Let’s understand cell passaging together
It is recommended to pass down the cells every 2 days. Overgrown cells will reduce the natural differentiation rate of cells. We have established a purification method to remove differentiated cells. Adhere to uncoated tissue culture plates to remove differentiated cells. The purification step is included in the following method.
1. Remove the culture fluid;
2. Washing with calcium and magnesium-free PBS (Gibco)
3. Add pancreatin EDTA (Gibco). Incubate at 37°C for 5 minutes.
4. Join ES. The culture medium inactivates pancreatin;
5. Transfer the cells to a 15 ml centrifuge tube and centrifuge for 3 minutes;
6. Remove the supernatant, resuspend the cells in 2 ml ES medium and pipette at least 10-20 times. If the amount of medium added is small, it will be easier to break up the cell clusters. ES cells tend to aggregate into clusters, and it is important to carefully separate the cells during the passage. This may reduce the natural differentiation of cells.
7. Inoculate the cells in an uncoated tissue culture dish (use a culture dish of the same specifications as the one at the beginning) and place them in the incubator for 2 hours (differentiated cells will adhere during this time, while ES cells will remain suspended);
8. Transfer the cell-containing medium to a tissue culture dish coated with geltin. Pipette to ensure that the cells are dispersed (as mentioned earlier-ES cells tend to clump together)
9. It is recommended to pass down at a ratio of 1:4-1:10 (ATCC)
It is very important to maintain the passage ratio of cells. In our experience, a ratio of 1:8 is good and can minimize the natural differentiation of cells. When you use culture plates of different specifications for cell passage, you can use Table 1 to calculate the passage ratio.
Post time: Nov-18-2021