
risk of cell damage and ice crystal formation. Ideally, a
Cell culture has many problems, including culture con- cooling rate of 1°C/min is recommended. Afterwards,
tamination by bacteria, mycoplasma, and fungus. Also, cells must be stored at –70oC or lower (ideally in liquid
cells must be subcultured frequently to prevent over- nitrogen at –196oC). One must ensure that the liquid
crowding of cells. Furthermore, media must often be nitrogen containers carrying the canisters of cryovi-
change to prevent the build-up of contaminants and als are relatively full of nitrogen during the period of
toxins and to provide fresh nutrients as nutrients are storage.
used up quickly by rapidly proliferating cells. There-
fore, as soon as a small surplus of cells becomes avail- 27.4.9.2 Resuscitation of frozen cell lines
able from subculturing, they should be frozen as a seed
stock, protected and should not be made available When cryopreserved cells are needed for study, it is vital
for general laboratory use. Working stocks can be pre- to thaw cells correctly to maintain the viability of cells
pared and replenished from frozen seed stocks. If the and enable the culture to recover quickly. Some cryo-
seed stocks become depleted, cryo preserved working protectants, such as DMSO, are toxic above 4°C; there-
stocks can then serve as a source for preparing a fresh fore, it is essential that cultures are thawed quickly and
seed stock with a minimum increase in the generation diluted in culture medium to reduce their toxic effects.
number from the initial freezing. Cell lines in continu- With careful handling, 50–80% of the cells of a healthy
ous culture are also prone to genetic drift and are fated culture will survive freezing.
for senescence, and even the best-run laboratories can The following steps are recommended for getting best
experience equipment failure. As an established cell results:
line is a valuable resource and its replacement is expen-
sive and time-consuming, it is vitally important that it is 1. Thaw frozen cells rapidly (<1 min) in a 37°C water bath.
cryopreserved for long-term storage. 2. Dilute the thawed cells slowly using a pre-warmed growth
medium.
27.4.9.1 Storage by freezing 3. Plate thawed cells at a high density to optimize recovery.
Viability of viruses and bacteria is well preserved dur- 27.4.10 Commonly used equipment in
ing freezing, but original attempts to preserve animal animal cell culture
cells by freezing resulted in cell death. This was first
thought to be due to laceration of cell plasma mem- A typical cell culture laboratory should have the follow-
branes by ice crystals, but more recent evidence sug- ing main equipment for doing animal cell culture work:
gests that the cause may be osmotic changes during cell culture hood, CO2 incubator, inverted microscope,
freezing which give rise to irreversible changes in centrifuge, etc.
lipoprotein complexes in intracellular membranes. In
any case, mammalian cells are cryopreserved to avoid 27.4.10.1 Cell culture hood
loss by contamination, to minimize genetic change
in continuous cell lines and to avoid aging and trans- All cell culture manipulations must be performed asep-
formation in finite cell lines. Before cryopreservation, tically in laminar air flow hoods to keep the work area
one need to ensure that cells are healthy (>90% viabil- free any bacterial or fungal contamination. Therefore,
ity) and growing in the log phase. Further, the culture the cell culture hoods should never be used for bacterial
Chapter 27.indd 10 6/23/2014 2:39:09 PM

or fungal work. A laminar hood has a dual role in cell hazardous agents as it blows air directly at users, but are
culture: good for cultures. Both vertical and horizontal hoods
have continuous air flow that passes through a high effi-
•• It protects the tissue culture from the users (by pro- ciency particle air (HEPA) filter to remove particulate
viding a sterile environment). matter from the air. The hoods are also equipped with
•• It protects the user from the tissue culture (from pos- a short-wave ultra violet (UV) light source which needs
sible infection risk). be turned on for a few minutes before use to sterilize
the work surfaces of the hood. Also, the hood should
There are two types of laminar air flow hoods – vertical be turned on about 10–20 min before being used. It is
and horizontal (Fig. 27.5). In a vertical hood, the filtered important to keep the hood free of all clutter as they
air blows down from the top of the cabinet; in a hori- interfere with the laminar flow air pattern. Wiping of all
zontal hood, the filtered air blows out at the user in a work surfaces with 70% ethanol inside the hood before
horizontal fashion. The vertical hood is also known as a and after each use is a good practice.
biology safety cabinet (BSC) and is appropriate for work-
ing with infectious agents. Here, the infectious aerosols 27.4.10.2 CO2 incubator
generated in the hood are filtered out before they are
released into the surrounding environment. Depend- The CO2 incubator is designed to maintain sterility
ing on the nature of the infective agent being used in inside the chamber and keep a constant temperature,
cell culture, the BSCs are designated as class I, II or III. high relative humidity and an atmosphere with a fixed
The horizontal hoods are unsuitable for working with level of CO2. Thus, it reproduces an environmental
Hepa filter
Glass
screen
Work area
Prefilter
Blower
Stand
(a) Horizontal Laminar Hood (c) Horizontal Laminar Flow
Hepa Recirculating fan

exhaust filter
Hepa
filter
Prefilter
Glass
screen
Work area
Stand
Figure 27.5 Cell culture laminar hoods. (a) Horizontal
(b) Vertical Laminar Hood laminar hood. (b) Vertical laminar hood. (c) Horizontal
(d) Vertical Laminar Flow laminar flow. (d) Vertical laminar flow.
Chapter 27.indd 11 6/23/2014 2:39:09 PM

