Many cell line, easy to adapt to the serum free medium and protein-free media and to the environment request too high but difficult to adapt to change of other cells, which requires a more professional approach to adapt to change.
According to the characteristics of cell lines, there are two ways to adapt cells to the serum-free medium.
The first method is continuous adaptation/sequestering method;
The other approach is to adapt directly.
1. Continuous adaptation/sequestering method
The first method is continuous adaptation/sequestering.
Through a series of serum reduction steps, the cells were adapted to the serum-free medium.
This is a preferred method and does not make too much of a change in cell culture.
Continuous adaptive/sequential isolation method - method 1
The cells in the original medium are continuously passed through the following stages:
Phase 1:7 % supplement serum medium or 25% serum free medium
Phase 2:50 % of supplementary serum culture medium or 50% serum free medium
Phase 3:The medium or 75% of the serum was added to the medium or 75% of the serum
Phase 4:0 % no serum culture medium
Continuous adaption/sequestering method - method 2
(1) the serum was added to the serum without serum in the same concentration as in the original medium.
The cell density in the new culture system should be increased when the cells are transferred from the original medium containing serum to the serum-free medium.
Let cells have an adaptive transition.
(2) according to method 1, slow down the serum concentration, so that the cells have the time of adaptation at each stage.
(3) once the serum complement is reduced to 0, the cell culture will have several adaptive transitions before being used for analysis or other operations.
2. Direct adjustment
The other approach is to adapt directly.
In these cells, the cells were transferred directly from the serum culture medium to the serum-free medium.
A large number of cells are transferred directly to a new medium without serum or protein.
The cells must be in the medium term and the survival rate is 90 %.
Replace about 50% of the medium every 3 to 4 days to prevent the medium from becoming acidic.
The cell culture density is higher than normal, until the cultured cells need to be replaced daily.
At this point, the cells are amplified to multiple culture bottles.
Consider recommending
In the process of adaptation, cells are usually more sensitive to changes in PH and temperature.
Allow the cells to have at least one transition period at each stage.
Cell survival and cell density
The cells divide rapidly in the middle of the logarithmic period, and the survival rate is greater than 90% at the beginning of the adaptation process.
This is crucial.
When the cell needs to be passed on, it is recommended that a ratio of 1:2 or 1:3 should be adopted to maintain higher cell density while providing the cell growth factor that may help cells during isolation.
When the cell ADAPTS to the new culture conditions, it increases the cell transmission density.
Cell growth
If the cell stops growing during the adaptation process, the cell has more time to adapt to the culture medium.
For example, in phase 1 of method 1, a small amount of serum (1-3%) was added to the serum free medium.
This helps cells adapt to new media more easily.
Then, during the transition period, the amount of serum will be reduced slowly until the amount of learning is 0.
Suspension culture
In Meiatech, the serum-free medium and unprotein medium did not contain the cells found in serum.
In a transitional period, the wall cells will begin to leave the surface and become suspended cell lines.
The cells may also be altered by adaptation.
Monitoring the growth rate of the cell, as long as the cell can fuse with the new system, any morphological change is not important.
Cells clump
In the absence of serum culture, cells can form clumps easily.
In the culture of the generation, the clump is lightly broken and dispersed.
Transfer the culture from a stationary cell culture bottle to a rotating conical bottle, which may help prevent the formation of the cell plexus.
antibiotics
Since serum proteins are easy to bind to antibiotics, it is not recommended to use antibiotics in the absence of serum adaptation.
If serum protein is not present, antibiotic levels may be toxic to cells.
Preventive measures
Ensure that a large number of cells are stored in the original medium before starting to adapt.
If the cell does not survive to the next stage, preserve the culture bottle used throughout the process.
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