TYBSc Zoology Paper III Unit I Sem VI Biotechnology-I

1. Animal Cell & Tissue Culture
TYBSc Zoology Paper III Sem VI
Biren Daftary

2. Mammalian cell lines & characteristics
• Some multiply while some do not when kept within
in vitro conditions
• Cancer cells, epithelial cells and fibroblasts are e.g. of
multiplying cells
• Normal diploid cell = finite life span
• Continuous cell lines = grow & multiply indefinitely
• CCL = easier to handle because of less dependency
on serum & growth factors
• CCL = increased growth rates, less sensitive to env.
disturbances & can be grown in suspension culture

3. • Negative side of CCL : increased metabolic rates &
formation of inhibitory by-products
• All mammalian cells follow the cell cycle
• Normal mammalian cells that do not proliferate are
arrested in G1 phase
• For CCL if unsuitable conditions are present, they
undergo apoptosis
• In culture, cells act as unicellular organisms
• Growth and division α availability of nutrients in
sufficient quantities

4. • In suspension culture cells assume a spherical shape
with diameter 7-20 μm
• Mammalian cells lack a rigid cell wall and hence
vulnerable to changes in osmolarity, shear forces &
air bubbles

5. Primary Culture
Mass Culture
Clonal Culture

6. Mass Culture
• Large number of primary cells are obtained from the
organism
• They are added to a culture dish where they attach to the
bottom
• Some cells die, the others survive
• The ones which survive, they proliferate on the culture dish
• After a number of generations of proliferations, they form a
monolayer of cells
• These cells cover the entire bottom portion of the culture
dish
• Here different type of cells are obtained in culture, hence
different cell lines

7. Clonal Cell Culture
• From clonal cell cultures, pure cell lines are obtained
• A single cell is attached to a culture dish some distance
away from its neighbours
• If the single cell survives then it proliferates and forms a
colony on the culture dish
• All the proliferated cells are clones of the original cell

9. Media for cultivation of mammalian cells
• Cell culture media should supply nutrients similar to those
present in the blood stream
• Initial media derived from natural sources such as chick
embryos, blood serum, clots & lymph fluids
• Basis for a media is balanced salt solution
• Proper salt soln. = physiological pH, osmolarity for
maintaining cell viability
• For proliferation, glucose, aa & vitamins were added
according to req. of the cell line
• Hence different media formulations for different types of
cell lines

10. Serum containing media
• Most widely used biological fluid
• It is the liquid exuded from coagulating blood
• Obtained from adult human blood, placental cord blood,
horse blood or calf blood
• Calf serum and foetal calf serum most widely used
• Human serum sometimes used for human cell lines,
however must be free from virus
• Important to test different serum preparations for sterility
& toxicity before use
• EMEM with 5-20% serum serves as a good nutrient media

11. Advantages:
• Supplies growth factors, trace elements & lipids
• Enhances buffer capacity
• Chelates heavy metals
• Protects against proteolytic activity, shear forces & bubble
damage
• Use of serum allows for a single medium formulation for
many cell lines

12. Disadvantages:
• Dependency on its supply
• Lack of reproducibility due to variation in quality between
batches
• Risk of contamination of product with virion or prion
particles e.g. BSE
• In serum free media following supplements are added:
Insulin (growth factor), transferrin (Fe3+ carrier), selenium
(trace element), fatty acids, dexamethason, BSA
• Also contain trace elements like Zn, Mb, Ni

13. Blood plasma
• Most commonly used clots are plasma clots, been in use for
a long time
• Plasma is commercially available either in liquid or
lyophilized state
• It may be prepared in laboratory usually from the blood of
a male fowl
• However blood clotting must be avoided during the
preparation

14. Tissue Extracts
• Chick embryo extract is the most commonly used tissue
extract
• Bovine embryo extract is also used
• Other tissue extracts used are: spleen, liver, bone marrow,
leucocytes, etc. extracts
• Tissue extracts can be substituted by a mixture of aa and
certain other organic compounds
• The natural biological fluids are generally used for organ
culture
• For cell culture, artificial media with or without serum are
used