The inverted microscope allows viewing of cells from
the bottom because its optical system is at the bottom,
with the light source on top [Fig. 27.6(b)]. Observation
of cultures in this way gives an immediate idea of the
health and growth of cells. The lights of microscope
should be turned off when not in use and should be kept
covered to protect the optical parts from dust.
27.4.10.4 Centrifuge
CO2 inculator Inverted microscope A low-speed centrifuge is required for animal cell pel-
leting during thes ubculturing step. In most cases,
cells are centrifuged at ambient temperature. Nev-
Figure 27.6 Cell culture equipment. (a) CO2 incubator. (b) Inverted
microscope.
ertheless, a low operation temperature is desirable
to minimize exposure of cells to uncontrolled higher
temperatures.
c ondition very close to that of living cells. The incuba- 27.4.11 Commonly used culture wares
tor chamber has an airtight silicone gasket on the inner
door which keeps it isolated from the external environ- The need for sterility also applies to the glassware or
ment [Fig. 27.6(a)]. The animal cells are maintained in plastic ware used to prepare media and reagents and
an atmosphere of 5–10% CO2, which keeps the medium to grow cells. Metal and glass devices can be steri-
buffered with sodium bicarbonate/carbonic acid. lized by autoclaving while disposable plastic culture
Culture vessels have vents to allow for sufficient gas flasks and other plastic ware are sterilized by gamma
exchange. A pan of water is kept at all times in the incu- radiation. Sterilization methods have the goal of kill-
bator chamber to maintain a high relative humidity, ing contaminating organisms without affecting the
prevent desiccation and maintain the correct osmolar- surface of the flask or vessel. The animal cells are
ity of the culture medium. The incubator doors should usually grown and maintained in petri dishes, cul-
not be left open for very long during use. ture flasks or multi-well plates of various shapes and
sizes (Fig. 27.7) at an appropriate temperature and
27.4.10.3 Inverted Microscope gas mixture (typically, 37°C, 5% CO2 for mammalian
cells) in an incubator. There are numerous suppliers
In tissue culture vessels such as petri dish, the cells are of cell culture plastic ware such as BD Biosciences,
present at the bottom, with the culture medium above. Corning, Falcon and Nunc International. Note that
Figure 27.7 Cell culture plas-

tic wares and accessories.
Chapter 27.indd 12 6/23/2014 2:39:10 PM

27.5 Gene delivery in cells 13
the most expensive cell culture plastic ware does not For long-term gene expression, the transfected gene is
always mean the best choice for your cells. A plastic integrated into the host cell genome to make permanent
ware check is always worth doing with primary cells cell lines. A selection marker is used to identify cells that
as their requirements are more specific. However, for have successfully integrated the gene sequence of inter-
cell lines, one may start with the brand currently used est. The selection agent enriches and enables the growth
in the laboratory. of a subpopulation of cells where the exogenous genetic
material has been incorporated into the genome.
Transfection of DNA or RNA molecules into cultured
27.5 Gene delivery in cells mammalian cells can be accomplished using a variety
of methods and reagents. These methods include physi-
cal (electroporation, microinjection), chemical (cal-
Animal cell transfection is a commonly used method cium phosphate method, lipofection) and virus-based
to deliver exogenous DNA or RNA into cells to express (adenovirus, retrovirus) delivery systems. The impor-
them in cell culture. It offers an important mechanism tant parameters that affect the outcome of cell transfec-
for advancing the knowledge about the structure and tion include: (i) cell type, (ii) cell density, (iii) amount of
functions of animal genomes and understanding their DNA to be transfected, (iv) ratio between the transfec-
new traits through cell culture. There are several differ- tion reagent and the DNA, (v) incubation period of cells
ent ways to transfect mammalian cells, depending on with the DNA complex, (vi) post-transfection incuba-
the cell line characteristics, desired effect and down- tion time of cells, etc.
stream applications. These methods can be broadly
divided into two categories: those used to generate tran- 27.5.2 Methods of transient transfection
sient transfection and those used to generate stable cell
lines. Table 27.2 summarizes the important aspects of The various methods of transient transfection are as
stable and transient transfection methods. follows.
27.5.1 Transient versus stable 27.5.2.1 Electroporation

