1. Cell Culture and handling of the cell
lines
By
Shakira Ghazanfar
PhD Scholar
PIASA
20-04-2011

2. Contents
• Cell Culture
• Cell Lines
• Continues Cell Lines
• Subculture
• Handling of cell lines
• Cryopreservation of cell lines

3. Introduction
• Cell culture is the process by which
prokaryotic, eukaryotic or plant cells are grown
under controlled conditions. But in practice it
refers to the culturing of cells derived from
animal cells.
• Cell culture was first successfully undertaken
by R. Harrison in 1907

4. Cell Culture
Definition:
In vitro cultivation of cells at defined
temperature using an incubator &
supplemented with a medium containing cell
nutrients & growth factors is known as Cell
Culture.

5. Cell Culture
• Tissues Individual cells
trypsin & mechanical shaking
• Cells are washed, counted & suspended in a
growth medium.
• Growth medium – Minimum Essential Medium
(MEM): essential aminoacids, vitamins, salts,
glucose & bicarbonate in 5% CO2 with 5% fetal calf
or calf serum, antibiotics & phenol red indicator

6. MAIN TYPES OF CELL CULTURE
according to the cultivation period (and survival in culture)
• Routinely used for growing viruses
• Classified into 3 types:
– Primary cell culture – normal cells taken from body &
cultured, limited growth
1. Chick embryo fibroblast
2. Human amnion cell culture
– Diploid cell strains – cells of single type (fibroblast cells)
that can be sub cultivated for limited number of times,
mostly 50
1. WI-38: human embryonic lung cell
– Continuous cell lines – indefinite subcultivtion
1. HeLa: Human Ca of cervix cell line
2. HEP-2: Human epithelioma of larynx
3. Vero: Vervet monkey kidney

7. Classification
1. Primary Cultures
– Derived directly from tissue and cultured either as
• Outgrowth of tissue in culture
• Cut into single cells (by enzymatic digestion or
mechanical dispersion)
– Advantages:
• usually retain many different characteristics of the
cell in vivo
– Disadvantages:
• initially heterogeneous but later become dominated
by fibroblasts.
• the preparation of primary cultures is labor intensive
• can be maintained in vitro only for a limited period of
time.

8. Classification
2. Continuous Cultures
– derived from subculture (or passage, or transfer) of
primary culture
• Subculture = the process of dispersion and re-culture
the cells after they have increased to occupy all of the
available substrate in the culture
– can be serially propagated in culture for several passages
Types:
– There are two types of continuous cultures
• Cell lines
• Continuous cell lines

9. Types of continuous cultures
1) Cell lines
• short time life, die after approximately thirty cycles of
division
• usually diploid and maintain some degree of
differentiation.

10. Types of continuous cultures
• 2. Continuous cell lines :Most cell lines grow for a
limited number of generations after which they ceases
• Cell lines which either occur spontaneously or induced
or chemically transformed into Continous cell lines
• Characteristics of continous cell lines
Smaller, More Rounded, Less Adherent With a Higher
Nucleus /Cytoplasm Ratio
Fast growth
Reduced serum and anchorage dependence and grow
more in suspension conditions

11. Common cell lines
• Human cell lines
• -MCF-7 breast cancer
• HL 60 Leukemia
• HEK-293 Human embryonic kidney
• HeLa Henrietta lacks
• Primate cell lines
• Vero African green monkey kidney epithelial cells
• Cos-7 African green monkey kidney cells
• And others such as CHO from hamster, sf9 & sf21 from
insect cells

12. American Type Culture Collection.

13. Practical

14. Design and Equipment for the Cell
Culture Laboratory
• 1. Laboratory design- in a safe and efficient
manner
• 2. safety cabinets

15. Cell Culture Room
Close Small AC/Heater
ROOM FOR ANIMAL CELL CULTURE
sterile conditions (disinfection of the work surfaces,
microbiological safety cabinets)
Hood

16. Basic equipments used in cell culture
• Laminar cabinet-
• Incubation- Temperature is 37 C for mammalian cells, Co2 2-
5% & 95% air at 99% relative humidity.
• Refrigerators- Liquid media kept at 4 C, enzymes (e.g. trypsin)
& media components (e.g. glutamine & serum) at -20 C
• Microscope- An microscope with 10x to 100x magnification
• Cell culture tubes-
• Autoclave-

17. Laminar- flow hood

18. Laminar- flow hood
• The working environment is protected from dust
and contamination by a
• constant, stable flow of filtered air
• Two types:
 Horizontal, airflow blow from the side facing
you, parallel to the work surface, and is not
circulating;
 Vertical, air blows down from the top of the
cabinet onto the work surface and is drawn
through the work surface and recalculated

