a detailed review over the most important technique of animal cell culture used in todays era of vaccine development

1. By:
Ankit SharmA
M.Pharma 2nd Sem
Pharmacology
Bhupal Noble’s University
Udaipur (Raj.)

5.  First one is the use of Antibiotics that help in
reducing contaminations
 Second one is use of Trypsin to remove
adherent cells from vessel
 Third one is use of chemically defined medium
for enhancement in result

6. Cell culture refers to the removal of cells from an
animal or plant and their subsequent
growth in a favourable artificial environment. The
cells may be removed from the tissue
directly and disaggregated by enzymatic or
mechanical means before cultivation, or they
may be derived from a cell line or cell strain that
has already been already established

7. o Cell culture hood (i.e., laminar-flow hood or
biosafety cabinet)
o • Incubator (humid CO2 incubator recommended)
o • Water bath
o • Centrifuge
o • Refrigerator and freezer (–20°C)
o • Cell counter (e.g., Countess® Automated Cell
Counter or hemacytometer)
o • Inverted microscope
o • Liquid nitrogen (N2) freezer or cryostorage
container
o • Sterilizer (i.e., autoclave)

10.  Two types of media used in culture technique:
 1. Natural Media 2. Artificial Media
 Media is generally defined as the medium
which contains all the necessary elements
requires for the growth of cell or tissue culture.

11. pH:
 Most normal mammalian cell lines grow well at
pH 7.4, and there is very little variability
 among different cell strains. However, some
transformed cell lines have been shown to
 grow better at slightly more acidic environments
(pH 7.0–7.4), and some normal fibroblast
 cell lines prefer slightly more basic environments
(pH 7.4–7.7). Insect cell lines such as Sf9
 and Sf21 grow optimally at pH 6.2

12. 
 Carbon Dioxide
 The growth medium controls the pH of the culture and
buffers the cells in culture against changes in the pH.
Usually, this buffering is achieved by including an
organic or CO2-bicarbonate based buffer. Because the
pH of the medium is dependent on the delicate balance
of dissolved carbon dioxide (CO2) and bicarbonate
changes in the atmospheric CO2 can alter the pH of the
medium. Therefore, it is necessary to use exogenous
CO2 when using media buffered with a CO2-
bicarbonate based buffer,especially if the cells are
cultured in open dishes or transformed cell lines are
cultured at high concentrations.

13. 
 TEMPERATURE
 The optimal temperature for cell culture largely
depends on the body temperature of the host from
which the cells were isolated, and to a lesser
degree on the anatomical variation in temperature
(e.g., temperature of the skin may be lower than
the temperature of skeletal muscle). Overheating is
a more serious problem than underheating for cell
cultures; therefore, often the temperature in the
incubator is set slightly lower than the optimal
temperature. 36 -37’C optimal for growth.

14.  (1) Acquisition of the sample
 (2) Isolation of the Tissue
(3) Dissection and/or disaggregation
 (4)Culture after seeding into the culture vessel

15.  An attempt should be made to sterilize the site of the
 resection with 70% alcohol if the site is likely to be
 contaminated (e.g., skin). Remove the tissue aseptically
 and transfer it to the tissue culture laboratory in dissection
 BSS (DBSS) or transport medium (see Appendix I) as soon
 as possible. Do not dissect animals in the tissue culture
 laboratory, as the animals may carry microbial
contamination.
 If a delay in transferring the tissue is unavoidable, it can be
 held at 4◦C for up to 72 h, although a better yield will
usually
 result from a quicker transfer.

16.  Tissue can be isolated from any of these 3
means:
 1. Mouse Embryo
 2. Chick Embryo
 3. Human Biopsy Material

17.  Material Required:
 Sterile:
 DBSS: Dissection BSS (BSS with a high
concentration of antibiotics; see Appendix I) in 25-
to 50-mL screw-capped tube or universal container
 BSS, 50 mL in a sterile beaker (used to cool
 instruments after flaming)
 Petri dishes, 9 cm
 Pointed forceps
 Pointed scissors

18.  Kill the mouse by cervical dislocation and swab
the ventral surface liberally with 70% alcohol
 Tear the ventral skin transversely at themedian
line just over the diaphragm and, grasping the
skin on both sides of the tear, pull in opposite
directions to expose the untouched ventral
surface of the abdominal wall
 Cut longitudinally along the median line of the
exposed abdomen with sterile scissors, revealing
the viscera

19.  Dissect out the uteri into a 25-mL or 50-mL screw-
capped vial containing 10 or 20 mL DBSS
 Dissect out the embryos:
(a) Tear the uterus with two pairs of sterile forceps,
keeping the points of the forceps close together to
avoid distorting the uterus and bringing too much
pressure to bear on the embryos
(b) Free the embryos from the membranes and
placenta and place them to one side of the dish to
bleed.
 Transfer the embryos to a fresh Petri dish.

23.  Cells when surgically or enzymatically remove from
an organism and placed in suitable culture
environment will attach and grow are called as
“Primary Culture”
 These cells have a finite life span
 These culture has a very heterogenous population of
cells
 Subculturing of these cells leads to generation of cell
lines
 Cell lines have limited life span,they passage several
times before they becomes senescent
 Lineage of cells obtained from primary culture are
called as CELL STRAIN

24.  Several techniques are employed for this
culture:
 1. Fine dissection (Primary Explant)
 2. Mechanical Disaggregation
 3. Enzymatic Disaggregation

27.  It can be achieved by any one of these 3
methods:
 1. Warm trypsin
 2. Cold Trypsin
 3. Collagenase

32.  The need to subculture a monolayer is
determined by the following criteria:
 1. Density of Culture
 2. Exhaustion of Medium
 3. Time since last subculture
 4. Requirment for other procedures

34.  The best method for cryopreserving cultured cells
is storing them in liquid nitrogen in complete
medium in the presence of a cryoprotective agent
such as dimethylsulfoxide (DMSO).
 Cryoprotective agents reduce the freezing point of
the medium and also allow a slower cooling rate,
greatly reducing the risk of ice crystal formation,
which can damage cells and cause cell death.
 DMSO is known to facilitate the entry of organic
molecules into tissues. Handle reagents containing
DMSO using equipment and practices appropriate
for the hazards posed by such materials. Dispose
of the reagents in compliance with local
regulations.

35.  Freeze your cultured cells at a high conc. and at
as low a passage number as possible. Make
sure that the cells are at least 90% viable before
freezing. Note that the optimal freezing
conditions depend on the cell line in use.
 Freeze the cells slowly by reducing the temp. at
approx. 1°C /minute using a controlled rate
cryo-freezer. Always use the recommended
freezing medium. The freezing medium should
contain a cryoprotective agent such as DMSO
or glycerol. Store the frozen cells below –70°C;
frozen cells begin to deteriorate above –50°C

38.  Demonstration of the absence of cross-contamination
 Confirmation of the species of origin
 Correlation with the tissue of origin, which comprises the
following characteristics:
a) Identification of the lineage to which the cell belongs
b) Position of the cells within that lineage
 Determination of whether the cell line is transformed or
not
 Identification of specific cell lines within a group from
the same origin, selected cell strains, or hybrid cell lines,
all of which require demonstration of features unique to
that cell line or cell strain

39.  Areas where this technique is playing a major
role:
 Model systems for studying basic cell biology,
interaction between diseases causing agents
and cells
 Toxicity testing
 Cancer research
 Gene therapy and genetic engineering
 Virology