15. Complex Natural Media
1. Supplemented Hanks-Simms medium:
• 3 parts Hank’s balanced salt + 1 part Simm’s Ox serum ultra
filtrate
• Developed by Weller and co-workers (1952) in their work
with polio viruses
• The complete media used for tissue culture is:
Hanks-Simms soln. (85%) + Beef embryo extract (10%) + Horse
serum inactivated at 56˚C for 30 min. (5-20%) + Penicillin (50
μg/ml) + Streptomycin (50 μg/ml)

16. 2. Supplemented bovine amniotic fluid medium:
• Developed by Milovanic & co-workers
• Bovine amniotic fluid (37.5%) + Horse serum inactivated at
56˚C for 30 min. (20%) + Bovine embryo extract (5%) +
Hank’s balanced salt solution (37.5%) + Streptomycin (100
μg/ml) + Penicillin (100 μg/ml) + Mycostatin (100 μg/ml)

17. 3. Serum supplemented yeast extract medium:
• Yeast extract medium: 10 parts 1% Difco’s yeastolate soln. +
2.5 parts of 10% glucose soln. + 87.5 parts Hank’s balanced
salt solution
• Entire media: Yeast extract medium (76 parts) + Human
serum (20 parts) + 1.4% NaHCO3 solution (4 parts)
4. Serum supplemented lactalbumin hydrolysate & yeast
extract medium:
Earle’s saline containing lactalbumin hydrolysate (0.5%) +
yeast extract (0.1%) + Human or Ox serum (10-20%)

18. Growth Factors
• Mammalian cells were first cultured in blood clots
• All efforts to define the minimal requirements for cell
proliferation failed
• In spite of adding glucose, aa & vitamins, cells would cease
to grow without the addition of serum
• Mammalian cells deprived of serum stop growing &
become arrested usually between mitosis & S phase called
as G0 state
• Eventually it was found that serum provided essential
components which contain highly specific proteins called
‘growth factors’

19. • Most of these growth factors are required in very low
concentrations
• An important function of growth factor is to regulate
protein synthesis & rate of cell growth
• This is because most factors that stimulate cell proliferation
also stimulate cell growth
• Hence growth factors act independently on the cell growth
along with other essential nutrients like aa, vitamins and
glucose

20. Preparation of cells for culture
• Disaggregation of tissue and primary culture naturally or by
artificial practises
Disaggregation is achieved by following methods:
• Physical disruption: Cutting the tissue
• Enzymatic digestion: Trypsin, pappain, collagenase, etc
• Treatment with chelating agents: EDTA & citrate

21. Isolation of tissue
• Sterilization of the site with 70% Alcohol
• Removal of the required tissue
• Storage of the tissue in a refrigerator
• Finally transferring the tissue to BSS

22. Enzymatic disaggregation
1. Trypsinization:
a. Warm trypsinization:
• Tissue sample exposed to the warm trypsin (36.5˚C)
• The dissociated cells are collected every half hr.
• Trypsin is removed by centrifugation after 3-4 hrs.
b. Cold trypsinization:
• Tissue soaked in cold trypsin for 4-6 hrs to allow
penetration
• Afterwards, trypsin is removed and tissue is incubated at
36.5˚C for 20-30 min

23. • After trypsinization, serum is added to the cells to nullify
traces of trypsin which are left
• This is done because leftover trypsin can cleave cells if left
behind for long

24. 2. Disaggregation by collagenase
• Sometimes trypsin is too damaging to the tissues or
ineffective for fibrous tissues
• The intracellular matrix contains collagen, therefore
collagenase proves effective for several normal & malignant
tissues which are sensitive to trypsin
• Collagenase is used with finely chopped tissue in complete
medium
• After disaggregation, collagenase is removed by
centrifugation

