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Decoding Cell Digestion:Are Your Cells Ready?

Source: PricellaPublished: 2024-04-01

Cell culture is a crucial skill in the laboratory, requiring ongoing practice and refinement. While cell digestion is not overly complex, mastering it is vital for successful cell culture. Despite its seeming simplicity, many individuals encounter difficulties with this step. For instance, improper timing of cell digestion can lead to the deterioration or death of rapidly proliferating cells due to nutrient deficiency or cell contact inhibition. Such setbacks inevitably postpone experiments. Hence, thorough exploration and understanding of cell digestion are essential.

(Figure 1)

When should cells undergo digestion?

Typically, cells require digestion when they reach 80%-90% confluence. Before passaging adherent cells, it's essential to detach them from the culture vessel's surface using a digestion reagent. This process allows the cells to dissociate into individual suspended cells for transfer into a new vessel containing fresh culture medium.

There are primarily four methods of cell digestion:
1.Enzymatic digestion method

This method is the most commonly used, mainly employing trypsin. Typically, the concentration ranges from 0.25% to 0.5%. The digestion time varies depending on factors such as cell type and incubation temperature, usually ranging from a few minutes to several tens of minutes.

Generally, for monolayer adherent cells, 0.25% trypsin digestion at 37 degrees Celsius for 1-5 minutes is sufficient.

It's crucial to note that the presence of Ca2+, Mg2+, serum, and proteins can inhibit the activity of trypsin. Therefore, trypsin(Cat.No.: PB180225) formulations lacking Ca2+ or Mg2+ but containing EDTA exhibit higher activity.

Finally, trypsin digestion can be terminated using culture medium containing serum. Trypsin is sensitive to temperature and should be transported and stored at -20°C, and it should not undergo repeated freeze-thaw cycles.

2.Chelating agents

Chelating agents do not disrupt cell surface molecules; they only chelate with CAMs (cell adhesion molecules). Therefore, if you are detecting cell surface molecules, it is advisable, or even necessary, to avoid using enzymatic digestion methods.

The most commonly used chelating agent is EDTA(Cat.No.: PB180320), typically at a concentration of around 0.02%. It acts on cell-to-substrate interactions and also has some effects on cell-to-cell interactions.

Note that EDTA can significantly affect the pH value and is more soluble under weak alkaline conditions. Therefore, it is important to adjust the pH when preparing the solution. This method is relatively simple to operate and does not require the use of protein or serum to terminate digestion.

3.Physical methods

It involves directly tapping or scraping the cells off using a cell scraper.

4.Freezing method

This method is applicable primarily during cell passaging since it cannot detach cells from tissue. Its principle relies on cell contraction after freezing, prompting detachment from the culture vessel. Advantages include minimal cell damage, no requirement for terminating or washing the cells, and no need for additional digestion solutions. It's particularly suitable for cells that are not tightly adherent and delicate. However, a disadvantage is that small patches of cells may frequently detach.

What constitutes a satisfactory digestion status for cells?

(1)A good digestion for cells doesn't necessarily mean they are completely dispersed and singularly round in shape.

Typically, when observing adherent cells under a microscope, their mobility is a key indicator. If they are mobile, often exhibiting a sandy-like movement, it's usually adequate. Many people tend to over-digest or mechanically detach cells until they are completely separated, which is unnecessary. Generally, cell mobility indicates the disappearance of adhesion between cells and between cells and the culture vessel, with cells now distributed independently (though not necessarily widely dispersed). There's no need to wait until all cells are completely separated with large gaps between them under the microscope before halting the digestion process.

(2)Not all cells will become round after digestion. Some cells, such as U87 and U251 cells, do not become round after digestion; they may only exhibit a semi-sandy appearance.

(3)Even if some cells become round, there may still be detached cells, indicating incomplete digestion.

For example, with 7860 cells, digestion should continue until most of the cells detach from the bottom of the culture dish. Otherwise, it may be difficult to detach them mechanically. This situation typically occurs with cells that are difficult to digest.

(4)For some special cells, it may be necessary to undergo partial digestion. For some cells, such as LNCAP cells, blowing them into single cells may actually improve their condition, while clustering them can cause an immediate deterioration in cell status.

What are the main characteristics of over-digestion?

Over-digestion can result in considerable cell damage and death. After passaging, clumps of cells may float on the surface of the culture medium, and small white film-like substances may be visible to the naked eye. If these cell clumps are composed entirely of dead cells, they will not adhere to the surface. However, if viable cells are still present within the clumps, they will adhere to the surface, leading to uneven local cell density. This can ultimately lead to the formation of a central necrotic cell zone in that area.

How to handle over-digestion of cells?

Immediately neutralize with culture medium, use a pipette to pipette the cells, collect all the cells into a sterile centrifuge tube at 800 RPM for 3 minutes. Discard the supernatant, resuspend in fresh complete medium, and continue culturing in a new culture vessel. Cells in poor condition may die and detach during the culturing process, and they can be removed during media changes!

What does it indicate when cells cannot undergo digestion in certain situations?

(1)Certain cells strongly adhere to the substrate, resulting in residual cells after digestion and pipetting. Alternatively, cells may thrive in culture but the vessel hasn't been changed for too long. To preserve the majority of viable cells, digestion should be appropriately terminated.

(2)The digestion time varies among different types of cells. Typically, digestion for adherent cells takes about 2 minutes. However, for certain cells that prove challenging to digest and remain unaffected even after repeated digestion and pipetting, it's advisable to discontinue further attempts.

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