culturing animal cells in vitro

1. Animal Cell Culture
By Ruchira Agarwal
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REPORT ON

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What Is Animal Cell Culture ?
Growing cells removed from animal tissues or whole animals in vitro conditions by
providing them with proper nutrients and growth factors is called Animal Cell Culture.
Cultures normally contain cells of one type ( e.g. fibroblasts )
 Homogeneous population- All the cells in the culture are genetically identical (clone)
 Heterogeneous population- cells show some genetic variation
The cells are maintained as independent units and can divide via mitosis. The cell
population can thus continue to grow unless limited by some parameter e.g.
nutrient depletion or contamination

3. Animal cell culture has following applications:
 To investigate the normal physiology or biochemistry of cells. For example metabolic
pathways can be investigated by applying radioactively labelled substrates and subsequently
looking at products.
 To test the effects of compounds on specific cell types. These compounds can be metabolites,
hormones , growth factors or even potentially toxic or mutagenic substances.
 To produce artificial tissue by combining specific cell types in sequence. For example
production of artificial skin for use in treatment of burns.
 To synthesize valuable products (biologicals) from large scale cell cultures. The biologicals
include specific proteins or viruses that require animal cells for propagation. The quantity of
proteins generated in vitro conditions can be several folds the quantities found in vivo conditions.
Why Grow Animal Cells in Culture?
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 Consistency and reproducibility of results can be obtained by using cells of a single
type {preferably a homogeneous population (clonal)}.
For e.g., doing a biochemical analysis (to relate a particular metabolic pathway to a
certain cell type) would be possible in a culture containing a homogeneous cell population
which can be monitored for biochemical and genetic characteristics, but very difficult in a
tissue homogenate which would contain a heterogeneous mix of cells at different stages
of growth and viability.
 For Toxicological testing, the use of cell culture techniques allows a greater
understanding of the effects of a particular compound on a specific cell type (e.g.
hepatocytes) than testing in laboratory animals which can also be more expensive .
 While producing biological products on a large scale, contaminants like unwanted
viruses or proteins can be more easily avoided in a cell culture than in a pooled animal
tissue.
Advantages of Cell Culture

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List of biotherapeutics approved by FDA produced in Chinese Hamster Ovary cell lines

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• after a period of continuous growth, cell characteristics can change and may be
quite different from those originally found in the donor animal. Cells can adapt to
different nutrients.
• Cell culturing favors the survival of fast-growing cells which are selectively retained
in a mixed cell population. This can be a problem when using cultures to develop an
understanding of the behavior of cells in vivo
• . After a period of culture, the cells may be significantly different from those that are
highly differentiated in vivo where growth has ceased.
• Intracellular enzyme activities change dramatically in response to nutrient depletion
and by-product accumulation in a culture.
Disadvantages of Cell Culture

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Main
milestones
in cell
cultures

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• The surface for cell adhesion, growth, proliferation determine cellular
secretion activity of cells.
• Earlier glass surface was widely used, now in most of laboratories plastic
(usually polystyrene) lab ware is used for typical monolayer cultures.
• The surface of that cell culture vessels can be enhanced by coating with
proteins, such as collagen, gelatin, laminin, fibronectin that are components
of extracellular matrix.
• For enhancement polymers can also be used, for example, poly‐L‐lysine or
other commercial matrices.
Growth substrates

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• Media are composed of two main components: a basal nutrient medium and
supplements.
• The balanced salt solution, for e.g. , DPBS, HBSS, EBSS, form basis of complex
media.
• The supplements complete media with nutrients, proteins, amino acids,
buffering system and vitamins.
• The most popular media are:
 Dulbecco's Modified Eagle's Medium (DMEM)
 Eagle's Minimal Essential Medium (EMEM)
 Medium 199 (M199)
 Roswell Park Memorial Institute (RPMI–1640) or Lebovitz Medium (L‐15)
Culture Medium

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Supplements:
 Amino acids - essential for growth and cell proliferation, for example, cysteine,
L‐glutamine and tyrosine.
 Vitamins- For proper metabolism, cells require B vitamins (especially presence of B12
is essential), choline, folic acid, inositol, biotin
 Ions - Na+, K+, Mg2+, Ca2+, Cl‐, PO43‐, SO42‐, HCO3‐ affect osmolarity of culture
media.
 Carbohydrates & organic supplements- Glucose is mainly used as an energy source
but in some cell types galactose, mannose, fructose or maltose can be used. Also
supplemented with pyruvate, lipids ,TCA intermediates
 Trace elements - zinc, copper, selenium and tricarboxylic acids intermediates

