This presentation will help to understand the basics of mammalian cell culture. I have also covered the difference between adherent and suspension cell lines. I have also included the advantages and disadvantages of the cell line.

1. Mammalian Cell Culture
Shubham A. Chinchulkar
M.Tech (Pharm.)
National Institute of Pharmaceutical education
and Research (NIPER)
shubhamchinchulkar007@gmail.com
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2. Mammalian Cell Culture
 Cells, removed from animal tissue or whole animals, will continue to grow if supplied
with nutrients and growth factors
 The cells are capable of division by mitosis and the cell population can continue
growth until limited by some parameter such as nutrient depletion
Parent Cell
DNA replication
Two daughter cells
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3.  Cultures normally contain cells of one type (e.g. fibroblasts)
 A fibroblast is a type of biological cell that synthesizes the
extracellular matrix and collagen, produces the structural framework
(stroma) for animal tissues, and plays a critical role in wound healing
Fibroblasts are the most common cells of connective tissue in animals
 The most common marker used for fibroblasts is Vimentin ( Millipore MAB 3400)
 The cells in the culture may be:-
Genetically identical Genetic variation
Hair color
Height, hair
texture,
disease
immunity,
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4.  Genetically identical: A population of humans that has inhabited an island for
thousands of years with little migration to or from the island
 Genetically variation: Humans that have migrated from different regions of the
world and currently live together
Applications for animal cell cultures
 To investigate the normal physiology or biochemistry of cells (metabolic
pathways can be investigated by applying radioactively labeled substrates
and subsequently looking at products)
Vi-CELL XR Cell
Viability Analyzer -
Beckman Coulter
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6. The metabolite products in culture can be determined by Nova analyzer
The gaseous present in cell culture can be determined by Bio gas analyzer (BGA)
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7. The osmolality in cell culture can be determined by Osmometer
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8.  To test the effects of compounds on specific cell types e.g. effect of supplements
 To synthesize valuable products (Biologicals) from large-scale cell cultures
Advantages:
1. Consistency and reproducibility of results that can be obtained by using a batch of
cells of a single type and preferably a homogeneous population
2. Toxicology testing - use of cell culture techniques may allow a greater understanding
of the effects of a particular compound on a specific cell type and less expensive
testing cost
Cell line
Animal
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9.  In the production of biological products on a large scale, the avoidance of
contaminants such as unwanted viruses or proteins is important
Disadvantages:
1. After a period of continuous growth, cell characteristics can change and may be
quite different from those originally found in the donor animal
2. This adaptation of different nutrients involves changes in intracellular enzyme
activities
3. Culturing favors the survival of fast-growing cells which are selectively retained in
a mixed cell population
4. The changes in the growth and biochemical characteristics of a cell population
may be a particular problem when using cultures to develop an understanding of the
behavior of cells in vivo
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10. 4. Intracellular enzyme activities change dramatically in response to nutrient depletion
and by-product accumulation in a culture
Primary Culture Survival:
 In 1961, Hayflick and Moorhead - human embryonic cells – repeated subculture
for about 50 generations
 The finite number of generations of growth is a characteristic of the cell type, age
and species of origin and is referred to as the ‘Hayflick limit’
 The capacity for growth is related to the origin of the cells – embryonic tissue have
a greater growth capacity than those derived from adult tissue
 Each cells have an inherent growth biological clock and defined no. of divisions
from original stem cells, even if stored by cryopreservation the capacity for cell
division is not altered
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11. The biological clock:
 The growth capacity of normal cells was a mystery for a number of years until
some key observations were made regarding chromosomal length
 The caps (telomeres) at the end of chromosomes of human germline cells were
longer than somatic cells
 These caps known as telomeres are repeats of the nucleotide sequence
TTAGGG/CCCTAA and are shortened at each generation of growth of somatic
cells
 Semiconservative mechanism of DNA replication which operates in one direction
(5′ to 3' end) is responsible for cap shortening
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12. DNA replication operates in one direction (5′ to 3' end)
Replication fork generation
end of double-
stranded DNA
(nonworking)
At each mitotic division there is a small segment of DNA that is not replicated, thus
shortening the telomeric cap
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13. In germ cells the telomere is maintained at 15 kilobases apparently by a ribonucleoprotein
enzyme, telomerase
Telomerase is expressed in germ cells and has moderate activity in stem cells but is absent
in somatic cells
Finite lifespan of normal somatic cells is regulated by 10 or more ‘senescence’ genes that
suppress the expression of the telomerase gene
It causes the human telomeres to gradually shorten at a rate of around 100 base pairs per
cell division
If oncogenes are activated then cells escape the negative control of the cell cycle and re-
express telomerase
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14.  Bodnar et al (1998) described the introduction of human telomerase reverse
transcriptase (hTRT) into normal human cells (retinal pigment epithelial cells and
foreskin fibroblasts)
 Epithelial cells – mean population doubling of 54 generations increased to a value
of 73 generations
 Fibroblasts – there was an increase of the mean population doubling of 64 to 100
 This experiment established a causal relationship between telomere shortening and
in vitro cell senescence which is dependent upon a mitotic clock
Not capable for expression of
the telomerase enzyme
hTRT¯ hTRT+
Capability for expression of
the telomerase enzyme
human telomerase
reverse transcriptase (hTRT)
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15. The first products of animal cell technology:
 In 1949, Enders showed that the poliomyelitis virus could be grown in cultures of
human embryonic cells
 Produced from the de-activated virus, became one of the first commercial products
of cultured animal cells
 Initially it was isolated from primary cell culture, later primary monkey kidney cells
were replaced by human diploid lung fibroblast cells with defined characteristics
 Eagle’s Minimum Essential Medium (EMEM) - developed in the 1950s was the
first widely used culture medium – Advantages - consistency between culture
batches; ease of sterilization; reduced chance of contamination
 Bacteria do not have the appropriate metabolism to complete these modifications
which are necessary for full activity of many mammalian proteins
 Macromolecules as plasminogen activator, factor VIII and erythropoietin
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16.  Fusion techniques - 1960s by Harris and Watkins who allowed cells of different
origin to be fused to produce hybrids
 Kohler and Milstein in 1975 - hybridoma cells capable of the continuous
production of a single type of antibody
 Products from animal cells - viral vaccines; monoclonal antibodies; recombinant
glycoproteins
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17. Adherent Cell Culture
 Requires periodic passaging, but
allows easy visual inspection under
inverted microscope
 Cells are dissociated enzymatically
(e.g., Gibco™ TrypLE™ Express,
trypsin) or mechanically
 Growth is limited by surface area,
which may limit product yields
 Requires tissue-culture treated vessel
 Used for cytology, harvesting
products continuously, and many
research applications
Suspension Cell Culture
 Easier to passage, but requires daily
cell counts and viability
determination to follow growth
patterns; culture can be diluted to
stimulate growth
 Does not require enzymatic or
mechanical dissociation
 Growth is limited by concentration of
cells in the medium, which allows
easy scale-up
 Can be maintained in culture vessels
that are not tissue-culture treated, but
requires agitation (i.e., shaking or
stirring) for adequate gas exchange
 Used for bulk protein production,
batch harvesting, and many research
applications
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