Difference Between Warm and 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.

CONTENTS

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.

The contents are mixed and agitated by using centrifugation methods for the ease of the protocol and to speed the disaggregation process. Once the recommended time is achieved, the cells can be derived from the supernatant. The cells derived from the supernatant are then incubated at a particular temperature and time.

What is Cold Trypsinization?

Cold trypsinization is the other type of trypsinization that takes place under cold conditions. In this technique, the cells that are chopped and washed are placed in vials on ice and then soaked with trypsin. The period of soaking is much longer – about 6 – 24 hours.

After completion of the soaking procedure, the trypsin is removed from the cell lysate, and the tissue pieces are further incubated at 37 ⁰C for about 20 – 30 minutes. The disaggregation of the cells is brought about by repeated pipetting of the tissue mixture. This will allow the cells to dissociate from the membrane and come to the supernatant. Once the cells are in the supernatant, they are incubated and grown at a desired temperature and time period.

The cold trypsinization method has several advantages

• Higher yield of viable cells as the cell damage is minimized. The cell damage is minimized by not using centrifugation steps.
• Highly convenient method.
• Less laborious.

The main limitation of the cold trypsinization method is that large quantities cannot be used in one instance.

What are the Similarities Between Warm and Cold Trypsinization?

• Both Warm and Cold Trypsinization processes use the enzyme trypsin for disaggregation of cells.
• Both Warm and Cold Trypsinization processes are used in cell culture procedures for the disaggregation of cells.
• In both Warm and Cold Trypsinization treatment procedures, the cells are derived from the supernatant.

What is the Difference Between Warm and Cold Trypsinization?

Warm vs Cold Trypsinization
Warm trypsinization is the method of treating the cells with trypsin under warm conditions at a temperature of 36.5 – 37 0.	Cold trypsinization is the process of trypsin treatment that takes place under colder conditions preferably in ice maintaining very low temperatures.
Protocol
The chopped tissue pieces are maintained at 37 0C continuously throughout the procedure.	The chopped tissue pieces are initially maintained at ice-cold temperatures and then maintained at 37 0.
Temperature
Warm trypsinization occurs at 36.5 – 37 0	Cold trypsinization occurs at ice cold temperatures.
Time Consumed
Lesser time required for the entire process (around 4 hours) of warm trypsinization.	Longer time required (around 6 – 24 hours) for cold trypsinization.
Yield of Viable Cells
Low in warm trypsinization.	High in cold trypsinization.
Use of Centrifugation
Centrifugation is required for disaggregation of cells in warm trypsinization.	Centrifugation is not required in cold trypsinization.
Initial Tissue Quantity for Trypsinization
A larger quantity of tissue can be used in warm trypsinization.	A smaller quantity of tissue can be used in cold trypsinization.
Cell Damage
High due to centrifugation in warm trypsinization.	Less due to cold trypsinization.

Summary – Warm vs Cold Trypsinization

Trypsinization is the method of using the protein-degrading enzyme trypsin for the disaggregation and preparation of primary cell cultures during cell culturing process. There are two main techniques of trypsinization based on the temperatures used during the procedure. They are warm and cold trypsinization. Warm trypsinization is carried out at 37 ⁰C whereas cold trypsinization is carried out under ice-cold conditions. Although cold trypsinization takes longer time for completion, it is said to have a higher yield of viable cells. This is due to the fact that cell damage is minimized in cold trypsinization as it does not use vigorous centrifugation steps. This is the difference between warm and cold trypsinization.

Reference:

1.“Trypsinization.” Trypsinization – an Overview | ScienceDirect Topics. Available here 
2.“Primary Cell Culture: 3 Techniques (With Diagram).” Biology Discussion, 16 Oct. 2015. Available here  

Image Courtesy:

1.’Trypsin active site’By Fdardel – Own work, (CC BY-SA 3.0) via Commons Wikimedia