Method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and prepared combined immune cells

A DC-CIK, immune cell technology, applied in blood/immune system cells, biochemical equipment and methods, animal cells, etc., can solve problems such as tumor cell killing, reduce the probability of cell contamination, reduce exogenous pyrogen, The effect of enhancing tumor-killing activity

Inactive Publication Date: 2016-06-08
TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these types of cells have certain and clinical limitations, and cannot efficiently kill various tumor cells

Method used

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  • Method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and prepared combined immune cells
  • Method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and prepared combined immune cells
  • Method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and prepared combined immune cells

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preparation example Construction

[0049] Combined immune cells prepared by the above preparation method.

[0050] The present invention uses a complete set of immune cell culture medium and various cytokines to induce DC cells, CIK cells and NK cells separately under the condition of using autologous serum. And at an appropriate time point, the cells are mixed for culture and mixed application, so as to achieve the purpose of increasing the tumor killing activity of various immune cells.

[0051] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments: the immune cell culture medium is TexMACS immune cell culture medium produced by Miltenyi Company.

Embodiment 1

[0053] 1. Collect 120ml of peripheral blood, dilute it with 240ml of normal saline, and use Ficoll density gradient centrifugation to efficiently separate mononuclear cells.

[0054] 2. After separation, transfer the upper plasma layer to a 500ml centrifuge cup to obtain a total of 300ml diluted plasma, and inactivate it in a water bath at 56°C for 30 minutes. After inactivation, centrifuge at 12,000rpm for 10min, and filter the supernatant with a 0.4um filter membrane to obtain nearly 290ml of autologous serum, which is divided into 40ml / tubes for later use.

[0055] 3. Prepare cell induction medium for use:

[0056] DC cell induction medium: the immune cell culture medium contains autologous serum prepared in step 2 with a volume concentration of 8%, IL-4 of 150IU / ml and GM-CSF of 1000IU / ml;

[0057] CIK cell induction medium: the immune cell medium contains autologous serum prepared in step 2 with a volume concentration of 6% and IL-2 of 1000IU / ml;

[0058] NK cell induct...

Embodiment 2

[0072] 1. Collect 120ml of umbilical cord blood, dilute it with 240ml of normal saline, and use Ficoll density gradient centrifugation to efficiently separate mononuclear cells.

[0073] 2. After separation, transfer the upper plasma layer to a 500ml centrifuge cup to obtain a total of 310ml diluted plasma, and inactivate it in a water bath at 56°C for 30 minutes. After inactivation, centrifuge at 12,000rpm for 10min, and filter the supernatant with a 0.4um filter to obtain nearly 298ml of autologous serum, which is divided into 40ml / tubes for later use.

[0074]3. Prepare cell induction medium for use:

[0075] DC cell induction medium: the immune cell culture medium contains autologous serum prepared in step 2 with a volume concentration of 8%, IL-4 of 150IU / ml and GM-CSF of 1000IU / ml;

[0076] CIK cell induction medium: the immune cell medium contains autologous serum prepared in step 2 with a volume concentration of 6% and IL-2 of 1000IU / ml;

[0077] NK cell induction me...

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Abstract

The invention discloses a method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and the prepared combined immune cells. Ficoll density gradient centrifugation is used for efficient separation, a mononuclear cell is obtained, sufficient quantities of DCs, CIKs and NKs are obtained through cell culture bags and an immune cell induction culture system, and finally, the induced cells are cultured in a combined manner and applied to clinical treatment, so that a tumor killing effect is realized. The DCs, the CIKs and the NKs are subjected to induction culture respectively with adoption of TexMACS immune cell culture media produced by Miltenyi Biotec, autoserum, various cytokines and a combined culture technology, the cells are mixed for culture and application at certain point in time, application of fetal calf serum is avoided, the pollution rate of an exogenous pyrogen and an exogenous allergen is reduced, and the tumor killing activity of the finally mixed cells is enhanced simultaneously; with adoption of a cell culture bag technology, the cell contamination rate is reduced, and the method is suitable for clinical treatment and application.

Description

technical field [0001] The present invention relates to a method for preparing anti-tumor combined immune cells at the same time, especially a method that uses peripheral blood or umbilical cord blood as a raw material, cooperates with TexMACS immune cell culture medium produced by Miltenyi Company, and cooperates with autologous serum and various cytokines Components to obtain anti-tumor combined immune cells DC-CIK and NK. technical background [0002] Tumor immune cell therapy, as the fourth treatment method for tumors, is gradually being recognized by the medical community because of its safety, effectiveness and no side effects, and is being vigorously developed as a weapon for human beings to finally completely defeat tumors. At present, clinically commonly used immune cell therapy includes DC (Dendritic cell) cells, CIK (Cytokine induced killer) cells, NK (Natural killer) cells and so on. However, these types of cells have their definite and clinical limitations, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0784C12N5/0786
CPCC12N5/0639C12N5/0646C12N2501/22C12N2501/2302C12N2501/2304C12N2501/2312C12N2501/2315C12N2501/24C12N2501/25C12N2501/51C12N2501/515C12N2501/599C12N2502/1157
Inventor 韩洪起鲁振宇刘俊江张冰晶秦臻
Owner TIANJIN PURUI SAIER BIOLOGICAL TECH CO LTD
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