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Method for culturing and cryopreservation of CIK cells by applying serum-free lymphocyte culture medium and obtained CIK cells

A lymphocyte and cell culture technology, applied in the field of cell biology, can solve problems affecting culture, cell death, and low cell viability, and achieve the effect of improving cell activity and ensuring amplification efficiency

Inactive Publication Date: 2017-05-10
TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The number of directly frozen PBMC cells is small, and the cell viability is lower than that of the logarithmic growth cells. In addition, the complexity of the components of the frozen cells causes a large number of cells to die after recovery, which affects the later culture.
After the expansion is completed, the workload of cryopreserved cells is relatively large, and the viability of recovered cells cannot be well guaranteed

Method used

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  • Method for culturing and cryopreservation of CIK cells by applying serum-free lymphocyte culture medium and obtained CIK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] (1) Coat a T175 culture flask with 10ml of serum-free lymphocyte medium containing Retronectin (1ug / ml) and CD3mAb (5ug / ml) protein, CO 2 Incubate for 2 hours.

[0068] (2) About 60 ml of conventional umbilical cord blood was collected, centrifuged by Ficoll-Hypaque density gradient, and centrifuged at 2000 rpm / min for 25 min (the acceleration was adjusted to 1 and the brake was adjusted to 1 in the centrifugation parameters).

[0069] (3) Collect and number the upper and middle plasma in step (2) in a 50ml centrifuge tube, and place the collected plasma in a 56°C water bath for 30min to inactivate complement. After that, centrifuge at 3000rpm / min for 6min to remove the precipitates in the plasma, and transfer 27ml of the upper serum to a new centrifuge tube, and place it in a 4°C refrigerator for later use.

[0070] (4) Collect the buffy coat cells in step (2), wash twice, and finally obtain 4.6*10 7 a monocyte.

[0071] (5) Take the coated T175 culture flask. Acco...

Embodiment 2

[0081] (1) Coat a T175 culture flask with 10ml of serum-free lymphocyte medium containing Retronectin (1ug / ml) and CD3mAb (5ug / ml) protein, overnight at 4°C.

[0082] (2) About 60 ml of collected conventional peripheral blood was collected, centrifuged with Ficoll-Hypaque density gradient, and centrifuged at 2000 rpm / min for 25 min (the acceleration was adjusted to 1 and the brake was adjusted to 1 in the centrifugation parameters).

[0083] (3) Collect and number the upper and middle plasma in step (2) in a 50ml centrifuge tube, and place the collected plasma in a 56°C water bath for 30min to inactivate complement. After that, centrifuge at 3000rpm / min for 6min to remove the precipitates in the plasma, and transfer 33ml of the upper serum to a new centrifuge tube, and place it in a 4°C refrigerator for later use.

[0084] (4) Collect the buffy coat cells in step (2), wash twice, and finally obtain 8*10 7 a monocyte.

[0085] (5) Take the coated T175 culture flask. Accordin...

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Abstract

The invention discloses a method for culturing and cryopreservation of CIK cells by applying a serum-free lymphocyte culture medium and obtained CIK cells. The cell culture method can develop 3*10<9> lymphocytes in a short time without introducing animal derived serum; the cell cryopreservation method can maximally improve the ratio of cryopreserved target cells and the survival rate after cell recovery. The method provides convenience for culturing, storing and related clinical research of the CIK cells.

Description

technical field [0001] The invention relates to the field of cell biology technology, in particular to a method for culturing and freezing CIK cells using serum-free lymphocyte culture medium and the obtained CIK cells, which are a set of methods that can standardize production operations. Background technique [0002] The 2000 International Cancer Biotherapy and Gene Therapy Annual Conference pointed out that "immune cell therapy is the only method in modern technology that can completely eliminate cancer cells". Dreams are becoming reality, and cancer is gradually being conquered by us. Cellular immunotherapy is the fourth largest cancer treatment modality after surgery, radiotherapy, and chemotherapy. But aging and disease constantly challenge our immune system, and it becomes necessary to store young and normal autologous immune cells. The construction of immune cell banks around the world has reached a certain scale. [0003] The current mainstream cryopreservation m...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A01N1/02
CPCC12N5/0646A01N1/0221C12N2501/2301C12N2501/2302C12N2501/24
Inventor 熊亮张冰晶鲁振宇韩洪起秦臻刘俊江徐悦黄文敬
Owner TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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