How to Troubleshoot Suspension Cell Culture Problems

Most suspension cells have round bodies with no attached support. They grow in suspension and are easy to reproduce in large numbers. It is also not easy to culture suspension calls although there are fewer digestion steps in suspension cell culture than in adherent cells.

Therefore, this issue sorts out the common problems in the process of suspension cell transportation and culture, and lists the solutions for each problem for reference.

Common transportation problems and solutions

1. More cell debris

Treatment: Suspension cells are prone to cell debris when they are damaged by transportation or other factors. The debris can be removed by low-speed centrifugation (800~1000rpm, 3min); low-speed centrifugation also loses cells, so it is not applicable when the cell density is low, and when the cell density is low, it should be centrifuged at a normal speed, and cells should be retained in priority.

2. Cells are not round/cells morphology changed/cells agglomerated 

Treatment: When the suspended cells are affected by transportation or micro-environmental changes, deformation and agglomeration may occur. The cells can be restored to their original state in a week or so if the correct culture medium is used and the operation is correct.

Common culture problems and solutions

1. Cells are not round/cell morphology changed/cells agglomerated

Treatments as follows:

It is a normal phenomenon for a small amount of cells to aggregate and appear as clusters of grapes, especially low cell density. By resting (less operation, less observation), adding serum (5%), etc., the cell density can increase and gradually adapt to the environment and return to normal; By resting (less operation, less observation), adding serum (5%), etc., the increased cell density will gradually adapt to the environment and return to normal.

The serum brand might affect the state of cell growth; if a type of cell that will not clump together is raised all clumped together with very few dispersed cells, then the serum effect should be considered.

2. Slow or no cell growth

Treatments as flows:

Suspension cells are density-dependent. The cell state is better at high density and poor at low density, so the passage ratio should be controlled. When the cells are in poor condition, passaging can be done by replenishment or half-exchange of liquid to avoid loss of cells by centrifugation.

3. Cell adherence

Treatments as follows：

If the cell culture flask is left standing for too long, a small amount of adhesion will appear after the cells sink to the bottom. However the adhesion is very loose, and the culture flask can be lifted by blowing or patting the culture flask. This phenomenon is normal and no special treatment is required.

If the cells are very tightly adhered and the morphology has become similar to epithelial, it is necessary to consider whether it is caused by cell characteristics or other factors.

Avoiding bacterial contamination in daily operations and using high-quality serum are effective methods to prevent suspension cell adherence.

Cell Culture Tips

1. Suspension cells routinely recommended inoculation density as 3-5×10⁵cell/mL, growth to about 2×cell/mL can continue to passage; this density is recommended as a theoretical value, and actually fluctuates according to the characteristics of each cell.

2. Cultivating suspension cells requires a stable growth environment, less operation and less observation, which is more conducive to cell growth.

3. It is not recommended to frequently blow off the aggregated cells. When the density is high and the state improves, the cells will disperse by themselves; some cells grow in clusters as a characteristic, and blowing off is not conducive to growth. Please pay attention to the relevant culture instructions.

4. Some cells grow in clusters (NK92, NK92MI, JURKAT, H209, ATT20, etc.), and some cells themselves are not round (H9, etc.), which is a normal phenomenon and no special treatment is required.

5. Vertical flask culture can promote cell growth, but since the flask is taken out for observation, the cells will stick to the flask wall after a long period of observation, and cells will hang on the flask wall when it is erected again after observation.

However, without medium, the cells will soon die and remain on the wall, resulting in fewer and fewer suspended cells and more and more adherent cells.

Therefore, care should be taken to blow off the cells at the bottom of the culture flask when the culture flask is placed vertically to avoid cell residue.