transfection
The use of high-voltage pulses or electroporation to
Transient transfections are most commonly used to introduce DNA into cultured cells was first estab-
investigate the short-term impact of gene and/or pro- lished by Wong and Neumann (1982) for fibroblasts. It
tein expression on the cell cycle, cell physiology and is a highly efficient technique for delivering exogenous
metabolism or even reporter genes. Plasmid DNA, mes- nucleic acids to suspension cells and non-adherent
senger RNA (mRNA), short-interfering RNA (siRNA), primary cells. This technique uses electricity to create
short-hairpin RNA (shRNA) and microRNA (miRNA) transient pores (electropores) in the cellular membrane
are commonly used in transfection experiments. Dur- to enable the uptake of charged nucleic acid molecules
ing transient expression, however, the nucleic acid into the target cells [Fig. 27.8(a)]. A highcell mortality in
sequence is not integrated into the host cell genome. this case is a major drawback.
Therefore, the effect on target gene expression is tem-
porary (24–72 h for RNA probes, 48–96 h for DNA 27.5.2.2 Calcium phosphate method
probes).
This method was first used by Graham and van der Eb
in 1973 to introduce adenovirus DNA into mammalian
cells. Here HEPES-buffered saline solution contain-
Table 27.2 Transient versus stable expression of genes ing phosphate ions is mixed with a calcium chloride
Transient expression Stable expression solution containing the DNA to be transfected. When
the two solutions are combined, a fine precipitate of
Short-term expression Sustained expression on a
the positively charged calcium and the negatively
(usually 48 h) long-term basis
charged phosphate is formed, binding the DNA to be
Transfection by chemical Transfection by chemical
methods or electroporation methods or viral vectors transfected on its surface [Fig. 27.8(b)]. The suspen-
followed by selection for sion of the p recipitate is then overlaid on the cells to
antibiotic or drug resistance be transfected. The resulting calciumphosphate–DNA
Useful for evaluating gene Useful for continuous source of complexes adhere to the cell membrane and enter the
activity or their regulation a gene product or gain/loss cytoplasm by endocytosis. This method is extremely
of a gene function
simple to handle but not good for all cell types. Tran-
No genomic integration of Non-specific integration of sient hypertonic shock of the transfected cells with
transfected DNA transfected DNA in host cell
genome 10% glycerol or DMSO improves the transfection
efficiency.
Chapter 27.indd 13 6/23/2014 2:39:10 PM

− −
Lid
Ca++ −
Ca++
Cuvette
− −
− −
Cell suspension Ca++

Ca++
Electrode
Ca++
− + −
−
−
Ca++
DNA - Calcium
phosphate precipitate
(a) Electroporation (b) Calcium phosphate method
+
+ +
+ + N C
Cationic
+ + liposome
+
− − −
N N C
− − − − N C
DNA Mammalian cell
(c) Lipofection
Figure 27.8 Methods of transient transfection. (a) Electroporation. (b) Calcium phosphate method; lipofection.
27.5.2.3 Liposome-mediated transfection 27.5.3 Methods of stable transfection
Liposomes are synthetic analogues of the phospholipid The various methods of stable tranfection are as follows.
bilayer, the building block of the cellular membrane.
These transfection compounds share a number of char- 27.5.3.1 Microinjection
acteristics with their natural counterparts, including
the presence of hydrophobic and hydrophilic regions Microinjection delivers the genetic material directly
of each molecule, which allow for the formation of into the cell nucleus (Fig. 27.9). Using a light micro-
spheroid liposomes under aqueous conditions. In the scope and a fine needle (micromanipulator) guided
presence of free DNA or RNA, liposomes encapsulate into the nucleus, a small amount of DNA or RNA is
the nucleic acids to create an efficient delivery system. injected. However, this method is labor intensive and
The charge, composition and structure of the liposome requires trained personnel. Nevertheless, this method
define the affinity of the complex for the cellular mem- is extremely useful in the case of animal transgenesis
brane. The cationic liposomes were first introduced and gene knock-in and knock-out technology.
in 1987 by Felgner and coworkers. The liposomes cur-
rently in use typically contain a mixture of cationic and 27.5.3.2 Virus-mediated gene delivery
neutral lipids organized into lipid bilayer structures. (transduction)
Transfection complex formation is based on the inter-
action of the positively charged liposome with the nega- Viral vector is the most effective means of gene trans-
tively charged phosphate groups of the nucleic acid. fer to modify specific cell type or tissue and can be
Liposomes fuse with the cell m embrane and deliver manipulated to express heterologous genes. Several

their cargo inside [Fig. 27.8(c)]. The method is quite virus types are currently being investigated for use to
simple as well as reproducible, and the transfection effi- deliver genes to cells to provide either transient or per-
ciency is reasonably high. However, cell toxicity could manent transgene expression. The viral vectors in cur-
be a problem sometimes. rent use are based on different RNA and DNA viruses
Chapter 27.indd 14 6/23/2014 2:39:11 PM

15
27.5 Gene delivery in cells
Cell holding pipet Egg pronucleus
Negative
Positive
pressure
pressure
DNA injecting needle
e.g., microinjection of egg cell
Inverted microscope
Cell holding pipet

DNA injecting needle
Cell
manipulator DNA
micromanipulator
Control knob
Micromanipulator
Figure 27.9 Method of microinjection. Micromanipulator and microinjectionof egg cell.
that possess very different genomic structures and host vectors are shown in Fig. 27.10. Very often, a selection
ranges. Among viral vectors developed so far, retrovi- marker (either antibioticbased or fluorescent protein-
ral vectors represent the most prominent delivery sys- based) is used to select for cells that have been suc-
tem, asthese vectors have high gene transfer efficiency cessfully transduced with the virus. Once the genetic
and mediate high expression of heterologous genes. material is incorporated into the host genome, it relies
Members of the DNA virus family such as adenovi- on the host transcriptional machinery for expression,
rus, adeno-associated virus or herpesvirus have also while in the case of shRNA/miRNAprobes, the host
become attractive for efficient gene delivery. The viral RNAi machinery is required. However, viral vectors
genomes are modified to act as viral vectors to intro- have limited laboratory uses because the viral vector
duce foreign DNA into mammalian cells. The viruses integrates randomly into the genome and could yield
have their own mechanism for moving nucleic acids different results.
into cells and transduction by this route is usually very
efficient. The viral vectors are selected as gene deliv- 27.5.4 Development of stable cell line
ery vehicles based on their capacities to carry foreign
genes and their ability to efficiently deliver these genes The episomal DNA stability is often limited, resulting
combined with their efficient gene expression and inte- in a gradual loss of transfected DNA vector from the
gration into the target cell genome with the help of the cells. As DNA integration into host chromosomes is a
viral machinery. While in adeniviral vectors, the early rare event, stablytransfected cells need to be selected
E1 and E3 genes can be replaced, the entire gag, pol and cultured in different ways. A variety of systems for
and env genes of the retroviral genome can be replaced selecting transfected cells exist, including resistance to
with foreign DNA. The lost viral gene functions can antibiotics or expression of metabolic enzymes. After
be provided in trans using appropriate packaging cell gene transfer, cells are cultivated in a medium contain-
lines. The simplified maps of adenoviral and retroviral ing the selective agent. Only those cells which have
Chapter 27.indd 15 6/23/2014 2:39:11 PM

