When the frozen storage cells were defrosted, there were problems of low survival rate, large number of cell fragments and slow growth. What was the reason for the solution?
Now let's take a look.
May be able to give your experimental problem solution to find the answer!
The low survival rate
The possible reasons for the low survival rate and the recommended solution are as follows:
1. Cell lysis during thawing
The recommended solution: a certain amount of cell death can be expected, so the cell concentration should be high enough to take into account the loss.
The initial concentration is 106 to 107 cells/ml.
2. Problems in the thawing process
Recommended solution: under - 70 ℃ to 80 ℃ keep frozen cultures, save time for 1-5 days, but this is not saved the preferred method.
In 37 ℃ fully thawed should immediately began to develop.
3. Allergy to frozen fluids
The recommended solution: A. Complete or partial replacement of the medium to reduce the amount of frozen liquid in the medium.
After 24 hours, change the culture fluid can remove the refrigerant completely.
B. allow more time for the culture to recover.
Sometimes cells take a few weeks to form a single or dense suspension, depending on the age of the cell at the time of freezing or the number of times it is passed or where it is in the growth phase.
The optimal conditions for freezing are in the logarithmic period.
4. The age or age of the culture when the original cell is frozen
Recommended solution: defrost newly frozen cells.
The longer the cell is in the frozen state, the lower the survival rate.
Check the time and method of freezing cells.
The cell should be in a logarithmic period when freezing.
Large number of cell fragments
The possible causes and recommended solutions for large number of cell fragments are as follows:
1. The cell density is too low when freezing
Recommended solution: shortening the time of the defrosting process.
Will remove cells immediately after the cryopreserved tube into the water bath at 37 ℃, and shock to melt, then quickly move to preheat medium.
2. Improper freezing process
Recommended solution: defrost the total test tube of different freezing chamber.
Ensure that appropriate techniques are used in the freezing process and ensure proper use of appropriate and appropriate refrigerating fluid.
The possible reasons for the slow growth and the recommended solutions are as follows:
1. Cultivate the size of the bottle
Recommended solution: some cells tend to maintain a certain density in the medium.
Transfer the culture to a smaller culture bottle and increase the cell density;
For example, according to the volume of the medium, the flask from 75cm2 is transferred to a flask of 2 or 3 25cm2.
2. Low cell density
Recommended solutions: to improve the freezing storage density of frozen species in the future, or to use smaller culture containers, or to defrost multiple test tubes for cultivation.
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