Causes and Solutions of Six Major Culture Problems of Adherent Cells

StartAdherent cells should be attached to the wall of the culture (flask) vessel during culture. Once attached, the cells spread rapidly and then start mitosis, and soon enter the logarithmic growth phase.

Adherent cells are more prone to problems during culture due to the cumbersome culture process. The following summarizes six common problems in the culture of adherent cells, and details the causes and solutions, if you encounter these problems in culture, you can refer to the corresponding solutions.

All cells float up after passaging
Solution: Check whether the color of the culture medium used is purple (as shown in Figure 1), check whether the bottle cap is air-permeable, and whether the air-tight bottle cap is unscrewed.

                                                                                                Figure 1. Purplish Medium

Usually, if the medium is alkaline, the color will be purple, mainly because the phenol red in the medium turns yellow when it meets acid and turns purple when it meets alkali. The medium will become alkaline if the amount of medium is too small or the medium is stored for too long. It is recommended to divide the prepared complete medium into 50mL centrifuge tubes and use them up within a week, or store them in 15mL centrifuge tubes if the quantity is small.

Some cells float up after passaging
The reasons and solutions are as follows:
Too much cell density before passaging: Generally, the passaging operation can be carried out when cell confluence reaches 80%. Passaging needs moderate cell density, while a too high density of passaging will also lead to floating after passaging;
Poor cell status: can be improved by increasing the serum ratio and stabilizing the culture environment;
Blowing too many times to cause mechanical damage: Gentle blowing, stop blowing when cells are single particles, the blowing process to avoid bubble generation;
Inadaptability after medium replacement: Replace with the original medium, or mix it with the original medium before use, so that the cells have an adaptation period.

Cell deformation after passaging
The reasons and solutions are as follows:
Deformation, but the cells are still growing: It may be caused by different serum batches. The serum components are complex, and there are component differences between different batches and brands, which affect cell status. It is recommended to use the same batch of serum or mix it with the original serum before use when replacing the serum, so that the cells have a transition period;
Deformation, but the cells are not growing, and the fragments are increasing: It may be damaged in passaging, commonly caused by improper dissociation, or potential mycoplasma and other contamination resulting in cellular pathology. The dissociation time should be adjusted according to the cell status because excessive or insufficient dissociation can affect the cells. The culture process should be strictly aseptic, and the experimental environment should be as clean as possible to ensure that the cells are free of external contamination.

The cell growth cycle slows down, drifting while raising
The reasons and solutions are as follows:
1. The cell density is too thin or too dense when passaging: It is recommended that the cell density should be moderate, reaching about 80% for passage. Too thin a density will prolong the growth cycle;
2. Improper passaging operation: The cell dissociation time is determined according to the characteristics of the cells. Generally, the dissociation can be terminated by observing the cells shrinking and rounding under a microscope and a small amount of cells falling off. The cells that are difficult to dissociate can be dissociated in stages, and each time does not exceed 5 minutes; frequent replacement medium or washing the cells will also lead to the deterioration of the cell state, and the regular interval of changing the medium is 2-3 days.

Cells form clumps after passaging
The solution is as follows:
Inoculate at low density and extend the time of medium exchange to prevent cell shedding;
Use polylysine to coat the culture vessels to increase the firmness of cell attachment and reduce the probability of cell aggregation into clumps;
Re-plate and replace the newly prepared medium with a larger proportion of serum (increase the proportion by no more than 5%);
When inoculating, reduce the amount of medium (e.g. T25 plus 3mL) to speed up cell attachment, after the cells adherent (about 8-12 hours), and then replenish the medium to the normal amount.

Cell vacuolation
The reasons and solutions are as follows:
1.Normal cell activity: Some cells have normal autophagic behavior or other membrane vesicle activities without special treatment (e.g. Caco-2 cells);
2.Cell malnutrition caused by serum inadaptation, medium composition change, untimely medium change, etc.: It can be solved by replacing with a more suitable serum or timely changing medium, etc;
3.Cell damage caused by mechanical damage, high or low PH, etc.: Pay attention to culture conditions and manipulation.