Adenovirus5
E1 E3 LTR
ψ LTR
gag pol env
Deletion in E1
and/or E3 regions LTR
ψ LTR
Therapeutic gene(s)
Recombinant retrovirus
Therapeutic gene Transfection into

in transfer plasmid pakaging cells
Cotransfection in 293 cells (E1 in trans) Cell with helper provirus
gag pol env

Transcription
+ Translation
Recombinant Ad5 genome AAA

AAA
Capsid proteins Genomic RNA
1012 particles/ml
Non-integrative 106 particales/ml
All cell types Integrative
Cell specific
Recombinant
virus Empty Recombinant
particles virus
(a) Adenoviral Vector (b) Retroviral Vector
Figure 27.10 Scheme of viral vectors for gene delivery. (a) Adenoviral vector.(b) Retroviral vector.
integrated the p
lasmid survive, containing the selec- potent translational inhibitor in both prokaryotic
tion marker gene. Both dominant and recessive selec- and eukaryotic cells. Resistance to puromycin is
tion marker genes are used for establishing permanent conferred by the puromycin N-acetyl-transferase
cell lines: gene (pac) from Streptomyces. Puromycin has a fast
mode of action, causing rapid cell death at low anti-
•• Dominant selection markers: These marker genes biotic concentrations. Adherent mammalian cells
are usually of microbial origin and confer resistance are sensitive to concentrations of 2–5 µg/mL and
to the toxic effects of a drug or other pharmacologi- stable mammalian cell lines can be generated within
cally active compounds used for selection. Such 1week
markers are used to confer an advantage tomam- •• Recessive selection markers: A number of mamma-
malian cells that have been transfected with exog- lian cells that are deficient for enzymes of key meta-
enous DNA constructs. Dominance refers to the bolic pathways are suitable for developing stable cell
fact that a single copy of the marker is sufficient lines. Thecells are transiently transfected with plas-
to confer the resistant phenotype. For example, mids carrying genes for deficient enzymes and then
G418 is an aminoglycoside antibiotic produced by selected using appropriate substrate analogues.For
Micromonosporarhodorangea that blocks polypep- example, dihydrofolate reductase (DHFR) is a ubiq-
tide synthesis by inhibiting the elongation step in uitous cytosolic enzyme that catalyses the reduc-
both prokaryotic and eukaryotic cells. Resistance tion of 5,6-dihydrofolate to 5,6,7,8-tetrahydrofolate
to G418 is conferred by the neomycin resistance (THF). THF is an essential co-factor in several meta-
gene (neo) from Tn5, encoding an aminoglyco- bolic pathways, including purine and thymidylate
side 3’-phosphotransferase (Fig. 27.11). Selection biosynthesis. Methotrexate is a folate analogue that
in mammalian cells is usually with concentrations acts as a slow, tight-binding competitive inhibitor
ranging from 0.2 to 2 mg/mL. of DHFR. It acts by inhibiting cellular THF synthesis
Puromycin is another commonly used antibiotic and, in turn, cellular proliferation. Therefore, only
for making permanent cell lines. It produced by the those cells that express the DHFR gene survive, while
bacterium Streptomyces alboniger. Aspuromycin is other cells die.
an aminonucleoside and resembles an aminoacyl- Popular hosts for stable expression are Chinese
tRNA, it can inhibit protein synthesis by disrupting hamster ovary (CHO) cells, baby hamster kidney
peptide transfer on ribosomes, causing premature (BHK-21) cells, myeloma cells and human embryonic
chain termination during translation. It is also a kidney (HEK) 293 cells.
Chapter 27.indd 16 6/23/2014 2:39:11 PM

