Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy

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The need to deal with some of these differences demands enormous

efforts for the development of culture media (chemical environment) or

shear, mixing, viscosity and bubbling conditions (physical environment),

which should be optimized to result in an industrial process that can be

validated. Requirements to avoid contamination have led to the formula-

tion of serum-free media or even of protein-free, chemically deﬁned media

for the production of biopharmaceuticals (Grifﬁths, 1988).

The range of culture ﬂasks and reactor types employed is quite wide,

both for suspension and adherent cultures, from small Carrel’s or Roux’s

ﬂasks to roller bottles. Fixed- and ﬂuidized-bed bioreactors, air-lift

reactors and even stirred and aerated tanks with capacities up to 15 m

are

common in large plants producing monoclonal antibodies (mAbs) for

anticancer therapies (Adams and Weiner, 2005; Grifﬁths, 1988).

One of the main purposes of animal cell culture development was the

search for viral vaccines, initiated during the Second World War (1939–

1945), particularly for poliomyelitis. The names of Enders, Syverton, and

Salk are undoubtedly associated with the production of the inactivated

polio vaccine, approved in the USA in 1955 and produced on a large scale

in primary monkey kidney cells. Later, after a dispute between Hilary

Kaprowski and Albert Sabin, the attenuated vaccine against poliomyelitis

was licensed in 1962. At the end of this period, at the Wistar Institute,

Hayﬂick developed a cell line from embryonic tissue capable of replicating

more than 50-fold before becoming senescent (Hayﬂick and Moorhead,

1961). The cell was diploid, easy to freeze and to reactivate and did not

show any evidence of contamination by the viruses normally found in

monkey primary kidney cells. This cell line (WI-38) turned into the basis

for the production of human viral vaccines against poliomyelitis and

MMR (measles, mumps, rubella), while other cell lines were evaluated for

the production of veterinary vaccines, such as BHK (baby hamster kidney)

in the case of the vaccine against foot-and-mouth disease.

After this period, there was an accelerated use of animal cells. Namalwa

cells (Finter et al., 1991) were used to produce alpha-interferon by Well-

come in 1986. Vero cells (a cell line derived from monkey) were used for

anti-rabies vaccine. Hybridomas were considered acceptable for mAb

production and the use of genetically modiﬁed CHO (Chinese hamster

ovary) cells was authorized for the production of tissue plasminogen

activator (tPA) by Genentech in 1986.

Finally, three relevant aspects should be mentioned to clarify the

scientiﬁc, technological, and industrial position of biopharmaceuticals and

animal cells.

(i) Complex biopharmaceuticals, such as proteins, virus or virus-like

particles (VLPs), among others, produced by cellular and/or recombi-

nant technologies are characterized/deﬁned by their own production

processes. This means that analytical, biological and immunological

characterization assays are usually not considered sufﬁcient for pro-

duct marketing, given the complexity of the molecules. Therefore,

product licensing is based on the speciﬁc production process, which

cannot be altered. Process changes may require new licensing proce-

2 Animal Cell Technology

dures, and this makes the introduction of biosimilars in the market

more difﬁcult.

(ii) Some of these proteins (for therapy) or VLPs (for vaccines such as

hepatitis B) can be produced by yeast or even by Escherichia coli

because of the limited requirement for post-translational modiﬁcation

(see Chapter 6). The total market value of biopharmaceuticals pro-

duced by E. coli or yeast was surpassed by those produced by animal

cells only around 1996. In the last few years, market dominance in

favor of animal cells has increased signiﬁcantly.

(iii) Considering the complexity and instability of biopharmaceuticals, the

production process from animal cells has to be designed, modeled,

and optimized in an integrated form, taking into account the culture,

extraction, and separation (Cruz et al., 2002). This may be different

from what is normal in the production of simple biological com-

pounds such as antibiotics or vitamins.

The number of biological processes has increased tremendously in the

last 15 years. This has resulted in high expectation for an improvement of

quality of life and an increase in the volume of business related to products

obtained from animal cell culture technology, with the broad potential use

of these products for disease diagnostics, prevention, therapy, and cure.

Table 1.1 indicates some examples of approved therapeutic products

obtained through animal cell culture.

