Cell culture introduction

introduction to cell culture with laboratory instruments used

1. Cell culture
By Dr. Saba Ahmed
University of Sargodha
M phil Pharmacology

2. *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
Ross Harrison in 1907
*Roux in 1885 for the first time maintained
embryonic chick cells in a cell culture
Introduction

3. *First development was the use of antibiotics which
inhibits the growth of contaminants.
*Second was the use of trypsin to remove adherent
cells to subculture further from the culture vessel
*Third was the use of chemically defined culture
medium.
Major development’s in cell culture
technology

4. 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

Why is cell culture used for?

5. *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
Contd….

6. *In vitro cultivation of organs, tissues & cells at defined
temperature using an incubator & supplemented with a
medium containing cell nutrients & growth factors is
collectively known as tissue culture
*Different types of cell grown in culture includes
connective tissue elements such as fibroblasts, skeletal
tissue, cardiac, epithelial tissue (liver, breast, skin,
kidney) and many different types of tumor cells.

Tissue culture

7. *Cells when surgically or enzymatically removed from
an organism and placed in suitable culture
environment will attach and grow are called as primary
culture
*Primary cells have a finite life span
*Primary culture contains a very heterogeneous
population of cells
*Sub culturing of primary cells leads to the generation
of cell lines
Primary culture

8. *Cell lines have limited life span, they passage several
times before they become senescent
*Cells such as macrophages and neurons do not divide
in vitro so can be used as primary cultures

9. *Most cell lines grow for a limited number of generations
after which they terminate division
*Cell lines which either occur spontaneously or induced virally
or chemically transformed into Continuous cell lines
Continous cell lines

10. *-smaller, more rounded, less adherent with a higher
nucleus /cytoplasm ratio
* -Fast growth
*-reduced serum and anchorage dependence and grow
more in suspension conditions
* -ability to grow unto higher cell density
* -different in phenotypes from donor tissue
* -stop expressing tissue specific genes
Characteristics of continous cell
lines

11. On the basis of morphology (shape & appearance) or
on their functional characteristics. They are divided
into three.
*Epithelial like-attached to a substrate and appears
flattened and polygonal in shape
*Lymphoblast like- cells do not attach remain in
suspension with a spherical shape
*Fibroblast like- cells attached to an substrate appears
elongated and bipolar
Types of cells

12. *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 media

13. Confluency
*Once the available substrate surface is covered by
cells (a confluent culture) growth slows & ceases.

14. hep 3B - 70% confluency
Gaps

15. 100% confluency

16. After 24 h

17. 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

18. *Once the available substrate surface is covered by
cells (a confluent culture) 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, dipase, collagenase in
combination with EDTA breaks the cellular glue
that attached the cells to the surface
Why sub culturing.?

19. *Cells are cultured as anchorage dependent
or independent
*Cell lines derived from normal tissues are
considered as anchorage-dependent
grows only on a suitable substrate e.g.
tissue cells
*Suspension cells are anchorage-
independent e.g. blood cells
*Transformed cell lines either grows as
monolayer or as suspension
Culturing of cells

20. *Cells which are anchorage dependent
*Cells are washed with PBS (free of ca & mg ) solution.
* Add enough trypsin/EDTA to cover the monolayer
* Incubate the plate at 37 C for 1-2 mints
* 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
Adherent cells

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

22. *Calcium phosphate precipitation
*DEAE-dextran (dimethylaminoethyl-dextran)
*Lipid mediated lipofection
*Electroporation
*Retroviral Infection
*Microinjection
Transfection methods

23. 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

24. *Vial from liquid nitrogen is placed into 37 C water
bath, agitate vial continuously until medium is
thawed
*Centrifuge the vial for 10 mts at 1000 rpm at RT,
wipe top of vial with 70% ethanol and discard the
supernatant
*Resuspend the cell pellet in 1 ml of complete
medium with 20% FBS and transfer to properly
labeled culture plate containing the appropriate
amount of medium
*Check the cultures after 24 hrs to ensure that they
are attached to the plate
*Change medium as the colour changes, use 20%
FBS until the cells are established
Working with cryopreserved cells

26. 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)

27. 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)

28. 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..

29. *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

30. *Remove the growth medium, wash the cells by PBS
and remove the PBS by aspiration
*Dislodge the cells by trypsin-versene
*Dilute the cells with growth medium
*Transfer the cell suspension to a 15 ml conical tube,
centrifuge at 200g for 5 mts at RT and remove the
growth medium by aspiration
*Resuspend the cells in 1-2ml of freezing medium
*Transfer the cells to cryovials, incubate the cryovials at
-80 C overnight
*Next day transfer the cryovials to Liquid nitrogen
Freezing cells for storage

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

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

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

34. *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-
Basic equipments used in cell
culture

36. * 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
Laminar- flow hood

37. Laminar- flow hood

38. Laminar- flow hood

39. *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
Laminar- flow hood

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

41. Cell Culture Incubator

42. *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
Cell Culture Incubator

43. *4C
*-20C
*-80C
*Liquid N2 tank
Refrigerators and Freezers

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

45. Autoclave
A simple autoclave may be sufficient.

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

47. Anaerobic Jar

48. Multiwell plates

49. Cell Culture Bottles /
Tubes

50. Heating of media on heater

51. Centrifuge