How to determine cell status

When culturing cells, due to the different culture environments and the differences in cell morphology at each stage, there is no definite reference for comparison. Many teachers and students are unable to determine whether the status of the cells culturing is normal.

So how to determine whether the cell status is normal or abnormal? This issue will introduce in detail several commonly used estimating methods.

01 How to determine cell viability

Trypan blue staining

Trypan blue is a cell-reactive dye that is commonly used to detect the integrity of cell membranes and determine whether cells are alive. Cells that are inactive or have incomplete cell membranes can be stained blue by trypan blue while normal living cells have intact cell membranes that reject trypan blue and do not dye cells blue. Cells that lose activity or have incomplete cell membranes have increased membrane permeability and can be stained blue by Trypan blue.

Cells that lose activity or have incomplete cell membranes have increased membrane permeability and can be stained blue by Trypan blue. Living cells are not stained, while dead cells are stained blue.

Mcroscopy

Cell morphology was observed under a microscope:

Suspended cells

Living cells are translucent and shiny, while dead cells are dull and have fragmented membranes.

Adherent cells

Living cells are wall-adherent and have intact membranes, while dead cells can’t adherent to the wall. 

（Dead cells are in red circles; living cells are in green circles; arrows point to serum impurities.）

There is a saying that a "clear border" determines cell viability. It is believed that if the border is not clear, the state of the cell has deteriorated. However, the actual situation is also found that some of the borders of the cell membrane are very thin and are not clear under the microscope, which leads to a misjudgment. Therefore, the clear membrane border is not a standard, while the standard is based on the integrity of the cell membrane. If the membrane is intact, the cell is alive.

   （The borders of the cell membrane are so thin and transparent that they may not be visible under a yellow light microscope.）

02  How to determine abnormal cell morphology

1. What should normal cells look like?

Before suspecting changes in cell morphology, it is important to know what normal cells should look like. Each kind of cells has their own unique characteristics, including growth rate, shape, culture environment, etc.

2. In what aspects can abnormal cell morphology be determined?

The microenvironment of cells can cause changes in cell morphology. For example, serum directly affects cell morphology:

Micrograph of HK-2 cells, cultured in serum-free medium.

Micrograph of HK-2 cells, cultured in MEM +10%FBS

Not all morphological changes are abnormal. As can be seen from the above examples, the cell morphology in the two culture environments is different, but the status is not abnormal. Therefore, cell abnormalities need to be determined by different situations and the comprehensive cell growth rate:

When the culture medium is not changed

If there is an increase in cell fragmentation and black spots, a change in morphology, as well as a slowdown in growth without changing the culture medium, exogenous irritants should be considered to be the cause, and the cells might be contaminated. So it is necessary to investigate the source of the contamination and make targeted preventive measures.

When the culture medium is changed

1. If the cell morphology changes after replacing the culture medium, the cells can be cultured normally if the growth rate is normal.

2. If the cells are severely deformed or even less adherent after replacing the medium and cannot be passaged normally, intervention must be carried out, which can be improved by coating culture bottles to promote adhesion, increasing serum concentration, and mixing with the original medium to allow cells to acclimatize in transition.

3. If the cells react violently to the new environment and die directly, the culture system needs to be changed.

03  How to determine cell contamination?

Bacterial contamination

Bacterial contamination should be considered if one of the following characteristics is present:

1. Highly repetitive particles with diameters smaller than cells can be observed under high magnification of the microscope.

2. Bacterial particles continue to multiply rapidly.

3. The medium rapidly turns yellow or turbid.

Fungal contamination

Bacterial contamination should be considered if one of the following characteristics is present:

1. Mold colonies are visible in the medium.

2. Under the microscope, the edge of the foreign body appears like a broom edge or a branch.

Cross-contamination

Cell cross-contamination is relatively rare and difficult to identify. Unless two cells with very different morphological characteristics are cross-contaminated, it can be identified with the naked eye under a microscope. It mainly relies on identification technology, which can be identified through STR identification, isozyme and other methods.