1. Animal Cell Culture
Prepared by
Amit Shrestha
M- pharm(Pharmacology)
Mallige College Of Pharmacy

2. Institute of Animal Health and
Veterinary Biologicals
2

3. History
• History
• The 19th-century English physiologist Sydney Ringer developed salt solutions
containing the chlorides of sodium, potassium, calcium and magnesium
suitable for maintaining the beating of an isolated animal heart outside of the
body.[1] In 1885, Wilhelm Roux removed a portion of the medullary plate of
an embryonic chicken and maintained it in a warm saline solution for several
days, establishing the principle of tissue culture.[3]
Ross Granville Harrison,
working at Johns Hopkins Medical School and then at Yale University,
published results of his experiments from 1907 to 1910, establishing the
methodology of tissue culture.[4]
• Cell culture techniques were advanced significantly in the 1940s and 1950s to
support research in virology. Growing viruses in cell cultures allowed
preparation of purified viruses for the manufacture of vaccines. The injectable
polio vaccine developed by Jonas Salk was one of the first products mass-
produced using cell culture techniques. This vaccine was made possible by the
cell culture research of John Franklin Enders, Thomas Huckle Weller, and
Frederick Chapman Robbins, who were awarded a Nobel Prize for their
discovery of a method of growing the virus in monkey kidney cell cultures.
3Note:-The neural plate is a key developmental structure that serves as the basis for the nervous
system

4. Introduction:
• Cell culture is the complex process by which cells are grown under
controlled conditions, generally outside of their natural environment.
In practice, the term "cell culture" now refers to the culturing of cells
derived from multi-cellular eukaryotes, especially animal cells.
However, there are also cultures ofplants, fungi and microbes,
including viruses, bacteria and protists. The historical development
and methods of cell culture are closely interrelated to those of
tissue culture and organ culture.
4

5. 5
Purpose
Cell culture is essential for growth of viruses because they
are obligate intracellular parasites; they cannot replicate in
any cell-free medium, and they require living cells from a
suitable host within which to multiply. Animals such as mice
and embryonated avian eggs may be used for the propagation
of viruses, but for various reasons (time, cost, uniformity,
ease of handling, animal welfare, etc.), the propagation of
most viruses in cultured living cells is the method of choice
today.
 Cell culture is also used in recombinant DNA technology
and many in vitro assays.

6. Isolation
• Cells can be isolated from tissues for ex vivo culture in several ways.
Cells can be easily purified from blood; however, only the white cells
are capable of growth in culture. Mononuclear cells can be released
from soft tissues by enzymatic digestion with enzymes such as
collagenase, trypsin, or pronase, which break down the
extracellular matrix. Alternatively, pieces of tissue can be placed in
growth media, and the cells that grow out are available for culture.
This method is known as explant culture.
Collagenase Collagenases are enzymes that break the peptide bonds in collagen.
Pronase is a commercially available mixture of proteinases isolated from the extracellular fluid of Streptomyces griseus.
Activity extends to both denatured and native proteins leading to complete or nearly complete digestion into
individual amino acids.
tripsin  is a serine protease found in the digestive system of many vertebrates, where it hydrolyses proteins.
6

7. 7
Cell Cultures/ Dispersion
Cultures
• Types
• Primary cell cultures
• Diploid cell cultures
• Continuous cell lines

8. 8
Primary cell cultures.
When cells are taken freshly from animal tissue and placed in
culture, the cultures consist of a wide variety of cell types, most
of which are capable of very limited growth in vitro, usually
fewer than ten divisions. These cells retain their diploid
karyotype, i.e., they have the chromosome number and
morphology of their tissues of origin. They also retain some of
the differentiated characteristics that they possessed in
vivo. Because of this, these cells support the replication of a
wide range of viruses. Primary cultures derived from monkey
kidneys, mouse fetuses, and chick embryos are commonly used
for diagnostic purposes and laboratory experiments.
a cell line derived directly from the parent tissue.
Cells in primary culture have the same karyotype
and chromosome number as those in the original
tissue.
Can be subculture only one or twice e.g-primary
monkey, baboon kidney

9. 9
2. Diploid cell strains.
Which are derived from human fetal tissue and can be
subcultured 20-50 times e.g-human diploid fibroblast such as
MRC-5(human fetal lung fibroblast establish from normal lung
tissue of 14 week old male foetus)
Some primary cells can be passed through secondary and
several subsequent subcultures while retaining their original
morphological characteristics and karyotype. Subcultures will
have fewer cell types than primary cultures. After 20 to 50
passages in vitro, these diploid cell strains usually undergo a
crisis in which their growth rate slows and they eventually die
out. Diploid strains of fibroblasts derived from human fetal
tissue are widely used in diagnostic virology and vaccine
production.