19. Laminar- flow hood

20. Laminar- flow hood
• Routine maintenance checks of the primary
filters are required (every 3-6 months).
• They might be removed and discarded or
washed in soap and water.
• Every 6 months the main high efficiency
particulate air (HEPA) filter above the work
surface should be checked for airflow and
hole

21. Precaution Measure Inside The Hood
Incubator Gloves are
always worn
The pipettes are disposable
Lab coat

22. Cell Culture Incubator

23. Cell Culture Incubator
• It requires a controlled atmosphere with high
humidity and super controlled of CO2.
• The incubator should be large enough, like 50-
200 l have forced air circulation
• Temperature should be + 0.5oC
• It should be stainless steel, and easily cleaned

24. Autoclave
A simple autoclave may be sufficient.

25. Refrigerators and Freezers
• 4C
•-20C
•-80C
• Liquid N2 tank

26. Microscope
Large stage so
plates and flasks
can be used.
Magnification; 5X,
10X, 20X, 40X

27. Culture vessels and medium
for animal cell culture
Culture vessel
Culture Media
removes a tray
of stem cell
cultures from an
incubator

28. Anaerobic Jar

29. Multiwell plates

30. Cell Culture Bottles /
Tubes

31. Heating of media on heater

32. Centrifuge

33. Culture media
• Choice of media depends on the type of cell being cultured
• Commonly used Medium are GMEM, EMEM,DMEM etc.
• Media is supplemented with antibiotics viz. penicillin, streptomycin etc.
• Prepared media is filtered and incubated at 4 C
• Culture Medium
– Basic Composition of Media
• Inorganic salts
• Trace elements
• Buffering systems
– pH range should be 7,2 – 7,4
• Carbohydrates
• Aminoacids
• Vitamins
• Proteins and peptides
• Fatty acids and lipids
• Serum – fetal bovine serum
• Antibiotics

34. Before use
• Ultraviolet lights are used to sterilize
the air and exposed work surfaces in
laminar flow cabinets between use.
• Detergent
• 70% alcohol

35. Culture Morphology
• Suspension (as single cells or small free-floating
clumps)
• or as a monolayer that is attached to the cell culture
flask.
• The form taken by a cell line reflects the tissue from
which it was derived ...
• From blood tend to grow in suspension
• From solid tissue (lungs, kidney) tend to grow as
monolayer's.
• Attached cell lines can be classified as endothelial,
epithelial, or fibroblasts and their morphology tell the
area within the tissue of origin

37. Anchorage dependent or independent
• Cell lines derived from normal tissues are
considered as anchorage-dependent
grows only on a suitable substrate.
• Suspension cells are anchorage-
independent e.g. blood cells

38. Adherent cells
• Cells which are anchorage dependent
• Add enough trypsin/EDTA to cover the
monolayer
• Incubate the plate at 37 C for 5 mts
• Tap the vessel from the sides to dislodge the
cells
• Add complete medium to dissociate and dislodge
the cells
• with the help of pipette which are remained to
be adherent
• Add complete medium depends on the
subculture
• requirement either to 75 cm or 175 cm flask

39. Suspension cells
• Easier to passage as no need to detach them
• As the suspension cells reach to confluency
• Aseptically remove 1/3rd
of medium
• Replaced with the same amount of pre-
warmed medium

40. confluency
• Once the available substrate surface is
covered by cells (a confluent culture) growth
slows & ceases.

41. hep 3B - 70% confluency
Gaps

42. 100% confluency

43. After 24 h

44. confluency
• Cells to be kept in healthy & in growing state
have to be sub-cultured or passaged , It’s the
passage of cells when they reach to 80-90%
confluency in flask/dishes/plates
• Enzyme such as trypsin, collagenase in
combination with EDTA breaks the cellular
glue that attached the cells to the surface

45. Cell culture contaminants
• Two Types:
• Chemical-difficult to detect caused by
endotoxins, plasticizers, metal ions or traces
of disinfectants that are invisible
• Biological-cause visible effects on the culture
they are mycoplasma, yeast, bacteria or
fungus or also from cross-contamination of
cells from other cell lines

48. Safety

49. Why sub culturing.?
• Once the available substrate surface is covered by
cells growth slows & ceases.
• Cells to be kept in healthy & in growing state have to
be sub-cultured or passaged
• It’s the passage of cells when they reach to 80-90%
confluency in flask/dishes/plates
• Enzyme such as trypsin, collagenase in combination
with EDTA breaks the cellular glue that attached the
cells to the surface

