INTRODUCTION

A cell line is a permanently established cell culture which proliferates

indefinitely under given appropriate conditions. The oldest and commonly
used human cell line is HeLa. DNA fingerprinting using multi-locus probes, STR
profiling, L1 retro transposon marker and Temperature-sensitive SV40, large T
antigen are technologies for the identification and characterization of cell lines.
Cell lines cost less to maintain, are easier to genetically manipulate, handle and
store, moreover their immortal nature enables them to be continuously
cultured, distributed and studied in many labs. Cell lines are in vitro models to
study the pathway of malignant progression, induction of cellular apoptosis,
DNA methylation, histone modifications & tumour suppressor gene
expressions, cell culture cytotoxicity assay and so on. The collection of human
material for culture must be passed by the relevant hospital’s ethics
committee. Cell cultures should be handled in Class II biosafety cabinets and all
discarded materials should be autoclaved, incinerated or chemically
disinfected. The cell line arises from a primary culture at the time of the first
successful subculture. The term cell line implies that the cultures from it
consist of lineages of cells originally present in the primary culture. Lines differ
from cell strains in that they have escaped the Hayflick limit and have become
immortal. Most cell biologists believe that cell lines are already transformed,
abnormal, and the fact that they have circumvented the Hayflick limit and
become immortal makes them analogous to neoplastic cells. These cells tend
towards anaplasia, and are usually more undifferentiated than their normal
cell counterparts.
HeLa cells are one of the oldest and most commonly used human cell
lines. The line was derived from cervical cancer cells taken from Henrietta
Lacks, a patient who eventually died of her cancer on October 4, 1951.The cells
were propagated by George Otto Gey shortly before Lacks died in 1951 and
was the first human cell line to prove successful in vitro, which was a scientific
achievement with profound future benefit to medical research. Gey freely
donated both the cells as well as the tools and processes his lab developed to
any scientist requesting for them, simply for the benefit of science.
TYPES OF CELL LINES

Cell lines can be classified into two types, Finite and Continuous,
depending upon the life span of the culture.
Finite cell lines are characterised by a limited life span, have limited number of
cell generations (usually 20-80 population doublings), exhibit the property of
contact inhibition, density limitation and anchorage dependence, slow growth
rate and the doubling time is around 24-96 hours.
Continuous cell lines are those that are transformed under laboratory
conditions or in vitro culture conditions. These lines show the property of
ploidy (aneuploidy or heteroploidy), absence of contact inhibition, absence of
anchorage dependence, rapid growth rate and the doubling timeTypes of Cell
Lines
Cell lines can be classified into two types, Finite and Continuous, depending
upon the life span of the
culture. Finite cell lines are characterized by a limited life span, have limited
number of cell generations
(usually 20-80 population doublings), exhibit the property of contact inhibition,
density limitation and
anchorage dependence, slow growth rate and the doubling time is around 24-
96 hours. Continuous cell
lines are those that are transformed under laboratory conditions or in vitro
culture conditions. These lines
show the property of ploidy (aneuploidy or heteroploidy), absence of contact
inhibition, absence of anchorage dependence, rapid growth rate and the
doubling time.
PROPAGATION OF CELL LINES
For initiating a cell culture, an important factor influencing the
growth of cells in culture is the choice of tissue culture medium. Many
different recipes for tissue culture media are available and each laboratory
must determine which medium best suit their needs. Individual laboratories
may elect to use commercially prepared medium or prepare their own.
Commercially available medium can be obtained as a sterile and ready-to-use
liquid, in a concentrated liquid form, or in a powdered form. Besides providing
nutrients for growing cells, medium is generally supplemented with antibiotics,
fungicides, or both to inhibit contamination. As cells reach confluency, they
must be sub cultured or passaged for establishment. Failure to subculture
confluent cells results in reduced mitotic index and eventually cell death. The
first step in sub culturing monolayers is to detach the cells from the surface of
the primary culture vessel by trypsinization or mechanical means. The
resultant cell suspension is then subdivided, or reseeded, into fresh cultures
for further propagation. Secondary cultures are checked for growth, fed
periodically, and may be subsequently sub cultured to produce tertiary
cultures, etc. The time between passaging cells depends on the growth rate
and varies with the cell line.
CELL CULTURE MEDIA
Culture media consists of natural or synthetic media.