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 Serum -Serum is a complex mixture of proteins, source of minerals, lipids,
hormones, and growth and adhesion factors. Fetal bovine serum (FBS) and
newborn calf serum (NCS) are most common. For more specific cultures human,
horse or rabbit sera are used.
 Antibiotics and antifungal solutions with laminar flow hoods reduce the
frequency of contamination. In cell cultures most often penicillin, streptomycin
solutions are used. As the antimycotic agents the kanamycin or amphotericin B
are applied.
 Growth factors and hormones - are used especially in serum‐free media. factors
ensure cellular growth, division, and differentiation. The most popular are
fibroblastic growth factor (FGF), insulin‐like growth factor (IGF), vascular
endothelial growth factor (VEGF) or platelet derived growth factor (PDGF). The
most common hormones are hydrocortisol and insulin
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Following are the requirements:
 Oxygen
• Oxygen as a part of the gas phase is required for adequate cell physiology, function,
and differentiation.
• The oxygen requirements depend on the cell type.
• In general, low concentrations of oxygen are used depending on the dissolved oxygen
in culture media.
• Higher concentrations of oxygen can inhibit cell growth and metabolism.
• In some cases transformed cells can be anaerobic.
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Physical & chemical Properties
of cultures in vitro

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 pH
• For animal and human cells a pH is in the range of 7.0 ‐7.4 . Some differences can
be noticed for transformed cells (7.0–7.4), and in some cases cells require higher pH
levels, for e.g. , normal fibroblasts (7.4–7.7).
• In the range of pH= 6.5–7.0 cells stop growing, between pH 6.0–6.5 cells lose their
viability.
• The pH level can be checked by presence of phenol red in culture medium
 Osmolarity
• Cells exhibit rather wide tolerance to osmotic pressure which can influence growth
and cell function. In general osmolarity should be similar to the natural tissue
environment. The osmolarities between 260 mOsm/kg and 320 ± 10 mOsm/kg are
applicable.
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 Carbon dioxide and bicarbonate
The buffering system is essential to maintain proper pH. For establishing physiological
pH for cells CO2 is dissolved in the culture medium. CO2 establishes equilibrium with
HCO3‐ ions. The bicarbonate buffers not only show low toxicity, but also help in
glucose metabolism.
The other buffering system include use of HEPES buffer (toxic to some type of cells)
 Temperature
Most of cell lines are maintained at 37°C (earlier called “warm‐blood animal”
temperature), but temperature is determined by origin of tissue e.g, lower temperature
is usually used for skin and testicles cell cultures.
 Viscosity
The important factor for cell suspension cultured in stirred vessels or when cells are
dissociated after trypsinization.
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16. • Adherent cells are said to be anchorage-dependent and attachment to a
substratum is a prerequisite for proliferation. They are generally subjected to
contact inhibition, which means they grow as an adherent monolayer and
stop dividing when they reach such a density that they touch each other.
• Most cells, with the exception of mature hemopoietic cells and transformed
cells, grow in this way.
• Cultures in which cells grow attached to each other or to a substratum have to
be treated by a proteolytic enzyme to break the bond between cells and
substratum. Most commonly Trypsin is used
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Adherent culture

17. Suspension Culture
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• In contrast to anchorage-dependent cells, cells cultured from blood,
spleen or bone marrow adhere poorly if at all to the culture dish. In the
body, these cells are held in suspension or are only loosely adherent.
• Suspension cultures are easier to propagate, since subculture only
requires dilution with medium.
• These do not require trypsinization and are thus easier to harvest.

18. Primary Culture
 A primary culture is defined as one 'started from cells, tissues or organs taken
directly from organisms'.
 The major advantages of primary cultures are the retention of :
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(1) the capacity for biotransformation
(2) tissue-specific functions.
 Primary cultures have a limited life
span and changes in metabolism
and tissue specific functions will
occur with time in culture.

