Method for proliferating natural killer cells and culture medium composition

A technology of natural killer cells and expansion culture, applied in animal cells, vertebrate cells, blood/immune system cells, etc., to achieve the effect of low culture cost and significant cytotoxic effect

Active Publication Date: 2015-06-03
HANGZHOU S EVANS BIOSCI LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The purpose of the present invention is to overcome the defect that existing natural killer cell expansion methods require co-cult

Method used

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  • Method for proliferating natural killer cells and culture medium composition
  • Method for proliferating natural killer cells and culture medium composition
  • Method for proliferating natural killer cells and culture medium composition

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Embodiment approach

[0027] Wherein, the first culture medium and the second culture medium may or may not contain serum. According to a particularly preferred embodiment of the present invention, the first culture medium further contains 8-12% by volume of serum, so The second medium also contains 0.5-10% by volume of serum.

[0028] Wherein, the serum contained in the first culture medium and the second culture medium can be serum routinely used in cell culture, such as fetal bovine serum; according to a particularly preferred embodiment of the present invention, the serum is obtained from Autologous serum isolated from the whole blood used to isolate the lymphocytes.

[0029]Wherein, it is particularly preferred that the second medium used in step (2) also contains 6-10% by volume of serum; the second medium used in step (3) also contains 0.5-2% by volume of Serum; more preferably, the second medium used in step (2) also contains 7-9% serum by volume; the second medium used in step (3) also co...

preparation Embodiment 1

[0039] This preparation example is used to illustrate the preparation of lymphocytes and autologous serum.

[0040] Sodium heparin was dissolved in normal saline to obtain a 20 U / ml sodium heparin normal saline solution. Whole blood was mixed with 20 U / ml heparin sodium saline solution at a volume ratio of 5:1 and then centrifuged at 800×g for 5 minutes to obtain blood cells in the lower layer and plasma in the upper layer. Lymphocytes are then separated from the blood cells with a lymphocyte separation medium, specifically, the blood cells in the lower layer are suspended with 3 times the volume of normal saline and then slowly added to 1 times the volume of the lymphocyte separation medium (containing Ficoll400 and diatrizoic acid Meglumine, purchased from Tianjin TBD Company, product number is LTS1077) on the liquid surface, 400-600 * g speed down centrifugation 15-30 minutes, after centrifugation finishes, be divided into the residual plasma layer that is positioned at upp...

Embodiment 1

[0044] Amplify according to the following steps: (1) inoculate lymphocytes in the first culture medium to carry out the first expansion culture to obtain the first expansion culture product; (2) combine the first expansion culture product with the second The medium is mixed according to the volume ratio of 1:1 and the second expansion culture is carried out to obtain the second expansion culture product; (3) the second expansion culture product is replaced by the first expansion culture product and returned to step (2) The operation and the operation of step (2) are carried out 6 times in a cycle to obtain the amplified product.

[0045] Wherein, the inoculation concentration of inoculating lymphocytes in the first culture medium is 10 6 cells / mL medium. The first medium contains basal medium (the product number purchased from Invitrogen is the opTmizerTMCTSTM T-Cell Expansion SFM medium of A1048501, the same below) and 15mg / mL of L-glutamine, 150IU / mL of cyanine sulfate Ama...

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Abstract

The invention discloses a method for proliferating natural killer cells. The method comprises the following steps: (1) separating lymphocytes from whole blood; inoculating the lymphocytes in a first culture medium for first proliferation culture to obtain a first proliferation culture product; (2) mixing the first proliferation culture product with a second culture medium according to a volume ratio of 1 to (0.5-2) and performing second proliferation culture to obtain a second proliferation culture product; (3) replacing the first proliferation culture product with the second proliferation culture product, and repeatedly operating according to step (2) for 2-7 times. The invention also provides a culture medium composition. The culture medium composition contains a basal culture medium, L-glutamine, gentamicin sulfate, mannatide, interleukin 2 and interleukin 15. The proliferation method disclosed by the invention is a natural killer cell proliferation method which is simple, convenient, high in efficiency, easy to operate and relatively high in safety.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a natural killer cell expansion method and a culture medium composition. Background technique [0002] Natural killer cells (NK cells) are important immune regulatory cells for the body to resist malignant tumors and viral infections. NK cells can secrete perforin, NK cytotoxic factor (NK cytotoxic factor, NKCF) and tumor necrosis factor (TNF). Among them, perforin is a medium released by NK cell cytoplasmic granules to kill target cells. Perforin purified from cytoplasmic granules of NK cells can dissolve a variety of tumor cells in vitro, and anti-perforin antibodies can inhibit the killing activity. IL-2 increases the transcription of the perforin gene. IL-6 can promote the induction of IL-2 on perforin gene transcription. Serine esterase may activate perforin. Among them, NKCF can selectively kill and lyse target cells after binding to target cells. Among them, TNF ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 张强张荣胜李程项春生
Owner HANGZHOU S EVANS BIOSCI LTD
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