25. Mechanical disaggregation
• Much more quicker than enzymatic disaggregation
• Slicing of the tissue, and collecting the cells which spill out
• Cells are passed either through the sieves or reduced mesh
or forced out through a syringe & needle or repeatedly
pipetted
• Disadvantage: Can lead to mechanical damage to the cells
sometimes
• Advantage: Provides a good yield of cells in a short time

26. Separation of viable & nonviable cells
• The separated cells which are obtained are called as the
primary cells
• They grow well and when seeded on a culture proliferate
quickly
• The ones which grow and proliferate are called as ‘adherent
primary culture’.
• The ones which do not grow are removed during the
change of medium and are called as nonviable
• Primary cultures can also be maintained in suspension by
removing the non viable cells through centrifugation

27. Slide and coverslip culture
Advantages:
• Simple and relatively inexpensive
• Cells are spread out in a manner suitable for photography
and microscopy
• Cells grown on a coverslip can be fixed and stained to make
permanent slides
Disadvantages:
• Nutrients rapidly get exhausted
• Sterility cannot be maintained for a long period
• Only small amount of tissues can be cultured

28. Single coverslip with plasma clot
• Place a drop of 50% plasma in BSS using a sterile capillary
pipette in the centre of one or more coverslips
• Transfer the explant (tissue fragment) on to the plasma drop
• Add small amount of 50% embryo extract (prepared in serum) to
the coverslip and mix before clotting starts
• On the concavity of a cavity slide place small amount of
petroleum jelly
• Invert the slide over the coverslip and apply little pressure so
that the jelly sticks to the coverslip
• Allow culture medium to clot, turn over the slide & seal the
margins using paraffin
• Label and incubate at 37°C

29. explant

30. Double coverslip with plasma clot
• It resembles the single coverslip method
• A large depression slide is used and entire preparation is
attached to it by petroleum jelly and wax
• The small coverslip used is not in contact with the cavity at
any time
Technique:
• A small drop of BSS is placed on a large coverslip (40mm)
• A smaller coverslip (22mm) is placed over BSS in the centre
of large coverslip
• Then the remaining steps are the same as followed in single
coverslip method

31. Large
Coverslip
Cavity
Small
Coverslip

32. Flask Cultures
• Carrel flask technique is used for establishment of strain
from fresh explants of tissue because it has excellent
optical properties of microscopic examination
• Polystyrene flasks can also be used
• Two kinds of flask techniques:
a. Thick clot culture: It allows rapid growth suitable for short
term cultures
b. Thin clot cultures: It can be maintained for a considerable
period

33. Carrel Flasks for tissue culture

34. Polystyrene Flasks for tissue culture

35. Advantages of flask culture:
1. The tissue can be maintained in the same flask for months
or years
2. A large no of cultures can be easily prepared
3. A large amount of tissue can be grown with large amount
of medium

36. Preparation of Flask Cultures
• D3.5 Carrel Flasks are placed in a rack with their necks
flamed and pointing to the right
• Place a drop of plasma on the flask and spread it out in a
circle
• Transfer of explant into plasma with the help of a spatula
• Fix the explant in position after the clotting and add extra
medium
• Thick clots: 1.2ml of dilute plasma, thin clots: 1.2ml of
dilute serum is added & whole thing is left for clotting
• Flasks are gassed with gas phase (5% CO2 in air)

38. Renewal of medium:
• Old fluid is drawn out with the help of a pipette
• 1.2 ml of fluid medium is added as a replacement
• The flask is gassed as above
Transfer of culture:
• The culture grown in flask is to be removed and cut into
pieces
• These pieces are then used for replantation as usual

39. Test Tube Cultures
• Feeding, patching and transfer done as in other primary
explantation techniques
• It is a cheap technique used for preparing a large no of
cultures which can be placed in racks or roller drums
• Cultures on plasma clots in tt are prepared in the same way
as in flasks
• Even without a plasma clot, the tissue can be grown on the
wall of the tt
• Suspension culture can also be developed using tt