Efficient factor secreting type NK cell amplification method and application thereof
A NK cell and secretory technology, applied in cell dissociation methods, animal cells, vertebrate cells, etc., can solve the problems of low number and purity of NK cells, achieve efficient survival and expansion, increase the number of expansion, guarantee The effect of uniformity
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specific Embodiment approach 1
[0027] Specific embodiment one: the efficient method for expanding factor-secreting NK cells in this embodiment includes the following steps:
[0028] Step 1: Separating mononuclear cells from umbilical cord blood or peripheral blood;
[0029] Step 2: Sorting mononuclear cells with a sorting flow cytometer, the screening range is the uppermost 5% of the Q1 area, that is, CD56 strongly positive cells are obtained;
[0030] Step 3: Culture the strongly positive CD56 cells obtained in Step 2 at 37°C and 5% CO 2 Saturated humidity; the specific method is: inoculate CD56 strongly positive cells into a T25 bottle containing serum-free medium A for culture, collect the cells on the 3rd day of culture, and supplement them with serum-free medium A to continue the culture; The cells were transferred to T75 culture flasks at 5 days, and serum-free medium A was supplemented; after that, the liquid was replenished every 2 days, and the replenished liquid was GT-T551 H3 serum-free medium c...
specific Embodiment approach 2
[0038] Embodiment 2: The difference between this embodiment and Embodiment 1 is that the method for separating mononuclear cells in cord blood or peripheral blood in step 1 is: take a portion of cord blood or peripheral blood, centrifuge in a centrifuge tube, remove After the upper layer of light yellow plasma, use normal saline to replenish the original volume of the plasma, then take a new centrifuge tube, add lymphocyte separation medium, and add the diluted blood to the lymphocyte separation medium, and separate the diluted blood from the lymphocytes The liquid is layered and centrifuged. The lower layer is red blood cells, and the middle buffy coat is mononuclear cells. After removing part of the supernatant, suck the buffy coat into a new centrifuge tube, add an equal volume of normal saline, and centrifuge to remove the supernatant ,washing. Others are the same as in the first embodiment.
specific Embodiment approach 3
[0039] Embodiment 3: This embodiment differs from Embodiment 1 or Embodiment 2 in that: the Q1 region described in Step 2 is NK cells. Others are the same as in the first embodiment.
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