1.2 Types of animal cell cultures

The methods developed for obtaining and maintaining primary cultures

paved the way for animal cell technology. However, the huge growth and

expansion of this technology was possible only because new cell types

were established, namely diploid cells, hybridomas, and other continuous

cell lines. Animal cells in culture can be classiﬁed, according to their origin

Table 1.1 Examples of approved products obtained through animal cell culture

Product Protein Use Cell Approval year

Avonex

-Interferon Multiple sclerosis CHO 1996

BeneFix

Factor IX Hemophilia B CHO 1997

Epogen

Erythropoietin Anemia CHO 1989

Gonal-f

Follicle-stimulating

hormone

Female infertility CHO 1995

Herceptin

trastuzumab

mAb Breast cancer CHO 1998

Kogenate

Factor VIII Hemophilia A BHK 1993

Simulect

basiliximab

mAb Acute transplanted

kidney rejection

Murine myeloma 1998

Campath

alemtuzumab

Humanized mAb Leukemia CHO 2001

Xolair

omalizumab

Humanized mAb Asthma CHO 2003

Avastin

bevacizumab

Humanized mAb Colon or rectum

carcinoma

CHO 2004

Introduction to animal cell technology 3

and biology, into primary cultures and cell lines (nomenclature most

frequently employed in textbooks). However, depending on their applica-

tions, animal cells can also be grouped as follows.

(i) Cells producing proteins employed in the production of complex

therapeutics, subunit vaccines, and diagnostic products, such as

CHO, BHK, HEK-293, WI-38, MRC-5, SP2/0, NS0, and insect cells.

(ii) Cells producing viruses used in gene therapy and viral gene vaccines

(for instance, Vero, HEK-293, and PER.C6

cells).

(iii) Normal cells, tumor cells, and stem cells used in research and

development, speciﬁcally in the discovery of new products and for in

vitro study and toxicology models (e.g. nerve cells, ﬁbroblasts, Caco-

2, MRC-5, and endothelial cells).

(iv) Human cells for subsequent use in cell therapy and regenerative

medicine (e.g. embryonic and adult stem cells).

Primary cells are isolated directly from organs or tissues. Primary cells

are normally heterogeneous and better represent the tissue from which

they originate. These cells have a ﬁnite growth capacity and can be

subcultured for only a limited number of passages. Subcultured cells,

which have been selected to form a population of cells of a single type, are

designated cell lines, and can be ﬁnite or continuous. Finite cell lines (cells

capable of a limited number of generations before proliferation ceases) as

well as continuous cell lines can be propagated and expanded for the

production of well characterized cell banks, where they are preserved by

employing cryopreservation techniques (Doyle et al., 1994).

A normal tissue usually provides ﬁnite cultures, while cultures obtained

from tumors can result in continuous cell lines (immortal). Nevertheless,

there are many examples of continuous cell lines that are obtained from

normal tissues and are not tumorigenic, such as BHK 21 (baby hamster

kidney ﬁbroblasts), MDCK (Madin-Darby canine kidney epithelial cells),

and 3T3 ﬁbroblasts (Freshney, 1994, 2000). Immortal cell lines can occur

spontaneously (rarely) or after a transformation process (more often),

which can be induced by carcinogenic chemical agents, by viral infection,

or by the introduction in the cell genome of a viral gene or an oncogene

capable of overcoming senescence. Several of the differences between

normal and neoplastic or tumor cells are analogous to the differences

between ﬁnite and continuous cell lines, since immortalization is an

important component of the cell transformation process.

The main advantages of continuous cell lines are: (i) faster cell growth,

achieving high cell densities in culture, particularly in bioreactors; (ii) the

possible use of deﬁned culture media available in the market, mainly

serum-free and protein-free media; and (iii) the potential to be cultured in

suspension, in large-scale bioreactors.

The major disadvantages of these cultures are the accentuated chromo-

somal instability, the larger phenotype variation in relation to the donor

tissue, and the disappearance of speciﬁc and characteristic tissue markers

(Freshney, 1994).

Many examples of immortalization methodologies and techniques to

obtain continuous cell lines are described in the literature (Land et al., 1983;

4 Animal Cell Technology

MacDonald, 1990; Ruley, 1983), including those involving transfection or

infection with viral genes (for instance, the E6 and E7 genes of human

papilloma virus, and the SV40T simian virus 40 large T-antigen gene) or

virus (such as Epstein–Barr virus and retroviruses). Another strategy is to

create hybrid cells resulting from the fusion of a cell with a limited lifespan

with a continuous cell. This is the strategy used to obtain hydridomas

for antibody production, as discussed below (see also Chapter 17).

