Introduction to animal cell culture

1. ANIMAL CELL CULTURE –
INTRODUCTION
DR. ANU P. ABHIMANNUE
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
ST. MARY’S COLLEGE, THRISSUR
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ANU P.A., ST. MARY'S COLLEGE,
THRISSUR

2. INTRODUCTION
• Tissue culture is in vitro maintenance and propagation of
isolated cells, tissues or organs in an appropriate artificial
environment.
• The cells can be obtained directly from the host organism or
may be derived from a cell line or cell strain that has already
been established.
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3. DEVELOPMENT OF ANIMAL
CELL CULTURE
• The first mammalian cell cultures date back to the early
20th century to study the development of cell cultures and
normal physiological events such as nerve development.
• Ross Harrison in 1907 showed the first nerve fiber growth
in vitro.
• In the 1950s, animal cell culture was performed at an
industrial scale.
• Major development took place during polio epidemics
during 1940s and 1950s and the accompanying requirement
for viral vaccines.
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https://en.wikipedia.org/wiki/Ross_Granville_Harrison
Ross Granville Harrison (1870 –
1959), American biologist and
anatomist

4. TYPES
On the basis of the source of cells, animal cell culture is categorized into
two types:
• Primary cell culture
• Secondary cell culture
On the basis of the life span of culture, the cell culture are categorized into
two types:
• Finite cell culture
• Continuous cell culture
On the basis of the pattern of growth, the cell culture are categorized into
two types:
• Monolayer culture
• Suspension culture
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Primary cell culture
• Obtained straight from the cells of a
host tissue. The cells dissociated
from the parental tissue are grown
on a suitable container and the
culture thus obtained is called
primary cell culture.
• Such culture comprises mostly
hetergeneous cells
• Most of the cells divide only for a
limited time.
• However, these cells are much
similar to their parents.
Secondary cell culture
• Sub-culturing of a primary culture
by removing the growth media and
disassociating the adhered cells
produces secondary culture/cell-
line/sub-clone.
• Leads to genotypic and phenotypic
uniformity in the population.
• Most of the cells divide only for
prolonged time.
• However, as they are sub-cultured
serially, they become different from
the original cell.

6. Advantages & Disadvantages -
primary cell culture
PROS:
• Primary cell culture represent the best experimental models for in
vivo studies.
• Share the same karyotype as the parent.
• Express characteristics that are not seen in cultured cells.
CONS:
• Difficult to obtain
• Limited lifespans.
• Potential contamination by viruses and bacteria
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7. Advantages & Disadvantages -
secondary cell culture
PROS:
Useful for obtaining a large population of similar cells
Transformed to grow indefinitely.
These cell cultures maintain their cellular characteristics.
CONS:
Cells have a tendency to differentiate over a period of
time in culture and generate aberrant cells.
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8. SUSPENSION CULTURE
• Anchorage independent or non-adherent cells
• Do not attach to the surface of the culture
vessel
• Grow floating in the culture medium giving
rise to suspension culture
• Hematopoietic stem cells (derived from blood,
spleen and bone marrow) and tumor cells.
• Grow much faster does not require frequent
media replacement and can be easily
maintained
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http://www.techne.com/product.asp?dsl=206

9. MONOLAYER CULTURE
• Anchorage dependent/adherent cells
• Cells attach to solid/semi-solid substrate for
proliferation
• Cover the bottom of the culture vessel with
a continuous layer of cells, usually one cell
in thickness, forming monolayer cultures
• Fibroblasts and epithelial cells are of such
types
• Grow relatively slowly and require frequent
replacement of the medium.
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https://www.semanticscholar.org/paper/Three-dimensional-
cell-culture-systems-and-their-in-Edmondson
Broglie/b776327dc8212170baef1e24100eaa4c60246ddb/figure/
0

