Animal cell culture techniques , and their introduction

2. Primary culture and cell line
 A primary culture refers to the starting culture of cells,
tissues or organs, taken directly from an organism.
 It is the initial culture before the first subculture.
 Cell line is used for the propagation of culture after the
first subculture
 Primary cultures are usually prepared from large tissue
masses.
 Therefore it may contain a variety of differentiated
cells eg. Fibroblasts, lympocytes, macrophages,
epithelial cells
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3. Characteristics of primary culture
1. Embryonic tissues rather than adult tissues are
preferred for primary cultures. As these cells can be
easily disaggregated and yield more viable cells,
besides rapidly proliferating in vitro
2. The quantity of the cells used in primary culture
should be higher since survival rate is lower
3. The tissues should be processed with minimum
damage to cells for use in primary culture.
4. Proper selection of media is suggested
5. It is necessary to remove the enzymes used for
disaggregation of cells by centrifugation
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4. Techniques for primary culture
1. Mechanical disaggregation
2. Enzymatic disaggregation
3. Primary explant technique
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5. Mechanical disaggregation
 For soft tissues such as spleen, brain, embryonic liver,
soft tumor this technique is used.
 It involve careful chopping or slicing of tissue into
pieces and collection of spill out cells. The cells are
collected by any one of the following methods-
 Pressing the tissue pieces through a series of sieves
with gradual reduction in the mesh size
 Forcing the tissue fragments through a syringe and
needle
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6. Enzymatic disaggregation
 It is the mostly used technique when high recovery of
cells is required from a tissue.
 Enzymatic disaggregation can be carried out by using
trypsin, collagenase or some other enzymes.
 The term trypsinization is commonly used for
disaggregation by trypsin. Two techniques are there
1. Warm trypsinization
2. Cold trypsinization
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7.  Collagenase disaggregation has been successfully used
for human brain, lung and several other epithelial
tissues, besides various human tumors and other
animal tissues.
 Addition of another enzyme hyaluronidase , trypsin
promotes disaggregation
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8. Primary explant technique
 The tissue in basal salt solution is finely chopped and
washed by settlings. The basal salt solution is then
removed.
 The tissue pieces are spread evenly over the growth
surface. After addition of appropriate medium,
incubation is carried out for 3-5 days.
 Then the medium is changed at weekly intervals until
a substantial out growth of cells is observed.
 Now the explants are removed and transferred to a
fresh culture vessel
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9. Cell lines
 The term cell line refers to the propagation of culture
after the first subculture.
 In other words, once the primary culture is
subcultured it becomes a cell line. A given cell line
contains several cell lineages of either similar or
distinct phenotypes.
 It is possible to select a particular cell lineage by
cloning or physical cell separation or some other
selection method.
 Such a cell line derived by selection or cloning is
referred to as cell strain.
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10. Difference between finite and continuous cell line
PROPERTY FINITE CELL LINE CONTINUOUS CELL
LINE
GROWTH RATE SLOW FAST
Mode of growth monolayer Suspension or monolayer
yield low High
transformation normal Immortal, tumorigenic
Anchorage dependence yes No
Contact inhibition yes no
Cloning efficiency Low High
Serum requirement High High
Markers Tissue specific Chromosomal, antigenic
or enzymatic
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11. Organ culture
 In vitro culture and growth of organs or parts thereof
in which their various tissue components eg.
Parenchyma and stroma are preserved both in term of
their structure and function, so that culture organ
resembles closely with the organ in vivo then called
organ culture.
 New growth is in the form of differentiated structures
eg. Glandular structures in case of glands, bronchioles
in case of lungs
 Cultured organ should retain their physiological
features
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12. Techniques of organ culture
 Following are some techniques of organ culture:
1. Plasma clot or watch glass method
2. Raft method
3. Combination of 1 and 2
4. Agar gel
5. Grid method
6. Cyclic exposure to medium and gas phase
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13. Plasma clot or watch glass method
 Explant is placed and cultured on suitably prepared plasma
clot kept in watch glass.
 Plasma clot is prepared by mixing 3 drops of chicken
plasma and 1 drop of chick embryo extract (50 %)
 One or two such watch glasses are kept in Petri dishes lined
with cotton wool or moist filter paper to minimize
evaporation of clot. May or may not be closed with lid and
sealed with paraffin wax.
 Petri dish is then incubated at 37.5 0C. Fresh clots have
been provided every 2 to 3 days for avian tissues and every 3
to 4 days for mammalian tissues.
 Maximow single slide technique: it is the modification of
the above mentioned method
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15.  Advantages:
1. In expensive
2. Permits light microscope hence suitable to study hair
growth, fetal mouse skin differentiation
 Disadvantages:
1. Clot liquefies in vicinity of explants so explants can
become immersed in the medium
2. Short duration of culture (Less than 4 weeks)
3. Biochemical analysis is not possible due to
complexity of medium
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16. Applications
 To study morphogenesis in embryonic organ
rudiments
 To study action of hormones, vitamins, carcinogens on
adult mammalian tissues.
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17. Raft method
 In this method explants are placed on the raft of glass/
lens/paper/ rayon acetate, which is then floated on
serum in watch glass.
 Four or more explants are placed on single raft.
Floatability of lens paper/rayon acetate is enhanced by
it treatment with silicone
 COMBINATION METHOD:
 In this method explants are first placed on the raft of
glass or suitable material then place on plasma clot.
This prevents sinking of explant into liquefied plasma
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19. Agar gel method
 Medium is gelled with 1 % agar.
 Gels are then kept in embryological watch glass and
explant is then transferred on surface of agar, sealed
with paraffin wax.
 Sub cultured into fresh agar gels every 5 to 7 days.
Explants can be examined with stereoscopic
microscope .
 The technique is used to study many developmental
aspects of normal organs as well as tumors
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21. Grid method
 Grid utilizes 25 mm X25 mm pieces of suitable wire
mesh or perforated stainless steel sheet, whose edges
are about to form 4 legs of about 4 mm height.
 Skeletal tissues are kept directly on grid while soften
tissues are firstly kept on raft and then on grid.
 Then grid is kept in culture chamber filled with fluid
medium up to the grid.
 Chamber is flushed with O2/CO2 to maintain
required O2 concentration.
 It is used to study growth and differentiation of adult
and embryonic tissues.
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23. Cyclic exposure to medium and gas
phase
 Useful for long term (4-5 months) culture of human
adult tissues. 2 to 8 explants per dish are attached at
bottom of plastic culture dish and covered with fluid
medium.
 Dishes are enclosed in chamber containing suitable
gas mixture, mounted on rocker platform for cyclic
exposures of medium and gas.
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24. Advantages:
 Explant remain comparable, so suitable to study
physiological studies
 May replace whole animals in experimentation, as results
from them are easy to interpret.
 APPLICATIONS:
 To study pattern of growth, differentiation, development of
organ rudiments
 To study action of drugs and carcinogens on organs
 Development of tissues called tissue engineering for
implantation in patients eg. Artificial cartilage, artificial
skin.
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