Preparation method of antitumor adoptive immune cells and prepared immune cells

An immune cell and anti-tumor technology, which is applied in the field of preparation of immune active cells and anti-tumor adoptive immune active cells, can solve the problems of large side effects of treatment and difficult cell sources, and achieves increasing cell proliferation multiples, improving induction effect, and improving The effect of the overall tumoricidal effect

Inactive Publication Date: 2012-01-18
深圳市中美康士生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the international tumor immunotherapy, it has experienced lymphokine-activated killer cells (LAK cells), anti-CD3 monoclonal antibody-induced killer cells and tumor infiltrating lymphocytes (TIL cells). The source is difficult (such as TIL) or the side effects of treatment are relatively large (such as LAK), so there are fewer and fewer studies and reports

Method used

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  • Preparation method of antitumor adoptive immune cells and prepared immune cells
  • Preparation method of antitumor adoptive immune cells and prepared immune cells
  • Preparation method of antitumor adoptive immune cells and prepared immune cells

Examples

Experimental program
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Effect test

Embodiment 1

[0041] The collection and separation of embodiment 1 peripheral blood mononuclear cells (PBMC):

[0042] Aseptically extract 40-45ml of peripheral blood, add it into a 50ml centrifuge tube with heparin saline according to the blood culture operation, shake gently, and fully anticoagulate. Dilute 1:1 with normal saline and put into two 50ml centrifuge tubes. Slowly add (25ml lymphocyte separation medium, 4 50ml centrifuge tubes) on the interface of the separation medium. Centrifuge at room temperature, 2000rpm: 20 minutes. Aspirate the PBMCs, transfer them into four new 50ml centrifuge tubes, add normal saline to 50ml, and blow gently with a straw to mix. Centrifuge for 20 minutes and discard the supernatant. Resuspend the cells in 4 centrifuge tubes with normal saline, combine them into 1 centrifuge tube, add normal saline to 50ml, mix well, take 20ul of samples and count. Centrifuge and wash 50ml of the cell suspension twice at 2300rpm for 10 minutes; discard the supernat...

Embodiment 2D

[0043] Example 2 DC induction and amplification:

[0044] Gently blow the above-mentioned cells adhered to the wall for 3 hours with a pipette, suck out the non-adhered cells and the culture medium, transfer them into a 50ml centrifuge tube, and centrifuge at 2300rpm for 10 minutes. The supernatant was added back to the plate along the side. Add GM-CSF 1000 μg / ml; IL-4 1000 μg / ml, culture at 37° C., 5% CO2. After that, add factors (GM-CSF, IL-4) and AIM-V medium every day to adjust the cell density to 1*106 / ml. After culturing until the 7th day, add autologous tumor tissue lysate and dendritic cell maturation-promoting factor to the culture dish, so that the final concentration of lysate protein is 50 μg / ml, and the final concentration of maturation-promoting factor is 500 μg / ml. On the 8th day, add GM-CSF 1000μg / ml; IL-4 1000μg / ml, cultured at 37°C, 5% CO2, matured on the 9th day, and collected DC.

[0045] Preparation method of autologous tumor cell lysate:

[0046] Draw...

Embodiment 3

[0047] Induction and expansion of embodiment 3 CIK cells:

[0048] Dissolve 1 tube of Retronectin 2.5 mg in 250 ml of normal saline in advance to make a coating solution with a final concentration of 10 μg / ml. Take 10ml of coating solution and put it on T-75cm 2 in a culture flask and let stand overnight. On the second day, the coating solution was discarded, and the culture flask was washed twice with physiological saline to obtain a culture flask coated with Retronectin factor.

[0049] The unattached cells after adherent culture in Example 1 for 3 hours were resuspended with 10ml of AIM-V medium, transferred into a T-75cm2 culture flask coated with Retronectin, added IFN-r1000μg / ml, and incubated at 37°C , 5% CO2 culture environment to continue the culture. On the second day, 2 μg / ml of CD3 McAb, 1 μg / ml of CD28 McAb, and 1000 μg / ml of IL-2 were added to the culture flask. After that, add medium and cytokine IL-2 every day, and adjust the cell density in the culture fla...

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Abstract

The invention discloses a preparation method of antitumor adoptive immune cells. The method comprises the following steps of: sampling and separating a single peripheral blood karyocyte; and adding a cell factor for inducting and culturing to obtain a cytokine induced kill cell (CIK), wherein the cell factor comprises CD28. In a CIK induced system, the cell factor CD28 is induced, so that the inducing effect is enhanced, and the cell proliferation multiple is increased simultaneously.

Description

Technical field: [0001] The invention relates to the field of biotechnology, in particular to a method for preparing anti-tumor adoptive immune active cells and the prepared immune active cells. Background technique: [0002] Although tumor is an ancient disease, in the past half a century, tumor has gradually become a frequently-occurring and common disease, and it is the first and second cause of death among residents, seriously threatening human health. [0003] Tumor biological therapy is the fourth major treatment method after surgery, radiotherapy and chemotherapy. Since the development of traditional surgery, radiotherapy and chemotherapy has entered a plateau, people are paying more and more attention to the biological treatment of tumors. In the past 10 years, the biological treatment of tumors has been greatly developed, and its important role in improving the quality of life of patients and reducing the recurrence rate has been more and more recognized and valued...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/0783
Inventor 李晓祥
Owner 深圳市中美康士生物科技有限公司
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