1. Natural Media The natural media are the natural sources of nutrients
sufficient for growth and proliferation of animal cells and tissues. The
Natural Media used to promote cell growth, fall in three categories:
Coagulants, Biological fluids and Tissue extracts. Coagulants, such as
plasma clots, are now commercially available in the form of liquid
plasma kept in silicon ampoules or lyophilized plasma. Plasma can also
be prepared in the laboratory by taking out blood from male fowl and
adding heparin to prevent blood coagulation. Biological fluids such as
serum is one of the very important components of animal cell culture
which is also the source of various amino acids, hormones, lipids,
vitamins, polyamines, and salts containing ions such as calcium, ferrous,
ferric, potassium, etc. It also contains the growth factors which promote
cell proliferation, cell attachment and adhesion factors. Serum is
obtained from human adult blood, placenta, cord blood, horse blood
and calf blood. The other forms of biological fluids used are coconut
water, amniotic fluid, pleural fluid, insect haemolymph serum, culture
filtrate, aqueous humour, from eyes, etc. Tissue extracts for example
embryo extracts from tissues such as embryo, liver, spleen, leukocytes,
tumour, bone marrow, etc. are also used for culture of animal cells.

2. Synthetic media are prepared artificially by adding several organic and

inorganic nutrients, vitamins, salts, serum proteins, carbohydrates,
cofactors, etc. Different types of synthetic media can be prepared for a
variety of cells and tissues to be cultured. Synthetic media are of two
types- serum containing media (media containing serum) and serum-
free media (media without serum).
Examples of some media are: Eagle′s Basal Medium (BME), developed by
Harry Eagle, is one of the most widely used of all synthetic cell culture media.
There are several “basal” media described by Eagle that vary slightly from one
another. The Tissue Culture Association recommends the use of the name
“Eagle′s Basal Medium” to describe the formula developed to support HeLa
cells. BME, when properly supplemented, has demonstrated wide applicability,
for supporting monolayers growth of a wide variety of normal and transformed
cell lines. BME is the predecessor of Eagle′s Minimum Essential Medium (MEM)
and Dulbecco′s Modified Eagle′s Medium (www.sigmaaldrich.com).Examples
are: BME (Basal Medium Eagle) with Earle's Salts and all its modifications; BME
with Hank's Salts and all its modifications; BME without salts &sodium
bicarbonate; Dulbecco's Modified Eagle Medium Base (DMEM) and all its
modifications; MEM Eagle, Base (Minimum Essential Medium) with L-
Glutamine, without salts & sodium bicarbonate; MEM Eagle, with Earle's Salts
and all its modifications etc.
REQUIREMENTS OF CELL CULTURE
Among the essential requirements for animal cell culture are special
incubators to maintain the levels of oxygen, carbon dioxide, temperature,
humidity as present in the animal’s body. In most of the mammalian cell
cultures, the temperature is maintained at 37oC in the incubators as the body
temperature of Homo sapiens is 37oC.Fluctuation of temperature out of
normal ranges can lead to permanent damage of cells. For example, when
incubating, the optimum growth of mammalian cells is at 37 ± 1oC. Most
media maintain the pH between 7 and 7.4. A pH below 6.8 inhibits cell growth.
The optimum pH is essential to maintain the proper ion balance, optimal
functioning of cellular enzymes and binding of hormones and growth factors to
cell surface receptors in the cell cultures. The regulation of pH is done using a
variety of buffering systems. Most media use a bicarbonate-CO2 system as its
major component. When conditions are too acidic or basic, poor cell growth
and permanent cell damage can occur. For best results keep mammalian cells
at pH range of 7.2 ± 0.2. Monitor cultures periodically and perform media
changes as needed, because the pH of a medium will change as cells take up
nutrients and as the levels of respiratory by-products increase. A change in
osmolality can affect cell growth and function. Salt, Glucose and Amino acids in
the growth media determine the osmolality of the medium. All commercial
media are formulated in such a way that their final osmolality is around
300 mOsm. For culture vessels to have loose caps for gas exchange and
humidity levels is also important. Low humidity can lead to the evaporation of
water from the medium resulting in concentrated salt levels and conditions
that cause cell lysis. A pan of water placed in the bottom of the incubator
usually is sufficient to supply the proper humidity level. You may want to add
clean water and change the water daily. Mammalian cell culture media
typically contain buffering systems that require a CO2 atmosphere. Severe pH
shifts can result if adequate CO2 levels are not maintained. 5% CO2 is usually
sufficient in most culture systems; however, this level may need to be adjusted
to accommodate or enhance the growth of certain cell lines. It is also
important to leave the flask caps of these cultures open slightly to allow
adequate gas exchange.
