What is the Difference Between Warm and Cold Trypsinization

The main difference between warm and cold trypsinization is that warm trypsinization is involved in the incubation of tissues with warm trypsin at 36.50 °C whereas cold trypsinization is involved in the soaking of tissues in cold trypsin at 4 °C followed by the incubation at 36.50 °C. Furthermore, damage to the cells is high in warm trypsinization while cold trypsinization minimizes the prolonged effect of warm trypsin on the tissue.

Warm and cold trypsinization are the two techniques of trypsinization, a method of enzymatic disaggregation of tissues used in the primary cell culture. Here, trypsin is a proteolytic enzyme which breaks down the proteins responsible for adhering the cells in cultures.

Key Areas Covered

1. What is Warm Trypsinization
     – Definition, Method, Importance
2. What is Cold Trypsinization
     – Definition, Method, Importance
3. What are the Similarities Between Warm and Cold Trypsinization
     – Outline of Common Features
4. What is the Difference Between Warm and Cold Trypsinization
     – Comparison of Key Differences

Key Terms

Adhering Proteins, Cold Trypsinization, Enzymatic Disaggregation, Primary Cell Culture, Trypsin, Warm Trypsinization

What is Warm Trypsinization

Warm trypsinization is one of the two types of trypsinization methods used in the enzymatic disaggregation of tissues while preparing them for primary cell culture. It directly uses warm trypsin at 36.50 °C for the disaggregation. First of all, the chopped tissue is washed with DBSS (dissection basal salt solution) and then is added to a flask with warm trypsin. The dissociated cells are in the supernatant and at each 30 minutes intervals, the content of the flask is stirred in order to recover dissociated cells. Then, fresh trypsin is added to the mixture. And, this procedure can be continued for 3-4 hours while recovering dissociated cells. Finally, trypsin can be removed from the mixture by centrifugation. 

Figure 1: Warm Trypsinization

Furthermore, warm trypsinization is the most effective method of cell harvesting since it uses warm trypsin directly. Also, this procedure can be completed within a few hours. However, the yield of the viable cells is less due to the adverse effect of warm trypsin.

What is Cold Trypsinization

Cold trypsinization is the second method of trypsinization. It is less effective but yields a high amount of viable cells. One of the drawbacks of cold trypsinization is the time taken for the completion of the procedure. Generally, the prepared tissues are first soaked with cold trypsin at 4 °C for about 6-24 hours. Then, the recovered tissue is incubated with residual trypsin at 36.50 °C for 20-30 minutes. Repeated pipetting helps to disperse the disassociated cells. 

Figure 2: Cold Trypsinization

However, as mentioned before, the risk of damaged cells due to the prolonged exposure to warm trypsin is minimal in this method. Therefore, this method shows improved cell viability in culture. Also, due to the void of centrifugation, this method is suitable for a conventional laboratory. Nevertheless, the major drawback of this method is that it cannot be used for large quantities of tissue at once.

Similarities Between Warm and Cold Trypsinization

• Warm and cold trypsinization are the two techniques of trypsinization, which use trypsin for enzymatic disaggregation of adherence proteins in cell cultures.
• Both methods can be used while preparing tissues for primary cell cultures.
• Furthermore, both methods use warm trypsin at 36.50 °C at some point.
• Besides, they help to isolate cells.

Difference Between Warm and Cold trypsinization

Definition

Warm trypsinization refers to one of the two methods of trypsinization involved in the incubation of the tissue with trypsin at 36.50 °C followed by the removal of trypsin by centrifugation while cold trypsinization refers to the other method of trypsinization involved in the soaking with trypsin at 4 °C followed by the incubation with trypsin at 36.50 °C. Thus, this is the main difference between warm and cold trypsinization.

Usage of Warm Trypsin (36.50 °C)

One other major difference between warm and cold trypsinization is that the tissue is treated with warm trypsin for 3-4 hours in warm trypsinization while the tissue is incubated with warm trypsin for 20-30 minutes.

Centrifugation

Centrifugation is another difference between warm and cold trypsinization. Centrifugation is essential in warm trypsinization to remove trypsin from the sample while centrifugation is not required in cold trypsinization.

Effectiveness

Also, warm trypsinization is more effective while cold trypsinization is less effective.

Importance

Besides, their significance is another difference between warm and cold trypsinization. Warm trypsinization can cause damage to the cells in the tissue while the adverse effect of warm trypsinization has been removed in cold trypsinization.

The yield of Viable Cells

The yield of viable cells is another difference between warm and cold trypsinization. Warm trypsinization yields less amount of viable cells while the yield of viable cells is high in cold trypsinization.

Time

Furthermore, warm trypsinization requires less time to complete the procedure while cold trypsinization requires more time.

Applications

Moreover, warm trypsinization is used for tissues containing extracellular matrix and fibrous connective tissues while cold trypsinization is used for soft tissues like embryonic organs.

Conclusion

Warm trypsinization is a method of trypsinization, which uses warm trypsin at 36.50 °C to treat tissue in order to degrade adherence proteins. Though it is an effective method of enzymatic disaggregation of tissues, it yields a less amount of viable cells due to the adverse effect of warm trypsin. In comparison, cold trypsinization is the second method of trypsinization, which involves soaking the tissue with cold trypsin at 4 °C prior to the incubation with warm trypsin. This method is less effective, but it yields a large number of viable cells. Therefore, the main difference between warm and cold trypsinization is the usage of warm trypsin and the importance of each method.

References:

1. “Trypsinization.” Scribd, Scribd, Available Here.