The hybridomas were, to a large extent, responsible for the biotechnol-

ogy ‘‘explosion’’ towards the end of the 1970s, opening perspectives for

remarkable advances in both the immunotherapy and diagnostic areas. In

1975, Ko

hler and Milstein demonstrated that, despite the impossibility of

cultivating differentiated B lymphocytes in vitro, it was possible after their

fusion with immortal myeloma cells. The hybrid cell lines (hybridomas)

can grow continuously and produce and secrete immunoglobulins. Since

all the immunoglobulin produced derives from a single type of cell, the

antibody is monoclonal and is directed against only one epitope. Although

the initial studies performed by Ko

hler and Milstein were restricted to the

production of mouse mAbs, soon thereafter the production of antibodies

from other species, including human, became possible.

As mentioned before, another relevant key development for the progress

of animal cell culture technology was the WI-38 human diploid cell line

obtained by Hayﬂick and Moorhead in 1961, since previously the options

were the use of primary cultures or of heteroploid cell lines (derived from

tumors or from cells that acquired tumor-like characteristics in culture).

Because of that, heteroploid cells were not acceptable for the production

of compounds for human applications, and therefore primary cultures

from other species were employed (such as primary monkey kidney cells).

After the development of diploid cell lines, i.e. with a diploid karyotype,

a new concept of cell line emerged, since these cells are considered

‘normal’ cells. They undergo senescence and die in culture after a ﬁnite

number of generations (around 50 generations for WI-38 cells). The

disadvantages of these cells in culture are that they grow slowly, not

reaching high cell densities, present relatively low productivity, are highly

dependent on support adhesion for growth, and consequently are not

easily cultured in suspension. Nowadays, the diploid cell line most

frequently employed is MRC-5, and its use is particularly important for

the study of cell aging mechanisms.

The animal cell lines mentioned above are more extensively discussed in

Chapters 2, 17, 18, 20, and 21.

1.3 Use of animal cells in commercial production

1.3.1. Animal cell proteins in human diagnosis and therapy

mAbs are currently the most important class of pharmaceutical proteins in

terms of market volume. Given their enormous biological speciﬁcity it is

not surprising that their ﬁrst clinical applications were the so-called

immunoassays, such as the ELISA-type assays, for in vitro diagnosis. After

1980, however, mAbs also started to be used in association with radio-

Introduction to animal cell technology 5

active markers, in imaging methods, such as immunoscintillography. In

addition, with higher doses of the radioactive agent used for tumor

detection, it became possible to treat cancer. This is the ‘magic bullet’

concept initially proposed by Paul Ehrlich at the end of the 19th century.

In this application, the antibody directs the radioactive product only to

cancer cells, which express large amounts of surface tumor antigens.

Instead of radioactive compounds, lymphokines or toxins can also be

associated to the antibodies to cause the death of tumor cells.

The ﬁrst therapeutic antibody approved (Orthoclone

OKT-3

Muromonab CD3, 1986) was indicated not for cancer treatment, but for

controlling acute rejection of transplanted organs (kidney, heart, and

liver). Nowadays, other clinical indications such as asthma, rheumatoid

arthritis, psoriasis, and Crohn’s disease are treated with mAbs (see

Chapter 17) (Antibody Engineering and Manufacture, 2005; Monoclonal

Antibodies and Therapies, 2004; Hot Drugs, 2004; Walsh, 2004).

Many recombinant proteins that are not antibodies are also on the

market for distinct applications (see Chapter 16). Examples are factor VIII

for hemophilia A treatment (Bayer, 1993, produced from BHK cells),

erythropoietin as an anti-anemic agent (Amgen, 1989, produced from

CHO cells) and -interferon for the treatment of multiple sclerosis

(Biogen and Serono, 1996, produced from CHO cells).

Nevertheless, in the last 20 years, the relevance of clinical diagnosis has

increased signiﬁcantly, forming the basis for the pharmaceutical strategy

known as pharmacogenomics, which could in the future enable a complete

customization/individualization of pharmaceuticals. A direct connection

between diagnosis and treatment could make it possible to introduce

individualized products that were eliminated for general use due to side

effects that were severe, but occurred only in a limited number of patients.