IDENTIFICATION AND CHARECTARISATION OF CELL LINES
DNA fingerprinting using multi-locus probes It has become established
as the most powerful technology for the identification and characterization of
mammalian cell lines. However, care must be taken in the technical
preparation and statistical evaluation of the DNA banding patterns. Identity
tests rely in general on the detection of genetic differences among individuals.
Short-tandem-repeat (STR) profiling It is the method of choice for cancer
cell line identification. STR profiling is a well established technique that
unambiguously characterises a number of different loci in the human genome
and provides a reference standard for human cell lines. Thus, it is the method
recommended by ‘The American Type Culture Collection’ (ATCC) for cell line
authentication. Likewise, STR-profiling is well established in DNA-based
forensics and paternity testing. STR loci consist of short, repetitive sequence
elements, 3–7 base pairs in length. These repeats are well distributed
throughout the human genome and are a rich source of highly polymorphic
markers.
L1 retrotransposon marker A novel marker is used for HeLa cell line
identification . L1 retrotransposons are active, autonomous mobile elements
that are very common in the human genome, comprising 17% of the human
genome sequence. Human-specific L1s (L1Hs) have been inserted into our DNA
since the origin of the species and thus are readily employed as population
and individual specific genetic markers with a number of useful characteristics.
Temperature-sensitive SV40 large T antigen Bone marrow-derived
stromal cell lines (TSB) were established from temperature-sensitive SV40
large T-antigen transgenic mice and were characterized for their supportive
activity for osteoclast differentiation. Subsequently, their osteoblastic
differentiation potential was examined in terms of expression of osteoblast
marker genes and the ability of bone nodule formation.
ADVANTAGES OF CELL LINES
Cell lines are suitable models to study the biology of many diseases e.g.
small cell lung cancer and many important contributions would have been
impossible without a large comprehensive panel of cell lines. These lines may
be suitable for the selection of the best in vitro regimen to treat individual
patients from whom the lines were derived, a hypothesis currently being
tested in Branch. Finally, in vitro studies already suggest newer, more rational
approaches to tumour control. Cell lines cost less to maintain and they are
easier to genetically manipulate. The main advantage of using cell lines for
such research is the immortal nature of the cell lines, enabling them to be
continuously cultured, distributed and studied in many labs and to act as a
reliable platform for comparison of results, before advancing research to the
next level. Another advantage of performing research on cell lines is the ease
of handling and storage. Cell lines are cultured in flasks under well-controlled
nutritional and environmental conditions and this ensures a greater degree of
reproducibility in the results. Apart from the ease in maintenance and access,
cell lines offer a convenient platform for genetic manipulation of cells. Cells can
be stored indefinitely in liquid nitrogen and can be used when needed. Cell
lines offer a realistic platform to knock-down or over-express genes of interest.
Due to the tremendous benefits of working on cell lines, they have
dramatically contributed to basic research in cancer biology. The in vitro cell
line model was predictive for non-small cell lung cancer under the disease-
oriented approach, for breast and ovarian cancers under the compound-
oriented approach, and for all tumour types together. They exhibit a relatively
high degree of homogeneity and are easily replaced from frozen stocks if lost
through contamination. There is considerable ethical pressure on scientists to
reduce or eliminate the use of animals in laboratory research, and primary cell
culture may be one way forward, especially in preclinical drug testing. Another
hot area which depicts a huge advantage for using cell lines is to predict how
patients will respond to chemotherapy regimens.
DISADVANTAGES OF CELL LINES
Major disadvantage is misidentification or cell line contamination. Cells
isolated from a breast tumour may behave differently in culture as compared
to their response when they are a part of a tissue/organ, because the cell–cell
interactions that exist in tissue are lost in vitro. Another disadvantage is the
slow population doubling time and the finite lifespan before senescence; often
cells will survive two or three passages only. For some experimental
techniques large numbers of cells are required, which can sometimes be a
limiting factor because this often cannot be achieved until after several
passages. Furthermore, cell lines are prone to genotypic and phenotypic drift
during their continual culture
Production of Recombinant Protein Therapeutics in
Mammalian Cell Lines
Recombinant protein production using mammalian cells offers several
advantages over microbial systems. For example, mammalian cells are able to
secrete the protein product, hence, obviating the need for cell
homogenization. Furthermore, they are able to perform post-translational
modifications, like glycosylation, which are necessary for the efficacy of protein
drugs targeted as human therapeutics. The challenge of improving protein
yield and quality can be met through various approaches. For this, three
strategies are currently developed: Firstly, to generate cell lines that can
produce high yields of recombinant proteins. The cell lines are of non-tumour
origin, they are immortalized by a function not oncogenic in human and they
are from an ethically accepted and easily accessible cell source. Since the cell
can be easily adapted to growth in serum-free and chemically defined medium,
they fulfill the requirements of biopharmaceutical production process. Second
factor that has a significant impact on recombinant protein yield and quality is
the culture media. Mammalian cells require a combination of both nutritive
(e.g. sugars and amino acids) and non-nutritive (e.g. trace metals, vitamins and
co-factors) components to support cell growth and protein production. In
addition, the media environment has been shown to affect protein
glycosylation, which is an important aspect of protein quality and influences its
efficacy as a therapeutic. Finally, a potential method to improve the efficacy of
recombinant proteins produced through cell culture.
