Difference Between Warm and Cold Trypsinization | Warm vs Cold Trypsinization

Key Difference – Warm vs Cold Trypsinization

Warm and Cold Trypsinization are two methods used in enzymatic disaggregation of cells in animal cell culturing. The key difference between warm and cold trypsinization, as the names suggest, depends on the temperature at which the trypsin is added for cellular disaggregation. Warm trypsinization takes place under higher temperature conditions (36.5 – 37 0C) whereas cold trypsinization takes place under low-temperature conditions.

During the process of primary cell culturing of animal cells, there are three main methods utilized and have proved to be successful. The three methods include mechanical disaggregation of cells, enzymatic disaggregation of cells and the primary explant technique. The enzymatic disaggregation of cells leading to the isolation of cells and it is done by the protein-degrading enzyme trypsin. Therefore, this process is known as Trypsinization. Trypsinization can be done under two different conditions namely Warm Trypsinization and Cold Trypsinization. Warm trypsinization is the method of treating the cells with trypsin under warm conditions at a temperature of 36.5 – 37 0C. Cold trypsinization is the process of trypsin treatment that takes place under colder conditions preferably in ice maintaining very low temperatures.


1. Overview and Key Difference
2. What is Warm Trypsinization
3. What is Cold Trypsinization
4. Similarities Between Warm and Cold Trypsinization
5. Side by Side Comparison – Warm vs Cold Trypsinization in Tabular Form
6. Summary

What is Warm Trypsinization?

Trypsinization can be done to disaggregate the cellular components in order to isolate the cells to produce a primary cell culture. Trypsin is a protein degradation enzyme, and the enzyme mixture used in trypsinization can be either a crude extract or a purified product. The crude extract is said to be more efficient in protein lysis and cell disintegration as it contains other degradative enzymes.

Warm trypsinization is the most commonly used enzymatic method for cell disaggregation that takes place under higher temperature conditions. Prior to the treatment by trypsinization, the desired tissue is chopped into smaller pieces. It facilitates the easy disaggregation process. The chopped tissue is then washed in a special media known as the Dissection Basal Salt medium.

Upon the completion of the washing step, the cells are transformed into a flask containing the active enzyme, which is trypsin. As this technique implies a warm trypsinization protocol, the trypsin is placed at a temperature of around 37 0C for about four hours.

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