10. FINITE CELL LINES
• The cell lines which go through a limited number of cell division
having a limited life span are known as finite cell lines.
• These are slow growing cells (24–96 hours).
• Characterized by anchorage dependence and density limitation
• The cells passage several times and then lose their ability to
proliferate and become senescence.
• Cell lines derived from primary cultures of normal cells are finite
cell lines.
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11. CONTINUOUS CELL LINES
• When a finite cell line undergoes transformation and acquires the
ability to divide indefinitely, it becomes a continuous cell line.
• These are grown indefinitely as permanent cell lines and are
immortal.
• Indefinite cell lines could be in vitro transformed cell lines or
cancerous cells
• Can be grown in monolayer or suspension form
• Divide rapidly with a generation time of 12–14 hours
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12. CONTINUOUS CELL LINES –
PROPERTIES
• Less adherent
• Fast growing
• Less fastidious in their nutritional requirements
• Able to grow up to higher cell density and
different in phenotypes from the original tissue.
• Potential to be subcultured indefinitely.
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13. CONTINUOUS CELL LINES
ADVANTAGES
• Faster cell growth and
achieve higher cell densities
in culture.
• Serum-free and protein-free
media are easily available.
• They have potential to be
cultured in suspension in
large-scale bioreactors.
• Easy to manipulate and
maintain
DISADVANTAGES
• Chromosomal instability
• Phenotypic variation in
relation to the donor tissue,
• Change in specific and
characteristic tissue markers
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14. CONFLUENCY
Confluence refers to the percentage of the
surface of a culture dish that is covered by
adherent cells.
when cultured under appropriate conditions,
cells occupy all of the available substrate
i.e. reach confluence.
For a few days, it can become too crowded
for their container and this can be
detrimental to their growth, generally
leading to cell death if left for a long time.
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https://star-protocols.cell.com/protocols/107

15. PASSAGE NUMBER
• The passage number is the number of sub-cultures the cells have gone
through.
• Passage number should be recorded and not get too high.
• High passage number is not advised because
– Passaging procedure is relatively stressful for adherent cells as they
must be trypsinized. Hence, it is recommend not to passage cells more
than once every 48 h.
– High passage number is avoided to prevent cells undergoing genetic
drift and other variations.
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16. SPLIT RATIO
• Cell splitting is a means of increasing the capacity of a cellular system by
subdividing or splitting cells into two or more smaller cells.
• For slow growing cells a low split ratio is recommended.
• Fast growing cells may require a high split ratio.
• Most cells must not be split more than 1:10 as the seeding density will be
too low for the cells to survive.
• When the cells are approximately 80% confluent (80% of surface of flask
covered by cell monolayer) they should still be in the log phase of growth
and will require splitting/sub-culturing.
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17. ANU P.A., ST. MARY'S COLLEGE,
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https://twitter.com/kosheeka/status/1175391468430606336

18. GENERATION NUMBER
• It refers to the number of doublings that a cell population
has undergone.
• It must be noted that the passage number and generation
number are not the same, and they are totally different.
• Also known as, the population doubling (pd) number -
indicates the number of cell generations the cell line has
undergone i.e. the number of times the cell population has
doubled.
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19. SUBCULTURE
• The transferring of portion of cells to a new vessel with fresh growth
medium & more space and nutrients for the continual growth of
both portions of cells.
• Sub-culturing keeps cells healthy and in a growing state.
• The primary culture, when sub-cultured, becomes a cell line or cell
strain.
• Cell-line is grouped into two on the basis of the lifespan as finite or
continuous.
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20. CELL - LINES
• Cell lines are used for
• Vaccine production
• Therapeutic proteins
• Pharmaceutical agents
• Anti-cancerous agents.
• For the production of cell lines, human, animal, or insect cells may
be used.
• Chinese hamster ovary (CHO) is the most commonly used
mammalian cell line.
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21. CELL - LINES
• When selecting a cell line, a number of general
parameters must be considered, such as
• Growth characteristics
• Population doubling time
• Saturation density
• Plating efficiency
• Growth fraction
• Ability to grow in suspension.
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22. MORPHOLOGY OF CULTURED
CELLS
Different morphological structure of cells can be
observed in a culture:
1. Epithelium type
2. Fibroblast type
3. Lymphoblast type
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23. EPITHELIUM TYPE
• Polygonal in shape
• Regular dimensions
• Becomes flattened as they
attach to the substrate
• It grows as a continuous
thin layer forming
monolayer on solid
surfaces
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https://www.thermofisher.com/content/dam/LifeTech/migration/en/images/ics-
organized/references/cell-culture-basics/data-
image/180.par.20311.image.180.120.1.epithelial-like-gif.gif