BIOETHICS OF USE OF CELL LINES
A form will be required for the patient to sign authorizing research use of
the tissue, and preferably disclaiming any ownership of any materials derived
from the tissue. Each laboratory has its own biosafety regulations that should
be adhered to, and anyone in any doubt about handling procedures should
contact the local safety committee. When the sample arrives at the laboratory,
it should be entered into a record system and assigned a number. This record
should contain the details of the donor, identified by hospital number rather
than by name, tissue site, and all information regarding collection medium,
time in transit, treatment on arrival, primary disaggregation, and culture
details, etc. The set up and implementation of an adequate containment level
include a list of general and more specific work practices and containment
measures. Precautionary measures should be applied whenever handling
animal cell cultures. Much of these measures basically aim at reducing the risk
of contamination with adventitious agents by ensuring protection of both
operator and cell culture. Cells are reasonably considered free of adventitious
contaminating pathogens if a number of conditions are fulfilled. If cell cultures
are known to harbour an infectious etiologic agent or virus, containment
measures should be the same as that recommended for the etiologic agent.
Respect good microbiological practices, especially those that are aimed at
avoiding accidental contamination. Treat each new culture that is manipulated
for the first time in the laboratory facility as potentially infectious. The use of a
biosafety cabinet of class type II is strongly recommended until the cells have
been shown negative in sterility tests for bacteria and fungi. Cell cultures from
ill-defined sources should be handled under biosafety level 2 (L2) conditions. If
there is a reasonable likelihood of adventitious agents of higher risk class, the
cell line should be handled under the appropriate containment level until tests
have proven safety. Clean up any culture fluid spills immediately, work with
one cell line at a time, disinfect the work surfaces between cell lines handling,
aliquot growth medium so that the same vessel is not used for more than one
cell line. Avoid pouring actions that are a potential source of
cross-contamination.
FUTURE OF CELL LINE RESEARCH
In the last four decades, mammalian cell culture has matured from
being merely a research tool into being one of the foundations of the
biopharmaceutical industry, and its use is continuing to expand rapidly. In vitro
models are replacing animals in many tests and assays; its enormous potential
in the fields of stem cell and regenerative medicine has hardly started to be
realized; and its utility in research has grown even faster. Increasing number of
mammalian cell products in development is a driver for improved and faster
process development technologies. High volume demands are driving both
capacity and improvements in process efficiency. Research is going on for
continued process improvements that involve improvements in selection
procedures for finding highly productive clones. Scientists across the world are
trying to improve the cell lines that are engineered for improved growth,
product synthesis, energy metabolism, glycosylation characteristics. A whole
range of genomic and proteomic approaches are used to increase our
knowledge of cell physiology and to inform design of cells and processes.
Undoubtedly the mammalian cell culture is likely to be the key technology for
foreseeable future.
CONCLUSION
A large portion of genes implicated in the emergence and
progression of cancer have similar gene expression values in tumours and
cancer cell lines indicating the value of cultured cell lines in cancer research.
Cell lines are proposed as a good model for investigating gene therapy
approaches. The current strategies for the development of cell lines for the
recombinant expression of therapeutic proteins remain empirical to a large
extent. This is mainly due the lack of consistent methods to predict production
capacity and growth characteristics of clonal cell lines at production scale. In
this regard, the knowledge gained from genomic and proteomic studies is
expected to provide a much better understanding of the biochemistry and
physiology of mammalian cells, inform design of cells and processes.
Continued improvements in selection procedures are expected for finding
highly productive cell lines. Cell lines should be engineered for improved
growth, product synthesis, energy metabolism, glycosylation characteristics.
Product quality may be achieved through genetic engineering of the host or by
a more rational optimization of the culture conditions.
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