This would be acceptable provided that unequivocal diagnostics can be

developed to identify these patients. This process has already been adopted

sometime ago, for instance, to guarantee that patients who are allergic to

an antibiotic, such as penicillin, are not treated with it. However, indivi-

dualized medical treatment may become more and more frequent to insure

that expensive biopharmaceuticals are administered only to patients who

can beneﬁt from them and avoiding excessive health expenses. One

example is the biopharmaceutical ‘trastuzumab’ (Herceptin

, Genentech),

used for the treatment of a very aggressive breast tumor type that over-

expresses the epidermal growth factor receptor type 2 (HER2). This

situation occurs in only about 25% of breast cancer cases, so only these

patients are treated with trastuzumab.

The value of therapeutic mAbs marketed in 2004 was above US$ 13

billion. At the same time, the market value for the many presentation

forms of erythropoietin exceeded US$ 8 billion, and the global biopharma-

ceutical market surpassed US$ 50 billion.

It is worth emphasizing that all biopharmaceuticals mentioned here are

produced from mammalian cell culture. The protein production system

based on insect cells known as BEVS (baculovirus expression vector

system) is widely employed for the expression of a wide range of proteins,

but, due to regulatory issues, biopharmaceuticals produced by insect cells

are not yet in the market. However, some of them are being evaluated,

6 Animal Cell Technology

particularly where supramolecular structures designated VLPs are pro-

duced (see Chapter 18).

1.3.2 Cell therapy

In the last two decades, the knowledge about stem cells and, speciﬁcally,

their expansion and differentiation capabilities has grown signiﬁcantly.

These properties (expansion and differentiation) make stem cells unique

tools for the treatment of a wide range of diseases, for which traditional

therapies have failed or do not exist. Some of the current applications of

stem cells are listed in Table 1.2.

However, to use these cells in clinical protocols, it is necessary to

understand the nature and properties of stem cells originating from differ-

ent tissues, as well as the mechanisms that make them differentiate into

mature, functional cells (Mayhall et al., 2004).

The use of hematopoietic stem cells in bone marrow transplants has

paved the way for other therapies, including transplants of skin, pancrea-

tic, and brain cells (Bonner-Weir and Weir, 2005; Laﬂamme and Murry,

2005). The fact that stem cells have been identiﬁed both in systems with

high (e.g. blood and skin) and reduced (e.g. brain) regeneration capacity

suggests that speciﬁc stem cells could also be found in many other organs

and tissues. The isolation, expansion, and differentiation of these cells,

which could in the future make the treatment of degenerative monocellular

disorders (e.g. Parkinson’s disease) possible, provides a great challenge for

the ﬁeld of animal cell culture.

As an alternative to adult stem cells, embryonic stem cells can be used.

These are totipotent and can be obtained from the internal blastocyst cell

mass. Because of the capacity of these cells to generate any type of

functional cell, their manipulation and differentiation have gained in

signiﬁcance. In spite of recent advances (Daley, 2003; Hwang et al., 2004),

knowledge on the control of their differentiation and proliferation is still

lacking, but will be necessary to make the exploitation of all their

therapeutic potential turn into reality. Further discussion on cell therapy

can be found in Chapter 20.

Table 1.2 Examples of current applications of stem cells

Source Cell type Applications

Bone marrow Hematopoietic Cancer

Immunodeﬁciencies

Metabolic diseases

Hemoglobinopathies

Myocardial infarction

Neuronal embryonic tissue Neuronal Parkinson’s disease

Skin Epithelial Burns

Ulcers

Genetic skin diseases

Pancreas Pancreatic Diabetes

Introduction to animal cell technology 7

1.3.3 Tissue engineering

Tissue engineering is a recent discipline that is closely related to cell

therapy, but combines knowledge of molecular and cell biology with

traditional concepts from biomaterials engineering, bioreactors, biomecha-

nics, and controlled drug release, aiming at the development of new

tissues.

The success of tissue engineering depends on the combination of differ-

ent factors (Heath, 2000). The ﬁrst one is to have cells that are capable of

regenerating and, if necessary, an adequate supporting matrix. Recent

developments indicate that it may be possible that the cells themselves

manage to build the tissue structure, without the need for an external

matrix. Another requirement is an environment that is appropriate for cell

growth, differentiation, and eventual integration into the surrounding

tissue. Since cells are a critical factor, the source from which they originate

deserves special attention. Obtaining a sufﬁcient amount of the appropri-

ate cells is of extreme relevance for the regeneration of damaged tissues,

particularly in cases when the tissue does not have the ability to self-

regenerate or when the native regeneration mechanisms are not sufﬁcient.

