This presentation will help you to understand the basics of Animal cell culture along with its applicability in the diagnosis and treatment of cancer, and autoimmune diseases.

1. Introduction to Animal Cell
Technology
Shubham A. Chinchulkar (Regulatory Affairs)
M.Tech (Pharm.)
National Institute of Pharmaceutical education and
Research (NIPER)
shubhamchinchulkar007@gmail.com

2.  Animal cell culture is nowadays used successfully for the production of vaccines and recombinant glycoproteins
 New fields such as organ transplants, cell and gene therapy, in vitro toxicology and physiology, tissue analogs,
production of biopesticides, bioelectromechanical devices, and nanobiotechnology
 More than 500 biopharmaceutical products that are being evaluated in clinical trials, about half are produced by animal cell
culture
 Annual market of more than ten thousand million dollars with impressive growth rates
 First therapeutic product generated by animal cells, tPA (tissue plasminogen activator) was approved in 1986
 1987, the first therapeutic monoclonal antibody, OKT3 (Muromonab-CD3-immunosuppressant)
 50 products produced by in vitro animal cell culture have now been approved out of which approximately 60% are
monoclonal antibodies
 Since 1980s product concentrations have increased to attain more than 5 g/L of product
 Cell metabolism and physiology will help to understand better definition of culture media and eliminate undefined and
undesirable components, such as fetal bovine serum, chemically defined media became widely used
INTRODUCTION
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3.  Improvement in analytical methods and understanding of protein post translational modifications helps to understand
the relation between the bioprocess and the quality of the glycoproteins produced
 Because of all this development, practical, efficient, and robust culture of animal cells in bioreactors for consistently
producing safe and effective biopharmaceuticals
 The important aspects are :
A. Cloning and expression of heterologous proteins, culture media, metabolism, cell growth and death
B. Design, monitoring and control of bioreactors
C. Processes for separation of cells and products
D. Quality control of products, regulatory issues and intellectual property
 The genomics and proteomics will lead to obtained biopharmaceutical products having good efficacy and safety
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4. Differences between cells in an organism
(in vivo) and cells in culture (in vitro)
Tissue (In vivo)
• Tissues are three-dimensional
• Cells are subject to tension and compression
• Lymphokines and chemokines vary in
proportion and concentration
• The mechanisms for cellular differentiation
control in tissues
Cell culture (In Vitro)
• Cell cultures are of zero dimension
(monodispersed in suspension culture
• Not when in culture, with the exception of
artificial organs)
• This parameters normally do not vary
• In culture are distinct
Landmarks in the culture of animal cells
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5. To avoid contamination - Serum-free media or even of protein-free and chemically defined media for the production of
biopharmaceuticals
The range of culture flasks and reactor types employed is quite wide for both suspension and adherent cultures
Culture Flasks
Carrel’s Flasks Roux’s Flasks Roller bottles
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6. Bioreactor
Fixed Bed Bioreactors Fluidized-bed bioreactors Air-lift reactors
Packed
bed
reactors
Trickle
bed
reactors
It is defined by
countercurrent
phase flow
The liquid
phase is
sprayed from
the top while
the gas phase is
fed from the
bottom
It is defined by
concurrent
downward
phase flow
The liquid and
gas phases are
fed from the
top
Flooded
bed
reactors
The flow
of the gas
and liquid
phases
occurs
upward
In this type of reactor, a fluid (gas or
liquid) is passed through a solid
granular material (usually a catalyst
possibly shaped as tiny spheres) at high
enough velocities to suspend the solid
and cause it to behave as though it were
a fluid
An airlift reactor is one type
of bubble reactor with an
internal draft-tube that
promotes gas–liquid mass
transfer and mixing
Airlift reactors have been
largely used for algae culture
Bubbles are sparged into the
air-riser - liquid to flow
vertically - To counteract
these upward forces, liquid
will flow downward in the
down-come - better mixing
efficiencies 6

