NK cell culture medium, culture method and application of NK cell culture medium and culture method
A technology of cell culture and medium, applied in the direction of cell culture active agent, cell culture support/coating, animal cells, etc., can solve the problems of low purity, high production cost, complicated technical operation, etc., and reduce the risk of pollution , low cost effect
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Embodiment 1
[0083] The cultivation of embodiment 1 NK cell
[0084] Utilize the cell culture medium provided by the invention and the cell culture method to cultivate NK cells, and the culture process is as follows: figure 1 shown, including the following steps:
[0085] Step (a): Coat the Lymactin-NK antibody with a concentration of 0.6mg / mL in the cell culture flask, the coating condition is 37°C, and the incubation time is 2h;
[0086] Step (b): collect human peripheral blood, transfer the collected peripheral blood to a 50mL centrifuge tube, centrifuge at 20°C at 2000rmp for 10min to collect plasma for later use; use lymphocyte separation medium (corning) to separate and collect individual peripheral blood For nuclear cells, the collected mononuclear cells were washed three times with PBS and counted, and set aside;
[0087] Among them, the method of using lymphocyte separation medium to separate and collect peripheral blood mononuclear cells is as follows: 1. Add the blood sample a...
Embodiment 2
[0093] Example 2 Cell Expansion Fold Analysis
[0094] The cells were counted on the 0th, 12th, and 14th days of culture, counted after staining with trypan blue, and the expansion multiples and cell viability were calculated. The counting results were divided by the initial cell number to obtain the cell expansion multiples.
[0095] Result analysis: This method can detect the expansion of cells, the results are shown in Table 1 and figure 2 As shown, it can be seen from the results that the peripheral blood mononuclear cells can be expanded by more than 100 times after 14 days of culture, which can meet the number required for clinical treatment. Table 1 NK cell expansion multiple results are the statistical results of repeated experimental data.
[0096] Table 1 NK cell expansion multiple results
[0097] Training days (days)
Embodiment 3
[0098] Example 3 Cell Purity Analysis
[0099] Cells were collected on the 14th day of culture, washed 3 times with PBS, and the cell concentration was adjusted to 1.5×10 after washing. 5 / mL, add flow cytometry antibody (CD3-PE, CD4-FITC, CD16-PEcy5, CD25-APC, CD56-FITC, NKG2D-APC fluorescently labeled monoclonal antibody 10 μL), incubate at 4°C in the dark for 30 min, wash once with PBS, After resuspended in PBS, the cell phenotype was detected by flow cytometry.
[0100] Result analysis: the results are shown in Table 2 and Figure 3A , 3B , 3C, 3D and 3E, it can be seen from the results that the cell phenotype is CD3 - CD56 + , CD3 - CD16 + , CD3 - NK2D + , CD16 + CD56 + The proportion of cells were greater than 70%, among them, CD3 - CD56 + Cell percentage: 79.98%, CD3 - CD16 + Cell percentage: 79.49%, CD16 + CD56 + Cell percentage: 85.40%, CD3 - NK2D + Cell ratio: 82.47%. Cell phenotype is CD4 + CD25 + The proportion of cells were all less than 10%, wh...
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