27.6 Scale-up of animal cell culture process 17
Gene of interest
flasks (for suspension cultures) are used in scale-up of
CMV animal cell culture process:
promoter
SV40 origin •• Roller bottles: Roller bottles provide total curved sur-
f1 origin face area of the micro-carrier beads for growth. This
CMV-Neo vector system offers the following advantages over the static
Neor
Amp r monolayer culture: (i) provides an increase in the sur-
face area, (ii) provides constant gentle agitation of the
SV40 poly A medium and (iii) provides an increased ratio of sur-
Baterial origin
face area of medium to its volume, which allows better
Cell transfection gaseous exchange. Roller bottles are cylindrical ves-
sels that revolve slowly (between 5 and 60 revolutions
Cells
per hour). These tissue culture bottles can be used
in specialized CO2 incubators with attachments that
rotate the bottles along the long axis [Fig. 27.12(a)].
After each complete rotation of the bottle, the entire
cell monolayer has transiently been exposed to the
medium. The volume of medium need only be suf-
G418 selection
ficient to provide a shallow covering over the mon-
Colonies olayer. Typically, a surface area of 750–1500 cm2 with
200–500 mL medium will yield 1–2 × 108 cells.
•• Micro-carrier beads: Micro-carrier beads (~90–300
μm diameter) can be used in any application where
anchorage-dependent cells are to be cultured. These
are either dextran or glass based, and are available in
Expansion of culture
a range of densities and sizes. They are primarily used
to increase the surface area for cell growth, and thus,
the number of adherent cells in a culture vessel is very
high. For example, micro-carrier beads usually pro-
vide 0.24 m2 area for every 100 mL of a culture flask. As
the beads are buoyant, these can be easily used with
a spinner culture vessel [Figure 27.12(b)]. Further,
adherent cells can be grown to high densities with the
Gene integration and expression analysis
help of micro-carrier beads.
Figure 27.11 Development of stable cell lines by neomycin •• Spinner cultures: Spinner-type vessels are used for scal-
selection. ing up the production of suspension cells or anchor-
age-dependent cells attached to micro-carrier beads.
Spinner is a type of bioreactor having a suspended
Teflon impeller, stirrer or similar device to agitate the
medium when placed on a magnetic stirrer. The ves-
sel is usually made of glass or stainless steel, with port
27.6 Scale-up of animal cell holes to accommodate sensors, medium input and gas
culture process flow [Fig. 27.12(b)]. Commercial versions incorporate
one or more side arms for sampling and/or decanta-
Mammalian cells are cultured by a variety of methods tion. As cells are not allowed to settle to the bottom
at a range of volumes for the production of therapeutic of the flask, they can be grown to very high densities.
and diagnostic proteins. For commercial exploitation, Further, stirring of the medium improves gas exchange.
the cells are grown in a bioreactor or fermentation ves-
sels where physicochemical and biological factors are
maintained at optimum levels. The most suitable bio-
reactor used is a compact-loop bioreactor with marine 27.7 Quality control of
impellers. Here, glucose and glutamine are the main car- recombinant products
bon and energy source and the metabolic products that
affect cell growth are monitored by gas chromatography It is extremely important to do analytical and pharma-
or high-performance liquid chromatography (HPLC). cokinetic characterization of protein biopharmaceuti-
However, for batch cultures, mainly roller bottles with cals produced through rDNA technology before these
micro-carrier beads (for adherent cells) and spinner could be marketed. Specific assays have been designed
Chapter 27.indd 17 6/23/2014 2:39:12 PM

Microcarrier culture
Roller bottle
Gas vent
Inoculation
port
Teflon stirrer
Figure 27.12 Scale-up culture of animal

cells. (a) Roller incubator. (b)Spinner flask. (a) Roller Inculbator (b) Spinner flask
to establish the identity, quality, purity and quantity of •• C-terminal sequencing by MS

recombinant proteins, mAbs, vaccines, antigens, aller- •• Molecular weightdetermination of intact protein iso-
gens and biosimilars and perform pharmacokinetic and forms
toxicokinetic studies. •• Western blot analysis by specific antibodies
•• Quantitative amino acid analysis
27.7.1 Analytical procedures •• HPLC profiling
•• Quantification by LC-MS/MS with isotope dilution
Combinations of specific analytical procedures are per-
formed to ascertain different properties of bioproducts. The analytical characterization of antigens is even more
For example, the antibody characterization involves: relevant in the case of subunit or complex protein-
based vaccines. Further, it is important to do MS and
•• Heavy- and light-chain molecular weightdetermina- proteomics-based analysis for the identity, potency and
tion by mass spectrometry (MS) stability of each preparation, combined with documen-
•• Peptide mapping by MS of heavy and light chains tation of antigen concentrations and comparison of dif-
•• Sequencing of variable complementary-determining ferent batch preparations.
regions
•• N- and C-terminal sequencing of heavy and light 27.7.2 Pharmacokinetic analysis
chains bymatrix-assisted laser desorption ionization–
in-source decay(MALDI-ISD) Just for any other drug, pharmacokinetic studies are an
•• Edman N-terminal sequencing integral part of human clinical studies to evaluate the effi-
•• Electrophoretic analysis by 1D and 2D polyacryla- cacy and safety of recombinant protein biopharmaceuti-
mide gel electrophoresis(PAGE) cals. This is required to ascertain the relationship between
•• Analysis of common translational modification– administered dose, the observed biological fluid concen-
deamidation, oxidation, pyroglutamate, N-glycosylation tration of the drug and time. These methods are used
•• Quantification by liquid chromatography–mass spec- to measure the bio-product concentration in biological
trometry (LC-MS)/MS fluids (serum, plasma, urine) or target tissue collected at
•• Host cell protein identification assay different timepoints. These studies are performed in com-
pliance with good laboratory practice (GLP).
Likewise, antigen and vaccine characterization involves
establishing the identity and purity of the antigen in 27.7.3 Toxicokinetic analysis
question using the following methods:
Toxicokinetic studies are performed to understand
•• Mass spectrometric peptide mapping with high the relationship between administered drug dose
sequence coverage and toxicity. It is a regulatory requirement done in
•• N-terminal Edman sequencing accordance with OECD (Organization for Economic