7. Types of animal cell cultures
Animal cells in culture can be classified, according to their origin and biology, into primary cultures and cell lines
Cells producing proteins
employed in the production
of complex therapeutics,
subunit vaccines, and
diagnostic products
CHO, BHK, HEK-293, WI-
38, MRC-5, SP2/0, NS0, and
insect cells
Cells producing viruses
used in gene therapy and
viral gene vaccines
Vero, HEK-293, and
PER.C61 cells
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
Nerve cells, fibroblasts, Caco-
2, MRC-5, and endothelial cells
Human cells for subsequent use
in cell therapy and regenerative
medicine
Embryonic and adult stem cells
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8.  Primary cells are isolated directly from organs or tissues
 These cells have a finite growth capacity and can be subcultured for only a limited number of passages
 Continuous cell lines that are obtained from normal tissues and are not tumorigenic, such as BHK 21, MDCK
Advantages of continuous cell lines are:
 Faster cell growth, achieving high cell densities in culture, particularly in bioreactor
 The use of serum-free and protein-free media
 The potential to be cultured in suspension, in large-scale bioreactors
Disadvantages:
 Accentuated chromosomal instability
 The larger phenotype variation in relation to the donor tissue
 The disappearance of specific and characteristic tissue markers
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9. Immortalization methodologies and techniques obtain continuous cell lines
Involving transfection or infection with viral genes Create hybrid cells resulting from the fusion of a cell with a limited
lifespan with a continuous cell
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)
Strategy used to obtain hydridomas for antibody production
 The hybrid cell lines (hybridomas) can grow continuously and produce and secrete immunoglobulins
 Monoclonal Antibodies - Derives from a single type of cell & directed against only one epitope (antigen determinant)
 Diploid cell lines 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)
Disadvantages –
 Cells in culture are that they grow slowly
 Not reaching high cell densities
 Present relatively low productivity
 Highly dependent on support adhesion for growth, and consequently are not easily cultured in suspension 9

10. Use of animal cells in commercial production
1. Animal cell proteins in human diagnosis and therapy
 mAbs are currently the most important class of pharmaceutical proteins in terms of market volume
 First clinical applications were the so-called immunoassays, such as the ELISA-type assays, for in vitro diagnosis
 mAbs also started to be used in association with radio-active markers in immunoscintillography
 Higher doses of the radioactive agent used for tumor detection, it became possible to treat cancer
 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
 Immunosuppressant, Asthma, Rheumatoid arthritis, psoriasis, and Crohn’s disease are treated with mAbs
 Many recombinant proteins that are not antibodies - factor VIII for hemophilia A treatment, erythropoietin as an anti-
anemic agent, and β-interferon for the treatment of multiple sclerosis
 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 10

11. 2. Cell therapy
 Stem cells and, specifically, their expansion and differentiation capabilities has grown significantly
 Necessary to understand the nature and properties of stem cells originating from different tissues, as well as the
mechanisms that make them differentiate into mature, functional cells
 Bone marrow transplants, transplants of skin, pancreatic, and brain cells tem cells have been identified both in systems –
High regeneration capacity (bleed and skin) & reduced regeneration capacity (Brain)
 Alternative to adult stem cells, embryonic stem cells can be use
 These are totipotent and can be obtained from the internal blastocyst cell mass
 Capacity of these cells to generate any type of functional cell, their manipulation and differentiation have gained in
significance
 Knowledge on the control of their differentiation and proliferation is still lacking
Source Cell type Applications
Bone marrow Hematopoietic Cancer, Immunodeficiency's, Metabolic diseases Hemoglobinopathies,
Myocardial infarction
Neuronal embryonic tissue Neuronal Parkinson’s disease
Skin Epithelial Burns, Ulcers, Genetic skin diseases
Pancreas Pancreatic Diabetes
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12. 3. Tissue engineering (Development of new tissues)
Different
factors
responsible
for success of
Tissue
engineering
Manage to
build the tissue
structure,
without the
need for an
external matrix
Source of cell
Appropriate
Environment
for cell growth,
differentiation,
eventual
integration into
surrounding
tissues
Sufficient
amount of the
appropriate
cells
Cells that are
capable of
regenerating
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13.  The best source for cell , in principle, is the patient
 The cells obtained from patients if frequently very limited, when cell collection may cause death or in case of elderly
patient
 The first tissue engineering product – treatment of burns, consisted of keratinocytes cultivated in vitro - form a tissue
that is later transplanted to the patient
 Eg. Carticel - chondrocytes - treatment of cartilaginous defects caused by acute or repetitive traumas
Apligraf - treatment of venous ulcer
Challenges –
 Isolation of progenitor cells from the tissue of interest
 Expansion of pure cultures
 Understanding of the signals necessary to direct differentiation of a specific cell type, in order to accelerate the process
of repairing the tissue
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14. 4. Gene therapy and DNA vaccines
 The transfer of genes 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
 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
 Producing cells have the genetic information coding for all elements necessary for the construction of the virus
 Vector just contains the information necessary for the transfer of the therapeutic gene to the target cell
5. Applications of animal cells in the development of new products
 Sequencing of the human genome has allowed an increase in the number of therapeutic targets from around 500 to about
1000
 Genomics and proteomics allow the association of the genetic information to new proteins
 Understanding 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 14