10. 3. Continuous cell lines. Derived from tumors of human or animals
e.g- Vero, Hep2
Certain cultured cells, notably mouse fetal fibroblasts, kidney cells from various
mammalian species, and human carcinoma cells, are able to survive the growth crisis
and undergo indefinite propagation in vitro. After several passages, the growth rate
of the culture slows down; then isolated colonies of cells begin to grow more rapidly
than diploid cells, their karyotype becomes abnormal (aneuploid), their morphology
changes, and other poorly understood changes take place that make the cells
immortal. The cells are now "dedifferentiated," having lost the specialized
morphology and biochemical abilities they possessed as differentiated cells in vivo.
Continuous cell lines such as KB and HeLa, both derived from human carcinomas,
support the growth of a number of viruses. These lines and others derived from
monkey kidneys (e.g., Vero), mouse fetuses (L929), and hamster kidneys (BHK) are
widely used in diagnostic and experimental virology. Continuous cell lines have been
established from many types of vertebrate and invertebrate animal tissues and are
available from the American Type Culture Collection.

11. 11
Primary Cell Culture
• Sterilization of
Cubicle
• Preparation of Media
• Preparation of
dispersion solution
• Preparation of
Monolayers

12. 12
Sterilisation of Cubicle (compartment)
• Fumigation
• 35 ml of formaline and 17.5 g of potassium
permanganate per 100 cubic feet of air space
• Keep the cubicle closed for 65 hours
• Working Surface Sterilization
• Mop with 5% phenyl/ 1/40 solution of dettol
• Use 1 % Copper sulphate for fungal disinfection

13. 13
Cell Culture Media
• Balanced Salt Solution (BSS)
• Glucose
• Amino acids
• Vitamins
• Sodium bicarbonate
• Phenol red
• Fetal Calf Serum

14. 14
Culture Media
 Prepare stock antibiotic solution
 Weigh the required amount of Ready made Balanced Salt Medium and
add sterile water
 Add the required amount of antibiotic stock solution into BSS
 Adjust the pH of the solution using 2.8% / 7.5% Sodium bicarbonate
or 0.1 N Hydrochloric acid to 7.4
 Sterilize using 0.2 µ Seitz filter applying positive pressure.
 Add sterile heat inactivated fetal calf serum to make a final
concentration of 15%
 Store at 4o
C.

15. 15
Dispersion solution
• Weigh Calcium Magnesium free Buffer premix and add sterile water
• Add 0.1 % w/v Trypsin powder
• Adjust pH to 7.4 to 7.6
• Sterilize by Filtration through Seitz filter
• Store at - 20o
C

16. Primary Cell Culture
Chicken Embryo Fibroblast

17. 17
Materials
 Ten to Eleven days Embryonated Chicken Eggs
 Forceps, Scissors, Rubber bulbs
 Pipettes, Beakers, Centrifuge tubes, Flasks, Petri dishes, Trypsinisation flask
 Seitz filter assembly, Vacuum/air pump
 Mono-pan analytical balance
 pH meter
 Magnetic stirrer, Teflon coated magnetic stirrer, Cyclo-mixer
 Refrigerated Centrifuge
 Laminar Air Flow Apparatus

18. 18
Preparation of Chicken Embryo Fibroblast MonolayerPreparation of Chicken Embryo Fibroblast Monolayer
1. Sterilize Outer shell of the Eggs
2. Remove the Shell, Shell membrane and CAM
3. Wash embryo with BSS
4. Remove Head, Limbs and Internal Organs
5. Wash several times with BSS
6. Cut into small pieces of ~ 1mm thickness
7. Wash in BSS
8. Trypsinize /.
9. Filter with a cheese cloth
10. Centrifuge and pack cells
11. Resuspend in growth medium
12. Repeat step 10 and 11, two more times
13. Count the cells and adjust to a concentration of 10 6
cells per ml
14. Seed Culture tubes/ flasks/ Bottles and Incubate at 37 o
C for 48 to 72
hours.