Chapter 27.indd 18 6/23/2014 2:39:12 PM

27.8 Applications of animal cell technology 19
Co-operation and Development) guidelines to assess companies, followed by its marketing. This is the pri-
systemic exposure in toxicology studies, that is, in pre- mary means by which the developer of the drug can
clinical animal studies for setting human plasma con- recover the investment cost for development of the
centration limits and safety margins. Quantification biopharmaceutical. The first therapeutic recombi-
of systemic exposure is often represented by plasma, nant biopharmaceutical for human use was ‘human’
serum or blood concentration of the compound. Toxi- insulin. The recombinant human insulin (rHI), also
cokinetic studies also need to be performed in consist- known as Humulin, was developed by the US phar-
ence with GLP. maceutical giant Genentech and licensed to Eli Lilly
for manufacturing and marketing in 1982. The global
27.7.4 Purity analysis biopharmaceutical industry has come a long way since
the development of Humulin. Today, more than 300
When human proteins are expressed recombinantly in biopharmaceuticals have already been approved and
another organism, the purified protein always contains many more are in the late-stage clinical development,
trace amounts of contaminating host proteins from the including blood factors, colony-stimulating factors,
expression organism. Such process-related impurities enzymes, growth factors, growth hormones, immuno-
are identified and evaluated qualitatively and quantita- globulins, insulin, interferons, interleukins and mAbs.
tively using procedures such as1D sodium dodecyl sul- Some important milestones in the therapeutic use of
phate polyacrylamide gel electrophoresis(SDS-PAGE), recombinant protein biopharmaceuticals aregiven in
combined with silver staining of the gel, followed by Box 27.3, while those produced using animal cell cul-
protein identification using mass spectrometric peptide ture and rDNA technology are listed in Table 27.3.These
mapping. The twin procedures offer an attractive sup- drugs have not only advanced the prevention and treat-
plement to the enzyme-linked immunosorbent assay ment of a number of life-threatening diseases, but have
(ELISA)-based protein assay as these are less time- also provided the thrust for the continued success of the
consuming and do not involve development of specific pharmaceutical industry. The industry’s US$ 92 billion
antibodies. The 1D SDS-PAGE is helpful in visualizing figures and double-digit growth rate in recent years is
the individual host protein bands and allows batch- a testimony of the higher approval rate for investors.
to-batch comparison of the relative amounts. The pro- According to a global biopharmaceutical market report
tein bands are then cut out from the gel, digested with (International Market Analysis Research and Consult-
trypsin and the peptide mixture is analysed by MS. ing’s group report, 2010–2015), the sale of recombinant
Then, the Mascot database is used to establish the iden- protein pharmaceuticals is likely to touch US$ 167 bil-
tity of the individual host protein bands. This database lion by 2015, including a share of $79 billion for mAbs.
includes protein/peptide sequences from different host Next are some examples of recombinant therapeutics
systems, such asyeast, E. coli, sf9 insect cells, rice and that are currently available in the market.
CHO cells.
27.8.1 Blood coagulation factors
27.8 Applications of animal cell Haemophilia A is a common heritable genetic disor-

der where the body lacks the ability to produce factor
technology VIII, required for blood clotting, while haemophilia B or
Christmas disease is the second most common type of
Cell culture is one of the major tools used in cellular and bleeding disorder due to the deficiency of factor IX – a
molecular biology, providing excellent model systems to
study the normal physiology and biochemistry of cells
(e.g. cell cycle, metabolic studies), the effects of drugs
and toxic compounds on the cells, and mutagenesis and Box 27.3 History of therapeutic use of recombinant
carcinogenesis (e.g. apoptosis, genetoxicity). It is also biopharmaceuticals
used in drug screening and development, and large-
1982: Human insulin became the first recombinant protein to
scale manufacturing of biological compounds (e.g. be licensed.
therapeutic proteins and vaccines). The main advan- 1985: Human growth hormone wasproduced from recombi-
tage of using animal cell culture in the above applica- nant bacteria.
tions is the consistency and reproducibility of results 1986: Lymphoblastoid gamma-interferon (IFN)waslicensed.
obtained through batch culture of clonal cells. However, 1987: Tissue-type plasminogen activator (tPA) from recom-
some changes in the characteristics of these cells after a binant animal cells became commercially available.
period of continuous growth are an important concern. 1989: Recombinant erythropoietin came intoclincial trial.
When a biopharmaceutical is developed, the institu-
1990: Recombinant products came into clinical trial (HBsAg,
tion or company will apply for a patent. Then exclusive factor VIII, HIVgp120, CD4, GM-CSF, EGF, mAbs, IL-2).
or non-exclusive rights are granted to manufacturing
Chapter 27.indd 19 6/23/2014 2:39:12 PM

