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Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells

A technology of specificity and culture method, which is applied in the field of effectively expanding CIK cells and improving their specific tumor killing, can solve the problems of weak specific tumor killing ability, cumbersome, unsatisfactory CIK cell proliferation multiples, etc., so as to save operation , the effect of stimulating activity improvement

Active Publication Date: 2016-04-20
FUJIAN YINFENG STEM CELL ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for effectively amplifying CIK and improving its specific tumor killing ability, which solves the problem that the multiplication factor of CIK cells obtained at present is not ideal; during the induction process of CIK, it is necessary to continuously supplement various cytokines, which is relatively cumbersome ; The characteristics of CIK cell broad-spectrum tumor killing lead to its weak specific tumor killing ability and other shortcomings

Method used

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  • Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells
  • Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells
  • Method for effectively amplifying CIK cells and improving specific tumor killing capability of CIK cells

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Effect test

Embodiment 1

[0040] Mononuclear cells were obtained by separating with Ficoll lymphocyte separation medium, transferred to a T175 culture flask, added CD3mAb, CD28mAb, IFN-γ, IL-2, IL-15, and added IL-2 and IL-15 on the fourth day On the 6th day, the cells were divided into flasks. On the 8th and 9th days, the cells in the flasks were transferred to 1.8L cell culture bags, and IL-2 and IL-15 were added. On the 11th day, the cells were cultured in separate bags and supplemented with IL- 2 and IL-15, continue to induce expansion, harvest cells within 15-21 days. After incubating the harvested cells with an anti-EGFR×anti-CD3 bifunctional antibody at room temperature for 30 minutes, the cells were washed with PBS and collected to prepare a CIK cell preparation that specifically kills EGFR-positive tumor cells.

[0041] Aseptically collect 60ml of peripheral blood from healthy people or patients, transport it to the laboratory for preparation within 2 hours, and separate PBMC and in vitro cult...

Embodiment 2

[0057] 1. Mononuclear Cell (PBMC) Isolation

[0058] (a) Take 500ul of peripheral blood and use a blood cell analyzer to count and detect activity: mix peripheral blood with normal saline in equal volumes, then add 40ml of Ficoll lymphocyte separation medium and centrifuge to separate mononuclear cells; centrifugation parameters: 1600rpm, 20 degrees Celsius, 20 minutes;

[0059] (b) Take buffy coat, add normal saline to a total volume of 80ml, centrifuge and wash twice; centrifugation parameters: 2000rpm, 20 degrees Celsius, 8 minutes;

[0060] (c) The cells were resuspended in the serum-free basal medium, and the cell count was performed using a cell counter to calculate the cell yield.

[0061] 2. CIK induction culture

[0062] (1) After counting the isolated PBMCs, press 9×10 5 Cells / ml density inoculated in T175 culture flask, add final concentration of 500ng / mlCD3mAb, 1500ng / mlCD28mAb, 4400IU / mlIFN-γ, 4400IU / mlIL-2, 150ng / mlIL-15, supplement serum-free medium to 50ml. ...

Embodiment 3

[0072] 1. Mononuclear Cell (PBMC) Isolation

[0073] (a) Take 500ul of peripheral blood and use a blood cell analyzer to count and detect activity: mix peripheral blood with normal saline in equal volumes, then add 40ml of Ficoll lymphocyte separation medium and centrifuge to separate mononuclear cells; centrifugation parameters: 1600rpm, 20 degrees Celsius, 20 minutes;

[0074] (b) Take buffy coat, add normal saline to a total volume of 80ml, centrifuge and wash twice; centrifugation parameters: 2000rpm, 20 degrees Celsius, 8 minutes;

[0075] (c) The cells were resuspended in the serum-free basal medium, and the cell count was performed using a cell counter to calculate the cell yield.

[0076] 2. CIK induction culture

[0077] (1) After counting the isolated PBMC, press 10×10 5 Cells / ml were inoculated in T175 culture flasks, and the final concentration was 600ng / mlCD3mAb, 1600ng / mlCD28mAb, 4800IU / mlIFN-γ, 4800IU / mlIL-2, 160ng / mlIL-15, and serum-free medium was added to 5...

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Abstract

The invention provides a method for effectively amplifying cytokine-induced killer (CIK) cells and improving specific tumor killing capability of the CIK cells. The method comprises the following steps: separating to obtain mononuclear cells; transferring the obtained mononuclear cells to a culture flask; adding CD3mAb with the final concentration of 400-600 ng / ml, CD28mAb with the final concentration of 1400-1600 ng / ml, IFN-gamma with the final concentration of 4000-4800 IU / ml and IL-2 with the final concentration of 4000-4800 IU / ml, IL-15 with the final concentration of 140-160 ng / ml; on the fourth day, replenishing fresh culture solution including 600-1000 IU / ml of IL-2 and 20-30 ng / ml of IL-15; on the sixth day, culturing the cells in separated flasks; on the eighth and ninth days, respectively transferring cells of the flask to a cell culture bag of 1.8 L, and replenishing fresh culture solution including 600-1000 IU / ml of IL-2 and 20-30 ng / ml of IL-15; at the eleventh day, culturing the cells in separated bags, and replenishing fresh culture solution including 600-1000 IU / ml of IL-2 and 20-30 ng / ml IL-15; continuously inducing and amplifying till the cells are obtained from the fifteenth day to the twenty-first day; incubating the obtained cells for 30-40 minutes at the room temperature by utilizing anti-EGFR and anti-CD3 bifunctional antibodies, washing the incubated cells in normal saline, and collecting the cells, thereby obtaining specific tumor-killing CIK cell preparations.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for effectively expanding CIK cells and improving their specific tumor killing. Background technique [0002] Malignant tumors are the second killer of human health after cardiovascular diseases. In recent years, the incidence and mortality of tumors have been increasing year by year, and the incidence of tumors has become younger. With the rapid development of molecular biology and immunology and other disciplines, tumor biological immunotherapy has become the fourth therapy after surgery, radiotherapy and chemotherapy. Among them, tumor adoptive immune cell therapy has become a current research hotspot, which refers to the reinfusion of immune cells with anti-tumor activity expanded in vitro into the patient, thereby directly or indirectly stimulating the body's immune response to kill tumor cells. kind of cell therapy. It has the advantages of individuality,...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0638C12N2501/2302C12N2501/2315C12N2501/24C12N2501/998
Inventor 肖桂清苏爱盟
Owner FUJIAN YINFENG STEM CELL ENG
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