There are different cell sources for use in tissue engineering, each of

them presenting advantages and disadvantages. The best source, in princi-

ple, is the patient. The autologous cells so obtained are expanded in vitro

for a later transplant, with no risk of immune reaction. However, the

amount of cells obtained from the patient is frequently very limited,

especially in cases when cell collection may cause death or in the case of

elderly patients. An alternative is the use of stem cells, particularly those

that already present some degree of differentiation, but still retain some

pluripotency. Although rare, such cells can be propagated and can be used

later for reconstituting and repairing the damaged tissue.

The ﬁrst tissue engineering product to be approved targeted the treat-

ment of burns and consisted of keratinocytes cultivated in vitro, which

form a tissue that is later transplanted to the patient. Beyond this product,

others are already being commercialized for a wide range of applications.

Examples are Carticel

(marketed by Genzyme), which consists of chon-

drocytes employed in the treatment of cartilaginous defects caused by

acute or repetitive traumas; Apligraf

(Novartis), used in the treatment of

venous ulcer; and DACS

SC (Dendreon), utilized for reconstitution of

the immune system after chemotherapy.

In terms of the in vitro cell culture techniques required to obtain tissues,

the greatest challenges are somehow similar to those of cell therapy,

particularly when cells that are not completely differentiated are used. The

challenges include the isolation of progenitor cells from the tissue of

interest, the expansion of pure cultures, and the understanding of the

signals necessary to direct differentiation of a speciﬁc cell type, in order to

accelerate the process of repairing the tissue. Also in tissue engineering the

use of embryonic stem cells is possible, but the control of differentiation is

equally an area where a deeper knowledge is still required. It is necessary

to assure that the cells introduced into the patient are completely differ-

entiated, since even a small percentage of undifferentiated cells in a tissue

can evolve to produce teratomas.

8 Animal Cell Technology

1.3.4 Gene therapy and DNA vaccines

The introduction and expression of recombinant genes in somatic cells, or

gene therapy, is also an important application of animal cell culture. The

cells employed in this case may be used at two different levels: in the

production of vectors that will be later utilized ex vivo or in vivo or as

already transformed cells, which are used directly in vivo in the therapy of

diseases.

The transfer of genes has been demonstrated both for viral vectors

(such as retrovirus, adenovirus, and lentivirus) and nonviral vectors

(plasmid DNA and liposomes). Animal cells are used mainly in the

production of viral vectors, which present a much larger integration

efﬁcacy than non-viral vectors. In this case, the situation is quite similar

to the production of proteins. The cells can produce a virus constitu-

tively (retrovirus and lentivirus) (Merten et al., 2001) or serve as a host

for their production (adenovirus and adeno-associated virus) (Ferreira et

al., 2005). The production of viral vectors for gene therapy usually

requires signiﬁcant genetic modiﬁcations of the cells, since these vectors

must not be self-replicating, so as not to infect the patient upon therapy.

Thus, the producing cells have the genetic information coding for all

elements necessary for the construction of the virus, but the vector just

contains the information necessary for the transfer of the therapeutic

gene to the target cell. The vectors produced can later be used in vivo,

for instance in the treatment of tumors (Shah et al., 2003) or ex vivo for

the transformation of cells that will later be introduced into the patient

(Aiuti et al., 2003).

From a prophylactic perspective, the same viral vectors can be utilized

to transfer a gene coding for an immunogenic protein of a given patho-

genic agent (viral or bacterial). This is the case of the so-called DNA

vaccines, where an immunogenic protein begins to be produced by the

host organism and, thus, a situation similar to the case of an infection

occurs (Whalen, 1996). This type of vaccine is discussed more extensively

in Chapter 18.

