The effect of enzymes in sub culturing of adherent cells

Title: The effect of enzymes in sub culturing of adherent cells

Aim: To assess how trypsin affects adhering of cells in sub culture


Cell culture is a fundamental practice in biological research. It refers to the process by which prokaryotic, eukaryotic animal and plant cells are grown in vitro under maintained conditions (Schillberg et al., 2013). The maintenance of cells in vitro is complicated in that as they continue to multiply, they exhaust the nutrients in the media. Sub culturing is done to prevent dying of cells (Bhasin et al., 2013).There has been various discoveries in cell culture studies. The first development was the use of antibiotics to inhibit cell growth. The other major discovery was the use of trypsin to remove adherent cells. Today, chemically defined culture medium can be used in such experiments. For the cells to be effectively sub cultured, they have to be put into suspension, an aspect which only works well in presence of enzymes such as trypsin which prevents the cells from sticking to the glass surface. EDTA is a good chelating agent and does not result in the death of cells even if exposed for a longer period of time (Tice et al., 2000). The use of trypsin in sub culturing of cells is a precise method of preventing cell death in vitro.

This method is applicable in cell and tissue culture procedures especially when it is necessary to transfer cells from one vessel to another. Studies in cell biology, the interactions between the disease causing organisms and the host cells, toxicity testing of drugs and cancer research studies are all important research areas that depend on this technology. Previous studies have been conducted in virology and genetic engineering in which sub culturing has been essential (Bhasin et al., 2013). Studies in stem cell research has been done with attempts to grow cells to be used in therapy (Cooper et al., 2013). Research involving cells of animals and their response to other organisms is very necessary for studies in medicine as well agriculture. Drug manufacturing companies also depend on this information to predict the behavious of host cells in the presence of manufactured drugs. This study was done to show the effect of trypsin in sub culturing of cells.

Materials and Method


  • PBS
  • Porcine Trypsin 2mM EDTA (TE)
  • 2mMEDTA (E)
  • Pipettes and Pipetters CHO-K1 cells
  • Non Sterile and sterile tips


All the cells were kept under PBS media to prevent them from dying.

  1. All the materials which were to come into contact with the cells were pre warmed at 37oC
  2. The work area was aseptically prepared
  3. Cells were removed from the media
  4. The cells were washed twice with PBS by adding enough PBS to cover the cells and shaking the beaker gently. The waste was removed and the cleaning process repeated.
  5. About 250μl of EDTA was added to one set and the same amount of Trypsin EDTA added to another set of the experiment
  6. The set up was incubated at room temperature
  7. Cell suspension was checked after 2, 5, 10, 20 and 30 minutes. The results were as recorded in the table below.


The results of the experiment were as in the table below showing the number of floating cells over period time

Table 1: The number of floating cells over period time for the treatment A,B and C.



Treatment A


Treatment B


Treatment C


0 0% 0% 0%
2 30% 6% 0%
6 70% 10% 0%
10 85% 13% 0%
20 90% 15% 0%
25 100% 20% 0%

(TA) is Trypsin- EDTA / (TB) is EDTA / (TC) is control

From the table we see that the cells completely dissociate and quickly get smaller and start to constrict and has its final shape (round and fully floating and stick to each other as group) in treatment A at 25 min, while treatment B it took very long time to dissociate and it was very slowly. This suggests that the presence of trypsin in treatment A makes the cells to dissociate and move faster. There is increased attraction between the cells than between the cells and the vessel.


The experiment was set up to show the effect of trypsin and EDTA in the sub culturing of cells. From the results, it can be seen that in treatment A where there was both trypsin and EDTA, the movement of cells was very first. This is because trypsin breaks down the proteins which enable the cells to adhere to the vessel. Enzymes work best at optimum temperature of 37oC. Since the samples are incubated at the optimum temperature, enzyme trypsin will act on the surface of the cells breaking the proteins responsible for binding then to the vessel. The cells which have been subjected to this treatment appear rounded due to the effect of trypsin. EDTA on the other hand promotes cell to cell adhesion. This is the reason for fast movement of cells in treatment A where there is both trypsin and EDTA. Since the forces of attraction between the cells and the vessel are broken by trypsin and the adhesive forces between the cells are enhanced by EDTA, treatment A results to cells which appear round and sticking to each other. As time increases, trypsin continues to act on the cells making them smaller and constricted. Overexposure of the cells to trypsin can kill the cells.

On the other hand for treatment B where there is only EDTA, the forces of attraction between cells are enhanced while those of attraction between cells and the vessel are still strong. This makes movement very difficult hence the cells take more time to move out of the vessel. This is why only a smaller percentage (20%) of the cells had dissociated after 25 minutes. The cells take more time to do this because EDTA increase the adhesive forces between cells. Without trypsin in the treatment, the cells will still be sticking to the vessel since the cohesive forces are still very strong. Treatment A with both trypsin and EDTA is better in sub culturing of cells than EDTA alone.


In conclusion many cell biology studies involve sub culturing of cells. The use of trypsin EDTA and EDTA has been in practice depending on the desired outcome. Trypsin and EDTA combination is preferred where quick and robust results are desired. But care must be taken not to overexpose the cells to trypsin since it can kill the cells over long exposure. EDTA alone takes a longer time for cell dissociation but cannot harm the cells even if exposed for a longer period of time.


Schillberg, S., Raven, N., Fischer, R., M Twyman, R., & Schiermeyer, A. (2013). Molecular farming of pharmaceutical proteins using plant suspension cell and tissue cultures. Current pharmaceutical design19(31), 5531-5542.

Bhasin, A., Srivastava, M. P., Mohanty, S., Bhatia, R., Kumaran, S. S., & Bose, S. (2013). Stem cell therapy: a clinical trial of stroke. Clinical neurology and neurosurgery115(7), 1003-1008.

Cooper, A., García, M., Petrovas, C., Yamamoto, T., Koup, R. A., & Nabel, G. J. (2013). HIV-1 causes CD4 cell death through DNA-dependent protein kinase during viral integration. Nature498(7454), 376-379.

Tice, R. R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H., … & Sasaki, Y. F. (2000). Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental and molecular mutagenesis35(3), 206-221.



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