Dissatisfied with the cultured NK cells, is it a problem with the medium, or is it a problem with the kit?
Regarding the problem of NK cell culture, it is not only that the culture medium is not good or the kit is not good. Many customers encounter the problem of seeding density, the problem of the rehydration procedure, the plasma problem and the sample problem during the culture process, so the NK cell culture is affected. There are so many factors in the effect, so if the cell culture is not satisfactory, we should follow the following principles.
1, blood sample problems
First confirm the state of the sample. Theoretically, the peripheral blood sample should be separated within 6 hours of extraction. During this period, the cells are in the best state, so please confirm whether the sample is fresh before the culture and whether the blood is hemolyzed.
Excessive storage of blood samples can cause mononuclear cells to fail to induce activation.
If the blood is hemolyzed during transport, it may cause problems in plasma separation and cannot be added later.
If cryopreserved with cryopreserved mononuclear cells, confirm the viability and status of resuscitated mononuclear cells.
2, inoculation density problem
Please ensure that the density of inoculation is 1.5-2x106cells/m L. Too low density will lead to slow cell proliferation in the early stage and cannot meet the requirement of rehydration.
3. Plasma treatment and addition methods
(1) The plasma must be inactivated at 56 ° C for 30 min to remove excess fibrin. If possible, it can be centrifuged after freezing to ensure that the plasma added during cell culture does not have fibrin precipitated and adhered to normal cells.
(2) If there is more residual plasma, it can be replenished at a later stage, and it will have better culture effect for some samples with poorer conditions.
4, the principle of fluid replacement
NK culture is greatly affected by sample variability, so there is no fixed rehydration procedure to guide a variety of different samples.
Some samples are activated rapidly, and the growth is rapid. The rehydration is relatively easy and better, and some samples may be slower in the early stage. If the patient is in advanced stage, it is even more difficult, so it is difficult. For different samples, we should have different coping principles:
(1) The principle of rehydration after 7 days is that the cell density should be 0.5-0.8x106cells/m L after rehydration. If the density is not reached, please delay the rehydration after one day. Otherwise, when your cell rehydration density is too low, The sample can grow up, and the poor sample will gradually become apoptotic and cannot continue to be cultured.
(2) For poor samples, it is better to increase the number of plasma additions. For example, the standard procedure requires 3-4 times of plasma, so you can add 5 times if possible.
5, the reason for operation
(1) Temperature of the rehydration medium
The medium should be pre-warmed before rehydration, and the super-cooled medium will cause cell stress reaction and flocculation;
(2) Dramatic changes in the incubator environment
Abnormal incubators, large changes in temperature and carbon dioxide concentration can cause cell death and flocculation;
(3) Bacterial, fungal, and mycoplasma contamination
Cells contaminated with bacteria, fungi, and mycoplasma grow very slowly, even without growth;
(4) Factor kit has undergone repeated freezing and thawing
Repeated freezing and thawing will greatly reduce the activity of the factor, and problems such as activation, induction, and amplification will occur, and the optimal effect will not be achieved.
Our special intermediates are mainly a combination of our own actual product structure and product properties, including: 1. The product does not belong to any other product of our company intermediates, 2. Chemical reaction type is more special 3. Can be used to synthesize two or more kinds of API which roles is almost equivalent and difficult to separate the level. 4. This intermediate is somewhat less used for actual production or less practical use. We summarized the above four cases of intermediates temporarily as a special intermediate, if the special intermediate application is enlarge, we will adjust it according to the actual situation to change its "identity."
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