2. What is cell culture ?
• Cell culture is a technique which involves isolation of cells from
animal/plant body i.e. from their natural environment (in vivo) and
practicing to grow isolated cells in cell specific media in plastic flask
or petri dish in a controlled environmental artificial condition (in
vitro).
• Cell culture means to keep cells alive and grow in an in vitro
condition in a nutritive media which are widely used for research and
diagnosis of different pathogens and to understand the function and
mechanism of operation of many cells.
3. Types of cell culture
•Primary culture
Primary culture is the cell culture system that is formed by culture cells
directly obtained from tissue and proliferate them until they occupy all of the
available substrate (i.e. reach Confluence).
For primary culture, the first step is to isolate the tissue from organ and
splitting of cells from tissues. This can be achieved by trypsin treatment.
• Then grow cells in freshly prepared cell specific medium and incubate the
flask containing cells in incubator providing suitable environmental
condition for the growth of cells.
• After the first subculture, the primary culture is known as a cell
line or subclone.
4. •Secondary culture
• When cells from primary culture on confluency are isolated and
cultured in new media, it is called as secondary culture.
• Secondary culture is also known as subculture or splitting of cells.
• Subculture allows fresh nutrients and more space for the expansion
of the cells.
• Cells from primary culture are splits by trypsin/EDTA treatment.
• Trypsin which is a serine protease digest the extracellular protein or
matrix protein so that cells get free.
• EDTA which is a chelating agent chelates calcium ion because calcium
helps in cell adhesion.
5. Finite Vs Continuous Cell Line
• Normally, cells divide to a limited times, after that loose their proliferation
ability (senescence) these cell line are known as Finite cell line.
• Cell line which are chemically or virally transformed (transformation)
acquires the ability to proliferate indefinitely, these cell line is called
continuous cell line.
• Contact inhibition and anchorage dependence and low growth rate are the
characteristics of finite cell line.
• Absence of contact inhibition and anchorage dependence and high growth
rate are some attributes for continuous cell line.
• Finite cell line grow as monolayer culture i.e cells cover the bottom of the
culture vessel uniformally.
• Continuous cell line may be grow as monolayer or suspension culture (cells
do not adhere but remain suspended in culture media).
11. DMEM MEDIA COMPOSITION
Note- 10% DMEM media is required
for normal cell culture like Vero or
C6/36 or BHK-21 cell lines.
✓ So to make 10%, we have to add
FBS and streptomycin/penicillin
solution, now we have complete
DMEM MEDIA which is ready to
use for culturing
✓ All other components are already
added in media so no need to add
others.
12. Cell culture incubator
Major equipments required for cell
culture
Bio Safety cabinet
Sub-culturing of cells on confluency
16. Things to be done before starting cells passaging
1) First of all we have to give UV radiation to culture hood for sterilisation.
2) Media which was kept in 4°C should be remain on room temperature
before start.
3) Clean the Hood with 70% ethyl alcohol.
4) Then keep all necessary things in culture hood after rinsing them with
alcohol.
5) Discard should be there in hood to discard the spent media of culture
flask.
6) Check the confluency of cells first under microscope. Cells should be
healthy.
7) Never split the cells which are not confluent. Otherwise it will stress to
the cells. Cells are very fragile and sensitive so handle very gently and
carefully.
8) Always clean your hands with 70% ethyl alcohol.
9) Trypsin/EDTA which is required for cell splitting should be pre-warmed
at 37 °C.
17. Procedure for splitting/passaging of cell line from established
primary cell line (Like Vero cell line which is an adherent cell line)
1) Take the confluent flask and remove all the spent media using serological pipette.
2) Wash the monolayer of cells once with Phosphate buffer saline.
3) Then take 1 mL of Trypsin/EDTA solution and let it cover the monolayer completely.
4) Remove some trypsin solution.
5) Keep the flask at 37°C in BOD incubator for approx. one and half minutes.
6) Tap gently on the side of flask so that cells can dislodge.
7) Add about 2-3 mL of 10% complete DMEM media to flush off the cells that have got dislodged.
8) Centrifuge the tube containing the cells at 1500 rpm for 5 minutes.
9) Cell pellet will form.
10) Now dissolve the pellet in 2 ml fresh DEMEM media.
11) Count the cells by hemocytometer and take the cells accordingly into the new flask to continue the cell
line.
12) Check the flask after 24 hour for its quality and any contamination.
13) Split this flask at alternate day to maintain the cell line for long time (i.e. upto 30 passages).
18. How freezing of cells in done in Liquid nitrogen
• After trypsinisation of Confluent culture flask , forms pellet and
dissolve in DMEM media(as mentioned in previous slide).
• Count the cell to get an idea about cell density.
• Then add 2-3 million cells per cryovial on freezing media.
• Freezing media is basically DMEM media with 10% FBS and 10%
DMSO). DMSO is dimethyl sulphoxide which is a cryoprotectant and
prevent ice crystal formation.
• Keep the cryovial in -80°C and then next day in liquid nitrogen.
• In this way our cell line remains with us for a very long time.
19. Source of contamination in cell culture
• Contamination of cell line means something which is undesirable in the culture system but comes unknowingly.
• Contamination makes our culture media present in cell culture flask fuzzy. Normally it should be clear.
• Two reason may be possible for contamination either
• 1. Chemical contamination
• 2. Biological contamination.
• Chemical contamination includes:-
✓ DMEM media
✓ Phosphate buffer
✓ Trypsin/EDTA solution
✓ Incubator
• Biological contamination includes:-
✓ Bacteria
✓ Fungi
✓ Mycoplasma (generally observed)
✓ Cross contamination with other cell line (i.e When two cell lines are splitted back to back without giving any UV-
radiation, then chances are more).
20. References
• Oyeleye et al (2016). Basics of animal cell culture: Foundation for
modern science. Biotechnology and Molecular Biology Reviews. Vol.
11(2), pp. 6-16, May 2016.
• Yao, T and Asayama, Y. (2016). Animal-cell culture media: History,
characteristics, and current issues. Reprod Med Biol. 2017;16:99.