24. ANU P.A., ST. MARY'S COLLEGE,
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https://cellapplications.com/fibroblast
 These are angular shaped elongated cells.
 It form an open network of cells rather than tightly packed cells
 These are either bipolar or multi-polar
 Anchorage-dependent cells and often align into parallel assemblies
FIBROBLAST TYPE

25. LYMPHOBLAST TYPE
• Cultured cells show a
spherical outline
• They are typically
grown in suspension
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26. REQUIREMENTS – PLASTICWARE
AND GLASSWARES
• Most of the consumables are commercially available as single use, sterile
packs.
• The use of plastic-ware is preferred over recycling glassware because
– Cost effective
– Enables a higher level of quality assurance
– Removes the need for validation of cleaning and sterilization
procedures.
– Designed according to the needs. Pre - treated to provide a hydrophilic
surface for anchorage dependent cells or untreated for suspension
culture.
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28. SPINNER FLASK
• Flask for animal cell
culturing and it provides
better cell production and
viability than conventional
flasks
• Has a top Cap and 2 Angled
Sidearms
• Side baffles enhance
aeration and agitation of
flask contents
• impeller design ensures
optimal stirring
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https://www.sigmaaldrich.com/catalog/product/sigma/cls450
236l?lang=en&region=IN

29. ROLLER BOTTLES
Cylindrical vessels that revolve slowly (5-
60 revolutions per hour) enabling culturing
of adherent cells in large quantity.
Roller bottles are economical and provides
larger surface areas for the growth.
The gentle agitation prevents formation of
gradients in the medium
It also helps in superior gas exchange as
only a thin layer of medium covers the
cell.
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https://www.bioprocessonline.com/doc/cell-culture-roller-flask-system-cellroll-
0001

30. T-FLASK
Animal cell culture flask with an
upright lid for prevention of
contamination.
Usually has vented caps
(with breathable membrane) or even
closed caps are available
Non-Pyrogenic, DNase/RNase-FREE
It is available in various capacities like
T25, T75, T175 and T225.
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https://www.tpp.ch/page/produkte/02_a_zellkultur_flasche_wv.php

31. CELL CULTURE DISHES
Cell culture dishes are disposable/reusable
shallow containers
Dishes can be pre-treated/ untreated for the
growth of anchor-dependent/ suspension cells.
They come in a variety of sizes in single- or
multi-well formats, and they can be round or
rectangular.
made of borosilicate glass/polystyrene or
polycarbonate plastics and allows distortion-
free microscopic observation.
Lids provide consistent gas exchange while
offering protection from the environment.
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https://www.amazon.com/Culture-60x15mm-Treated-Sterile-
Sleeve/dp/B075JQL9HZ

