Animal free alternatives have increased in cell culture use in an effort to reduce variability and contamination present in FBS and other animal products used in culture. FBS is the prime example of an animal product used commonly for over 50 years to grow cells. While FBS is a clear animal product other products like trypsin are not typically considered an animal product.
Commercially available trypsin from Sigma (cat#4049) or Life Tech (25300-054) are both purified from pig pancreas (porcine pancreas). Trypsin is an animal product and should be considered as such when considering moving to an animal free system.
There are other practical reasons for considering alternatives to trypsin. Trypsin is used in cell culture as a means to cleave attachment proteins during passaging. Trypsin is a non-specific protease, which can cleave any protein with the correct amino acid sequence; consequently, cleavage is site specific not protein specific. While trypsin does cleave attachment proteins, it is not limited to those and will cleave all accessible proteins.
In cell culture conditions including serum there are excess proteins mostly bovine serum albumin (BSA). BSA limits the trypsin mediated cleaving of cell surface proteins. BSA also quenches the trypsin for future passages. These conditions are different when there is no excess animal protein in the culture. In serum free conditions trypsin can have a deleterious effect on the cells by cleaving the cell surface proteins and possibly leading to cell death. Since BSA is not present in animal free culture systems, there is no quenching agent and trypsin can bind to the cell and potentially be internalized 1. Furthermore some investigators have shown in serum free conditions trypsin can alter the gene expression of the cells 2.
Trypsin purified from pigs provides a vehicle for many potential contaminants. Trypsin purified from porcine pancreas can be contaminated. There are protein contaminants including other proteases. There are also potential viral contaminants 3,4 . When moving to a more controlled environment, like a serum free environment, eliminating sources of contaminants is critical.
Adherent cell culture is not only possible, but can be optimized in serum free conditions. This then raises the question of how to detach the cells for future passages. There are alternatives for trypsin including: Accutase® , a mix of recombinant proteases; TrpLE® , a recombinant form of protease; a citrate buffer based method. All of the methods are animal free and unlike trypsin, all of the methods have a controllable protease activity in serum free culture and subculture.
1. Hodges, G., Livingston, D. & Franks, L. The localization of trypsin in cultured mammalian cells. J. Cell. Sci. 12, 887–902 (1973).
2. Huang, H.-L. et al. Trypsin-induced proteome alteration during cell subculture in mammalian cells. J. Biomed. Sci. 17, 36 (2010).
3. Marcus-Sekura, C., Richardson, J., Harston, R., Sane, N. & Sheets, R. Evaluation of the human host range of bovine and porcine viruses that may contaminate bovine serum and porcine trypsin used in the manufacture of biological products. Biologicals 39, 359–69 (2011).
4. Oliveira, T. et al. Detection of contaminants in cell cultures, sera and trypsin. Biologicals 41, 407–414 (2013).
Cell-Ess® serum replacement is a chemically defined, animal component free cell culture media supplement that provides cells the essential fats they need. It doesn’t contain the unknown factors in FBS that introduce variables into your experiment. The researcher now has greater control over the plating factors, hormones and growth factors in play during the cell culture experiment.