19. • It is possible to develop populations of cells that can be passaged
indefinitely and that express a reasonably stable phenotype. These are
called continuous cell lines.
• Some cell lines have arisen spontaneously in normal cells being
passaged in culture, but the majority has been obtained by culturing
tumor cells.
• These have an infinite life span, divide more rapidly, do not require
attachment to the substratum for growth, and when reintroduced into
animals, they form tumors.
• Cell lines with these properties are sometimes referred to as
transformed cell lines. 19
Continuous Cell Lines

20. Basic Equipment and facilities
in Animal cell culture
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LABORATORY SETUP FOR CELL CULTURE

22. Aseptic Technique of Culture
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• In culturing animal cells, it is essential that all procedures are carried out
using aseptic techniques.
• laminar flow facilities or sterile rooms provide a suitable environment,
but even then aseptic techniques should be employed.
• If a nonsterile environment is used, then the reliance on aseptic
techniques is very high.
• Basic aseptic technique is to ensure that the work area is clear, swabbed
down regularly with 70% ethanol and that all the equipment used has
been sterilized. Clean laboratory coats are also essential.

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Techniques for disaggregation of tissue isolated
for primary culture-

24. Primary Explantation
 The original method developed by Harrison &
others for initiating a tissue culture.
 A fragment of tissue was embedded in blood
plasma or lymph, mixed with heterologous serum
and embryo extract, and placed on a coverslip
that was inverted over a concavity slide. This
technique is still used but has been largely
replaced by the simplified method ( shown in
figure)
 This technique is useful for small amounts of tissue
like skin biopsies in which there is a risk of losing
cells during mechanical or enzymatic
disaggregation.
 Its disadvantages lie in the poor adhesiveness of
some tissues and the selection of the more
migratory cells in the outgrowth.
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25. Mechanical
Disaggregation
(a)Scraping : Cutting action, or abrasion of cut surface,
releases cells.
(b) Sieving : Forcing tissue through sieve with syringe piston.
(c) Syringing : Drawing tissue into syringe through wide bore
needle or canula and expressing.
(d) Trituration by pipette : Pipetting tissue fragments up and
down
 Only soft tissues (e.g. spleen, embryonic liver, embryonic
and adult brain) & some human and animal soft tumors
respond well to this technique.
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26.  Coarsely chopped tissue is stirred in Trypsin until
fully disaggregated, with dissociated cells
collected at intervals, centrifuged, re-suspended
in medium, and stored on ice to be pooled later.
 It is useful for the disaggregation of large amounts
of tissue in a relatively short time, particularly for
chopped whole mouse embryos or chick
embryos.
 It does not work as well with adult tissue, in which
there is a lot of fibrous connective tissue, and
mechanical agitation can be damaging to some of
the more sensitive cell types like epithelium.
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Warm Trypsin Disaggregation

27. Cold Trypsin Disaggregation
 Tissue is soaked in trypsin at 4◦C for 6-
18h to allow penetration of the enzyme
with little tryptic activity. Following
procedure (in figure) the tissue requires
20-30 min at 37◦C for disaggregation.
 gives a higher yield of viable cells, with
improved survival after 24-h culture and
preserves more different cell types than
the warm method.
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28. Use of Collagenase
 Disaggregation in trypsin can be
damaging (e.g., to some epithelial
cells) or ineffective for very fibrous
tissue so attempts have been made to
utilize other enzymes.
 Because the extracellular matrix often
contains collagen, collagenase has
been the obvious choice
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29. Future of cell cultures
• animal cell technology will enlarge its applications, for e.g. , use of viral vectors for
gene therapy, vaccine technology, recombinant protein production for therapeutic
purposes.
• human cell cultures can also be used for personal therapies—gene therapies, tissue
engineering, transplantation of organs.
• In the future, more human diseases will be treated by new form of therapies based on
organ and tissue cultures.
• primary tumor cell cultures can give more accurate information about individual
cancer cases and support establishment of clinical setting.
• Future medicine will able to use widely stem cells [adult and as well human embryonic
stem cells (HESC)] for damage tissue replacement.
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30. REFERENCES
• Butler, M. (2004). Animal Cell Culture & Technology. London &
New York : BIOS Scientific Publishers
• Freshney, R. I. (2010). Culture of animal cells. Hoboken: Wiley &
Blackwell.
• Jedrzejczak-Silicka, Magdalena. (2017). History of Cell Culture.
• Unchern, Surachai. (2020). Basic Techniques In Animal Cell
Culture.
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