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Animal Cell Culture
Animal Cell Culture
Rohit Sagar
Assistant Professor
Deshbandhu college
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).
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).
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.
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).
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.