19. 19
procedure
• Disinfect the surface of the egg
over the air sac. With scissors
or the blunt-end of a forceps,
break the shell over the air sac
• Opening of egg through a
circular incision around the
airsac
•10- to 12-day-old embryonated eggs

20. 20
• Transfer the live
embryo to a petri dish
containing media

21. 21
• Collect more embryos

22. 22
• Remove the
appendages and
viscera

23. 23
• Collect the remaining
portion

24. 24
• Cut the embryo in to
small parts (about 1
mm)

25. 25
• Trypsinise in a flask
Add 10 ml of sterile warm
trypsin solution to fragments .

26. 26
• Keep in magnetic
stirrer for about 30
minutes

27. 27
• Centrifuge the cell
suspension

28. 28

29. 29
• Wash the cells 3 more
times.

30. 30
• Mix well in cyclo
mixer

31. 31
• Incubate for about 30
min at 4o
c

32. 32
• Dispense in cell
culture bottles

33. 33
• Incubate at 37o
c in a
CO2incubator

34. 34
• Preservation of Cultured Cells by Freezing
• 50 to 80% of the cells of a healthy culture will
survive freezing.
• . Cells must be stored at -70o
C or lower

36. 36
Ex vivo (Latin: out of the living) means that which takes place outside an organism. In science, ex vivo refers
to experimentation or measurements done in or on living tissue in an artificial environment outside the
organism with the minimum alteration of the natural conditions. The most common "ex vivo" procedures
involve living cells or tissues taken from an organism and cultured in a laboratory apparatus, usually under
sterile conditions and no alterations done for a few hours up to 24 hrs. Experiments lasting longer than this
using living cells or tissue are typically considered to be "in vitro". Ex-vivo conditions allow experimentation
under highly controlled conditions impossible in the intact organism, albeit at the expense of looking at the
tissue in its "natural" environment. One widely performed ex vivo study is the chick chorioallantoic
membrane (CAM) assay. In this assay, angiogenesis is promoted on the CAM membrane of a chick embryo
outside the organism (chicken). Ex vivo studies are usually performed in vitro, although the use of these two
terms is not synonymous.
In vitro (Latin for within the glass) refers to the technique of performing a given procedure in a controlled
environment outside of a living organism. Some may argue that in vitro refers to a process that is created in a
"test tube"; however, Robert Kail and John Cavanaugh on page 58 in the 4th edition of Human Development:
A Life-Span View cite that in fact the process is contained in a petridish. Many experiments in cellular
biology are conducted outside of organisms or cells; because the test conditions may not correspond to the
conditions inside of the organism, this may lead to results that do not correspond to the situation that arises in
a living organism. Consequently, such experimental results are often annotated with in vitro, in
contradistinction with in vivo.
What's the difference between ex vivo and in vitro?
Or is it the same thing

37. Institute of Animal Health and
Veterinary Biologicals
37
karyotype
the chromosomal constitution of the cell nucleus; by extension, the photomicrograph of
chromosomes arranged

38. 38
Diploid cultures vs. continuous cell lines
What is the difference between a diploid culture and a
continuous cell line?
-Diploid cultures have a finite lifespan. They can undergo a
maximum of 20-80 PDLs (Population Doubling Level) before
they senesce. Normal human cells, such as human skin
fibroblasts, are one example of diploid cells.
-Continuous cell lines are immortalized cell lines with an infinite
lifespan. These usually either come from tumor tissue or have
been deliberately immortalized or transformed. However, many
rodent cell lines spontaneously transform.

39. 39
Formulations of Media and Solutions
Saline A
Ingredient g/l
NaCl 8.0
KCl 0.4
NaHCO3 0.35
Glucose 1.0
Phenol red 0.05
Add distilled H2O to 1 liter. Filter sterilize. Saline A is usually prepared as a
10X solution and stored at -20o
C.
Saline-Trypsin-EDTA (STE)
Ingredient g/l
Trypsin (1:250) 0.5
EDTA (disodium salt) 0.2
NaCl 8.0
KCl 0.4
NaHCO3 0.35
Glucose 1.0
Phenol red 0.05
Add distilled H2O to 1 liter. Dissolve trypsin first by slowly stirring in ~200
ml H2O (avoid denaturation). Add remaining ingredients and filter sterilize.