For gene therapy, hematopoietic stem cells are the most common targets

due to their capacity for expansion and differentiation. The current

applications focus essentially on diseases caused by a localized genetic

deﬁciency. Excellent candidates for gene therapy are those targeting severe

combined immunodeﬁciency (SCID) (Cavazzana-Calvo et al., 2000) and

adenosine deaminase deﬁciency (ADA) (Aiuti et al., 2003), since both

affect blood cells and are usually treated through bone marrow transplants

(Noguchi et al., 1993). In these cases, the progenitor cells transformed

with the respective functional genes complement the immune system of

patients. See Chapter 21 for a more extensive discussion on gene therapy.

1.3.5 Applications of animal cells in the development of new

products

The majority of the products currently in use are formed by chemical

agents that can be potentially dangerous for human health. About 80% of

cancer cases are attributed to chemical products found in the environment

Introduction to animal cell technology 9

(industrial chemicals, cosmetics, food additives, drugs, dyes, etc.). Every

year thousands of new products are developed, which need to be ade-

quately tested for toxicity. In February 2001, the European Commission

adopted a ‘White Book’ deﬁning a European strategy for chemical

products. One of the aims refers to tests that do not use animals.

Nowadays, the ﬁeld of toxicology uses different in vivo models (mouse,

rat, rabbit, among others) to evaluate the toxic potential of chemical

compounds. These classical tests using animals present signiﬁcant limit-

ations regarding the applicability of the results to the case of humans, and

pose ethical problems due to the use of animals. The application of the

current European law implies the sacriﬁce of 12 million animals, corre-

sponding to A 20 billion by 2012.

The development of tests that use cells cultured in vitro instead of

animals could thus represent an important alternative in this area. Further-

more, the use of cell lines and primary tumor cells allows the evaluation of

the potential of anti-tumor compounds with no need for inducing tumors

in animals. The current protocols are well established for a variety of

tumors, for instance in the National Cancer Institute of the USA.

Conﬁdence in in vitro tests represents one of the greatest challenges. In

this type of test, the use of human cells in culture is especially promising

for the evaluation of acute and chronic toxicity. Current tests are based on

the growth-inhibiting potential of the chemical compound, as brieﬂy

presented in Chapter 2. The current methods include the determination of

proteins or ATP, or the incorporation of an indicator (neutral red), and

these analyses can be combined with an evaluation of cell morphology.

More recently, efforts have been made to use speciﬁc cells and incorporate

gene expression analysis in order to have a more precise determination of

sublethal toxicity to speciﬁc organs. At the same time, the development of

high-throughput systems has contributed to even greater possibilities for

this type of assay.

The sequencing of the human genome has allowed an increase in the

number of therapeutic targets from around 500 to about 1000, paving the

way for a qualitative improvement not only in the development of new

candidate products, but also in the efﬁcacy of the therapeutic product.

Recent advances in areas such as genomics and proteomics allow the

association of the genetic information to new proteins. Thus, the under-

standing of disease mechanisms and their relation to the genes involved

could be used to predict the possibility of acquiring diseases and also to

offer possible therapies. In this respect, it is expected that new technologies

and strategies involving animal cells will be developed, allowing the

determination of the security and efﬁcacy of new products with greater

accuracy. These will include tests for the bioavailability of products, their

eventual genomic impact and the regulation of target genes, among others,

with applications to research and the development of new pharmaceuti-

cals, nutraceuticals, or cosmetic products.

1.4 Conclusions

A signiﬁcant number of industrial cell lines (CHO, BHK, NS2/0) are

nowadays well characterized, forming the basis for mature technologies

10 Animal Cell Technology

for the production of recombinant proteins, for both therapeutic and

diagnostic purposes.

Other uses for obtaining more complex products, such as viruses for

vaccines, are also well established. However, the use of animal cell

technology for applications such as viral vectors for gene therapy, anti-

cancer vaccines, cell therapy, or regenerative medicine (organ and tissue

engineering), is in a much more initial phase, since more scientiﬁc work is

needed to provide improved technologies and respond to complex regula-

tory and ethical questions.

Finally, the most recent ‘stars’ of this area, the stem cells, are still in

their infancy, due to the greater biological complexity that they represent.

Their potential uses are so promising, however, that this will assure a great

and continuous effort to determine the controlled conditions needed for

expansion, complete differentiation, and storage, probably in hospital

environments.

If we add to all these applications and challenges the dominant position

of animal cell technology in the discovery and development phases of new

pharmaceuticals, it is easy to predict two or three decades of continued

expansion in this ﬁeld of knowledge and technology.

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Introduction to animal cell technology 11