Table 27.3 Protein pharmaceuticals produced from animal of patients with anaemia associated with renal fail-
cell culture ure. The recombinant human EPO (r-HuEPO) is now
produced using CHO cell lines. The use of r-HuEPO
Protein Animal cell Therapeutic use is advantageous over blood transfusion as it does not
line used
require donors or blood transfusion facilities and is free
Erythropoietin (EPO) CHO cells Anaemia of risk for transfusion-associated disease.
Factor VIII CHO cells Haemophilia A
Factor IX CHO cells Haemophilia B 27.8.3 Follicle-stimulating hormone
Follicle stimulating CHO cells Infertility
hormone (FSH) CHO cells GH deficiency
Follicle-stimulating hormone (FSH) is a glycoprotein
Human growth hormone hormone produced by the anterior pituitary gland. It
CHO cells Cancer therapy
(hGH) regulates the development, growth, pubertal matura-
CHO cells Stroke
Interleukin 2 (IL2) tion and reproductive processes of the human body
Hybridoma Cancer therapy along with luteinizing hormone. Low levels of FSH may
Tissue cells and autoimmune
plasminogenactivator diseases
result in failure of gonadal functions. Recombinant FSH
(tPA) produced in CHO cell lines is used commonly in infer-
Monoclonal antibodies tility therapy to stimulate follicular development, nota-
(mAbs) bly in in vitro fertilization (IVF) therapy, as well as with
interuterine insemination.
27.8.4 Growth hormone
serine protease of the blood coagulation system. Many
patients with haemophilia are treated with factor VIII or Human growth hormone (hGH) is a peptide hormone
IX. Traditionally, factors VIII and IX were isolated from produced by the anterior pituitary gland and respon-
the plasma. However, due to the short supply of plasma, sible for regulating normal body growth by stimulating
increased blood testing and quality demands on donor’s cell division and regeneration in the body. GH defi-
blood in the light of the potential danger from HIV, ciency is linked with growth failure and short stature in
hepatitis and some unknown viruses, it increasingly children and delayed sexual maturity. hGH is used for
became difficult to produce these factors from human the treatment of dwarfism. Initially, hGH was obtained
blood. Further, some of the patients treated with con- from the pituitary glands of deceased humans, before
ventional factor VIII or IX products developed inhibitors it could be produced from genetically modified animal
against these drugs. The production of factors VIII and cells. The quantity of hormone produced by human
IX using animal cell technology will ensure sufficient pituitaries was only sufficient to treat some, but not all,
virus-free supplies. patients. In addition, this production method involved a
In principle, another coagulation factor, factor VII, risk of transferring other infectious agents. The current
could also be used for treating haemophilia patients. As hGH production method using genetically modified
factor VII is available only in minute quantities in our animal cells enables the treatment of all patients and is
body fluids, it is a non-viable proposition to isolate it a much safer product.
from human blood. However, with the help of modern
recombinantDNA technology, it has become possibleto 27.8.5 Interleukin-2
produce this factor in mammalian cells. The recombi-
nant factor VII is expected to reach the market soon for Interleukin-2 (IL-2) is a type of cytokine produced by
human use. lymphocytes as a natural immune response in the
body. It is secreted in response to microbial infections
27.8.2 Erythropoietin to increase growth and activity of other T- and B-cells
and activate maturation of the immune response. The
Erythropoietin (EPO) is a glycoprotein hormone essen- recombinant form of IL-2 is used in the case of cancers,
tial for RBC production and wound healing. It stimulates chronic viral infections and adjuvant for vaccines.
the bone marrow to produce more red cells and, thus,
increase the oxygen-carrying capacity of the blood. Its 27.8.6 Tissue plasminogen activator
requirement increases in the case of anaemia. There-
fore, EPO is useful in the treatment of anaemia caused Tissue plasminogen activator (tPA) is a thrombolytic
due to cancer, chronic renal failure and even AIDS. Ear- (clot-dissolving) agent produced by the endothelial lin-
lier, 2500 Lof urine from patients with aplastic anaemia ing of blood vessels. It is a serine protease that cataly-
had to be processed to obtain small quantities of EPO. ses the conversion of plasminogen to plasmin, which
Now, with the aid of geneticallymodified cells, it is pos- triggers the dissolving blood clots. It is quite effective
sible to produce sufficient EPO to treat many thousands in patients of pulmonary embolism and myocardial
Chapter 27.indd 20 6/23/2014 2:39:12 PM