50. Rules for working with cell culture
Never use contaminated material within a sterile area
Use the correct sequence when working with more than one
cell lines.
• Diploid cells (Primary cultures, lines for the production of
vaccines etc.)
• Diploid cells (Laboratory lines)
• Continous, slow growing line
• Continous, rapidly growing lines
• Lines which may be contaminated
• Virus producing lines

51. Cell Culture Used
Areas where cell culture technology is currently playing a
major role.
– Model systems for
Studying basic cell biology, interactions between disease
causing agents and cells, effects of drugs on cells,
process and triggering of aging & nutritional studies
– Toxicity testing
Study the effects of new drugs
– Cancer research:
Study the function of various chemicals, virus & radiation to
convert normal cultured cells to cancerous cells

52. • Virology
Cultivation of virus for vaccine production, also used to
study there infectious cycle.
• Genetic Engineering
Production of commercial proteins, large scale
production of viruses for use in vaccine production e.g.
polio, rabies, chicken pox, hepatitis B & measles
• Gene therapy
Cells having a functional gene can be replaced to cells
which are having non-functional gene
Cell Culture Used

53. Advantage
Viral Vaccines:
Continuous Cell Lines
Cell Substrate Live Vaccines Inactivated
Vaccines
African green monkey kidney:
Vero Poliovirus Poliovirus
(Europe) (U.S.)

54. Disadvantages
• Cell characteristics can change
• Cell can adapt to different nutrients
• If mixed cells cultivated some types will
disappear.
• Activity of enzymes may altered by
environment.

55. Cryopreservation
Definition
Cryopreservation is a process where cells or whole
tissues are preserved by cooling to low sub-zero
temperatures, such as, −196 °C (the boiling point of
liquid nitrogen).
Liquid Nitrogen

56. Principles of cryopreservation
• Water in cell: Around 90% of water is free (water)
while the remaining 10 % bounds to other molecular
components of the cell (proteins, lipids, nucleic acids
and other solutes). This water does not freeze and
called hydrated water
– Removal of water is necessary during freezing to avoid ice
crystal formation, dehydration is limited to the free water
– Removal of hydrated water could have adverse effect on the
cell viability and the molecular function (freezing injuries)

57. Cryopreservation of Cell Lines
• The aim of cryopreservation is to enable stocks of cells to be
stored to prevent the need to have all cell lines in culture at
all times. It is invaluable when dealing with cells of limited life
span.
The other main advantages of cryopreservation are:
• Reduced risk of microbial contamination
• Reduced risk of cross contamination with other cell lines
• Reduced risk of genetic drift and morphological changes
• Work conducted using cells at a consistent passage number
(refer to cell banking section below)
• Reduced costs (consumables and staff time)

58. Successful Cryopreservation of cell lines
• There has been a large amount of developmental work
undertaken for successful cryopreservation of a wide
variety of cell lines of different cell types.
• The basic principle of successful cryopreservation is a
• Slow freeze
• Quick thaw.
• Cell lines should be cooled at a rate of –1ºC to –3ºC
per minute and thawed quickly by incubation in a 37ºC
water bath for 3-5 minutes..

59. • A high concentration of serum/protein (>20%)
should be used. In many cases serum is used at
90%.
• Use a cryoprotectant such as dimethyl sulphoxide
(DMSO) or glycerol to help protect the cells from
rupture by the formation of ice crystals.
• The most commonly used cryoprotectant is DMSO
at a final concentration of 10%.
Successful Cryopreservation of cell lines

60. Vocabulary
 Cell-Culture-In vitro cultivation
of cells at defined temperature
using an incubator &
supplemented with a medium
containing cell nutrients & growth
factors is known as Cell Culture.
 Culture – growth of
microorganisms in a special
medium; the process of growing
microorganisms in the laboratory.
 Anchorage-dependent- Cell
lines derived from normal tissues
are considered as anchorage-
dependent grows only on a
suitable substrate
 Primary Culture :Cells when
surgically or enzymatically
removed from an organism and
placed in suitable culture
environment will attach and grow
are called as primary culture
 Cell Lines: Sub culturing of
primary cells leads to the
generation of cell lines
 Cell Strain :Lineage of cells
originating from the primary
culture is called a cell strain
 Anchorage-independent-
Suspension cells are anchorage-
independent e.g. blood cells

61. Next practical
• Transformation
• See you next week!

62. Thanks