15.  Understanding 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
 Animal cells will be developed - determination of the security and efficacy of new products with greater accuracy
 Bioavailability of products, eventual genomic impact, the regulation of target genes, and development of new
pharmaceuticals, nutraceuticals, or cosmetic products
Conclusions
 Significant number of industrial cell lines (CHO, BHK, NS2/0) are nowadays well characterized and plays an important
role in the production of recombinant protein for diagnosis and therapy
 The use of Animal cell technology for applications such as viral vectors for gene therapy, anticancer vaccines, cell
therapy, or regenerative medicine is in initial phase
 Stem cells stars of this area represent greater biological complexity hence still in infancy
 Continuous efforts are required to determine the controlled conditions needed for expansion, complete differentiation, and
storage probably in hospital Environments
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16. Glossary
 Muromonab-CD3 (trade name Orthoclone OKT3, marketed by Janssen-Cilag) is an immunosuppressant drug given to reduce acute
rejection in patients with organ transplants.[1][2] It is a monoclonal antibody targeted at the CD3 receptor,[3] a membrane protein on the surface
of T cells. It was the first monoclonal antibody to be approved for clinical use in humans
BHK (Baby hamster kidney) cells line most (Mesocricetus auratus, commonly known as Syrian golden hamsters)
 BHK 21 (BHK Strain 21) fibroblast cell line was derived from baby hamster kidneys of five unsexed, 1-day-old hamsters
 Most widely used line is a subclone (clone 13) of BHK21, usually called as BHK 21(C13), which has been involved in the commercial
manufacture of foot and mouth disease vaccines over a twenty year period
 BHK-21 cells are susceptible to human adenovirus D, reovirus 3, vesicular stomatitis virus (VSV)
 BHK cell line is available for both stable expression and transient expression of recombinant proteins
 Produce the coagulation Factor VIIa and Factor VIII
 Factor VIII is probably the largest, most complex and most challenging recombinant protein therapeutic to manufacture since it possesses
a. 25 potential N-linked glycosylation and
b. many O-glycosylation sites
c. 6 tyrosine sulfation sites
d. 7 disulfide bonds
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17. HEK293 Cell Lines
The HEK293 cell line was originally generated by the transformation of normal human embryonic kidney (HEK) cells following
exposure to sheared fragments of human adenovirus type 5 (Ad5) DNA
Normal human
embryonic kidney
(HEK) cells
HEK293
Exposure to sheared fragments of human adenovirus type 5 (Ad5) DNA
E1A adenovirus gene is expressed in these cells
participates in transactivation of some viral promoters
produce very high levels of
Advantages
 Producing adequate post-translational modifications
 Being easily grown in suspension serum-free culture
 Having quick and easy reproduction and maintenance
 Being highly efficient at transfection and protein production
 Being suitable for both stable expression and transient expression of numerous proteins
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18. HEK293 cell line is further transformed with
the Epstein Barr virus (EBV) nuclear antigen 1
(EBNA1)
Transforming HEK293 with the simian
virus 40 (SV40) large T antigen
HEK293T
(Derivative for HEK293) Derivative HEK293E
Polyethyleneglycol fusion of
HEK293-S and a human B-cell line
HKB-11 cell line
 The most outstanding advantage of HKB-11 is high-level protein production without the formation of aggregate, which
can be a problem in other human cell lines
 Immunoscintigraphy is a nuclear medicine procedure used to find cancer cells in the body by injecting
a radioactively labeled antibody, which binds predominantly to cancer cells and then scanning for concentrations of
radioactive emissions
 Protein toxins such as Pseudomonas exotoxin, diphtheria toxin, and ricin may be useful in cancer therapy because they are
among the most potent cell-killing agents
 In cell biology, a lymphokine-activated killer cell (also known as a LAK cell) is a white blood cell that has been
stimulated to kill tumor cells. If lymphocytes are cultured in the presence of Interleukin 2, it results in the development of
effector cells which are cytotoxic to tumor cells
 Chondrocyte - a cell which has secreted the matrix of cartilage and become embedded in it 18