32. Apart from the specific glasswares, animal cell culture make
use of the common apparatus like
• Measuring jar
• Tubes/vials
• Tissue grinder/homogenizer
• Media bottle
• Flasks etc..
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33. ADVANTAGES
• Physiochemical and physiological condition: Role and effect of pH,
temperature, O2/CO2 concentration, and osmotic pressure of the culture
media can be altered to study their effects on the cell culture
• Metabolism of cell: To study cell metabolism, investigate the physiology
and biochemistry of cells.
• Cytotoxic assay: Effect of various compounds or drugs on specific cell
types such as liver cells can be studied.
• Homogenous cultures: These cultures help study the biology and origin of
the cells
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34. ADVANTAGES
• Valuable biological data from large-scale cell cultures: Specific
proteins can be synthesized in large quantities from genetically
modified cells in large-scale cultures.
• Consistency of results: Reproducibility of the results that can be
obtained by the use of a single type/clonal population.
• Identification of cell type: Specific cell types can be detected by the
presence of markers such as molecules or by karyotyping.
• Ethics: Ethical, moral, and legal questions for utilizing animals in
experiments can be avoided.
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35. DISADVANTAGES
• Expenditure and expertise: This is a specialized technique that requires
aseptic conditions, trained personnel, and costly equipment.
• Dedifferentiation: Cell characteristics can change after a period of
continuous growth of cells in cultures, leading to differentiated properties
compared to the original strain.
• Low amount of product: The miniscule amount of mAB and recombinant
protein produced followed by downstream processing for extracting pure
products increases expenses tremendously.
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36. DISADVANTAGES
• Contamination: Mycoplasma and viral infection are
difficult to detect and are highly contagious.
• Instability: Aneuploidy chromosomal constitution in
continuous cell lines leads to instability.
• In addition, this system cannot replace the complex live
animal for testing the response of chemicals or the impact
of vaccines or toxins.
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37. APPLICATIONS
• Vaccines Production
– cells are widely cultured on a large scale to produce vaccines for
many diseases like rabies, chickenpox, hepatitis B, polio and
measles.
• Virus cultivation and study
– It is easy to observe cytopathic effects and easy to select
particular cells on which the virus grow as well as to study the
infectious cycle. Cell lines are convenient for virus research
because cell material is continuously available.
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38. • Cellular and molecular biology
– providing excellent model systems for studying the normal
physiology and biochemistry of cells (e.g., metabolic studies,
aging), the effects of different toxic compounds on the cells, and
mutagenesis and carcinogenesis.
• In Cancer Research
– Normal cells can be transformed into cancer cells by methods
including radiation, chemicals, and viruses. These cells can then
be used to study cancer more closely and to test potential new
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39. • Gene therapy
– Cells having a functional gene can be replaced to cells which are
having non-functional gene, and for which the cell culture
technique is used.
• Immunological studies
– Cell culture techniques are used to know the working of various
immune cells, cytokines, lymphoid cells, and interaction
between disease-causing agents and the host cells.
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40. • Cell lines are also used in
– In-vitro fertilization (IVF) technology
– Recombinant protein development
– Drug selection and improvement.
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41. NAMING A CELL LINE
• Proper Nomenclature of Cell Lines is vital
– Because number of cell lines has dramatically increased
– To maintain individuality of cell lines
– Avoid confusion and duplication of cell line names and identities.
– For consistent usage in publications.
• Cell lines are designated with an alphanumeric code for their identification.
• For instance, the code NHB 2-1 represents the cell line from normal human
brain, followed by cell strain (or cell line number) 2 and clone number 1.
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42. • The usual practice in a culture laboratory is to maintain a log book or
computer database file for each of the cell lines.
• While naming the cell lines, it is absolutely necessary to ensure that each
cell line designation is unique so that there occurs no confusion when
reports are given in literature.
• At the time of publication, the cell line should be prefixed with a code
designating the laboratory from which it was obtained
– NCI for National Cancer Institute
– Wl for Wistar Institute
– MCF for Michigan Cancer Foundation
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43. REFERENCES
• https://www.abcam.com/ps/pdf/protocols/cell_culture.pdf
• https://www.corning.com/catalog/cls/documents/application-notes/CLS-AN-042.pdf
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7325846/
• https://microbeonline.com/animal-cell-culture-introduction-types-methods-
applications/
• https://academicjournals.org/journal/BMBR/article-full-text-pdf/7A2154261212
• https://www.qiagen.com/us/service-and-support/learning-hub/molecular-biology-
methods/animal-cell-culture/
• https://microbeonline.com/animal-cell-culture-introduction-types-methods-
applications/
• https://www.thermofisher.com/in/en/home/references/gibco-cell-culture-basics/cell-
culture-protocols/maintaining-cultured-
cells.html#:~:text=Subculturing%2C%20also%20referred%20to%20as,cell%20line%
20or%20cell%20strain.
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