Concluding remarks 21
infarction, provided it is given within a few hours after for treating cancer using a toxin or an enzyme pro-drug
symptoms begin. In different multicentric clinical trials, coupled to a specific antibody. The specific antibody
it has been shown that the impact of such thrombolytic binds to specific antigens on cell surface, leading to the
therapy is significant. tPA is another product that could accumulation of toxin or pro-drug in tumour cells and
not be previously made in sufficient quantities to treat their eventual death.
these patients. However, through biotechnological pro-
duction of recombinant tPA in animal cell cultures, it
has been made possible to produce sufficient quanti-
ties of tPA for clinical treatment of patients. So far, more
Concluding remarks
than 5,00,000 patients worldwide have been treated
with recombinant tPA. The benefits for the patients are Animal cell culture is a powerful tool in the hands of
higher survival rates and higher reperfusion. There are biologists. It has not only allowed us to do fundamen-
fewer side effects of tPA as compared withother throm- tal studies such asunderstanding of cell metabolism
bolytic agents or invasive methods. and cell cycle control but is also very useful in the test-
ing effects of drugs and toxins, and production of many
27.8.7 Monoclonal antibodies important diagnostics and therapeutics. The animal
cells can be cultured and scaledup to provide large
mAbs antibodies are antibodies with a single speci- quantities of the product of interest. Some of the thera-
ficity that can be produced in vitro by culturing the peutics would never have been available for clinical use
hybridoma cells or in vivo as mouse ascites tumours. because they could not be extracted from the natural
Because of their specificity, mAbs are most suited as sources in good quantities. Besides, mammalian cells
diagnostic agents for laboratory tests and were among offer special advantage of producing biopharmaceuti-
the first products of the modern biotechnology to enter cals with correct post-translational modifications, mak-
the market. At present, hundreds of such products ing them most suitable to get desired pharmacological
are available. Their application ranges from detect- effects without being immunogenic. Biopharmaceuti-
ing pregnancy to blood group typing prior to blood cal industries are currently facing the challenge of pro-
transfusion as well as detection of hormone deficien- ducing biological recombinant medical products in a
cies. The therapeutic applications of mAbs are just cost-effective manner, while meeting the safety stand-
starting, and many applications are being evaluated ards formulated by the regulatory agencies. Recent new
in the clinic. Developments with respect to therapeu- developments in the area such as better methods of
tic mAbs have already made significant advancements gene delivery into cells andbetter protocols for estab-
in the treatment of cancer, AIDS and other diseases. lishing stable cell lines combined with new tools for
Some products are already on the market, foe example, scale-up culture have given a major thrust to animal cell
mAbs for the treatment of transplant rejection and for biotechnology. The protein biopharmaceutical industry
the diagnosis of some cancers. Another example of the has been growing at an amazing pace in recent years,
therapeutic use of mAbs is the ‘magic bullet’ concept suggesting that it holds a bright future.
Web resources
http://www.atcc.org/
http://flashserver.sac.ac.uk/biology_sac/cellbiol_index.htm
http://www.scribd.com/doc/427679/Introduction-to-Cell-and-Tissue-
Culture
References
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technique. J Exp Med 1911;13:387–396. et al.Lipofection: A highly efficient, lipid-mediated DNA-transfection
Earle WR, Schilling EL, Bryant JC, Evans VJ. The growth of pure procedure. ProcNatlAcadSci USA 1987;84:7413–7417.
strain L cells in fluid-suspension cultures. J Nat Cancer Inst Goff S, Berg P. Construction of hybrid viruses containing SV40 and
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Eagle, H. Nutritional needs of mammalian cells in tissue culture. monkey cells. Cell 1976;9:695–705.
Science 1955;122:501–514.
Graham FL, van der Eb AJ.A new technique for the assay of
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Ham RG.Clonal growth of mammalian cells in a chemically defined McLimans WF, Giardinello FE, Davis EV, Kuchera CJ, Rake GW.
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Harrison RG.Observations on the living developing nerve fiber.
Proc Soc Exp Biol 1907;4:140–143. Mueller G, Graessmann A, Graessmann G. Mapping of early SV40
Harrison RG.The outgrowth of the nerve fiber as a mode of specific functions by microinjection of different early viral DNA
protoplasmic extension. J Exp Zool 1910;9:787–846. fragments. Cell 1978;15:579–585.
Hayflick L, Moorhead PS.The serial cultivation of human diploid cell Rous P, Jones FS. A method for obtaining suspensions of living
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cells. J Exp Med 1916;23:549–555.
Ian Freshney R.Culture of Animal Cells: A Manual of Basic
Technique and Specialized Applications. Hoboken, NJ: Wiley- Shamblott MJ, Axelman J, Wang S, Bugg EM, Littlefield JW,
Blackwell; 2010 Donovan PF, et al. Derivation of pluripotent stem cells from
cultured human primordial germ cells. Proc Natl Acad Sci USA
Lewis MR, Lewis WH.The cultivation of tissues from chick embryos 1998;95:13726–13731.
in solutions of NaCl, CaCl2, KCl and NaHCO3.Anat Rec 1911;5:
277–293. Thomson JA, Iskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ,
Marshall VS, et al. Embryonic stem cell lines derived from human
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Animal cells as host

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of the production processes. Biopharmaceutical indus-

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Box 27.2 Importance of animal cell culture

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(a) Viral transformation of cells

c-MYC HBx+c-Myc Control siRNA Specific siRNA

(b) Colony formation assay (c) Xenograft model

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27.4.3.2 Suspension culture

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27.4.6 Typical experimental protocol

A typical experimental protocol for cell culture is as

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(a) Horizontal Laminar Hood (c) Horizontal Laminar Flow

Hepa Recirculating fan

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Figure 27.7 Cell culture plas-

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27.5.1 Transient versus stable 27.5.2.1 Electroporation

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Cell suspension Ca++

(a) Electroporation (b) Calcium phosphate method

DNA Mammalian cell

27.5.2.3 Liposome-mediated transfection 27.5.3 Methods of stable transfection

Chapter 27.indd 14 6/23/2014 2:39:11 PM

Cell holding pipet Egg pronucleus

DNA injecting needle

e.g., microinjection of egg cell

Cell holding pipet

Figure 27.9 Method of microinjection. Micromanipulator and microinjectionof egg cell.

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Therapeutic gene Transfection into

Cotransfection in 293 cells (E1 in trans) Cell with helper provirus

gag pol env

Recombinant Ad5 genome AAA

(a) Adenoviral Vector (b) Retroviral Vector

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Figure 27.12 Scale-up culture of animal

to establish the identity, quality, purity and quantity of •• C-terminal sequencing by MS

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27.8 Applications of animal cell Haemophilia A is a common heritable genetic disor-

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27.4.6 Typical experimental protocol

27.5.1 Transient versus stable 27.5.2.1 Electroporation

27.5.2.3 Liposome-mediated transfection 27.5.3 Methods of stable transfection

27.8 Applications of animal cell Haemophilia A is a common heritable genetic disor-