In-vitro preparation method of NK cells

A kind of NK cell and cell technology, applied in the biological field, can solve the problems of limited curative effect, insufficient source of NK cells, difficult industrial production, etc., and achieve the effect of high NK cell activity, significant tumor killing effect, and increased survival rate

Active Publication Date: 2015-06-17
奥思达干细胞有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to address the problems and deficiencies in the prior art, and provide a method for in vitro preparation of NK cells induced by embryonic stem ce

Method used

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  • In-vitro preparation method of NK cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 In vitro culture of embryonic stem cells:

[0034] 1. Put the embryonic stem cells stored in liquid nitrogen in a 37°C water bath to dissolve quickly, and the cell suspension changes from solid to liquid.

[0035] 2. Inoculate the above-mentioned cell suspension into gelatin-pretreated culture dishes, and place them at 37°C, 5% CO 2 , Cultured in an incubator with 95% saturated humidity, cultivated for 2-3 days, and after the cell confluence reached 85%-90%, it was digested and passaged with digestive juice.

[0036] 3. Passage to a new culture dish according to 1:3-1:5, replenish the culture medium and continue the culture.

[0037] 4. Digest and passage with digestive juice every 2-3 days.

Embodiment 2

[0038] Example 2 Inducing embryonic stem cells to differentiate into hematopoietic stem cells

[0039] 1. Prepare frozen OP9 cells 1 day before use in a volume of 1.5×10 5 The cells / mL cell concentration was inoculated into 6-well culture plates pre-coated with gelatin.

[0040] 2. On the second day, centrifuge the embryonic stem cells, discard the supernatant, resuspend in the differentiation medium, inoculate in a 6-well plate with OP9 cells in advance, and place at 37°C, 5% CO 2 , 95% saturated humidity incubator culture, and add cytokines 5ng / mL FL, 20ng / mL IL-15, 20ng / mL IL-6, 10 ng / mL IL-7, 10ng / mL KL.

[0041] 3. After culture 1d, transfer the differentiating ES to a new ultra-low adsorption culture dish, and supplement the above cytokines.

[0042] 4. After culturing for 2-3 days, collect the cells by centrifugation, add PBS solution to wash the cells, remove the supernatant after centrifugation (1500r / min, 10min), add PBS solution to resuspend the cells, and centri...

Embodiment 3

[0043] Example 3 Separation and purification of CD34 by immunomagnetic beads + cell

[0044] 1. Divide the collected cells into 1×10 8 Mononuclear cells were suspended in 300 μl PBS buffer, 100 μl Fc receptor blocker was added, and 100 μl anti-CD34 immunomagnetic beads were added to it, mixed evenly, incubated at 4°C for 30 min, and then centrifuged and washed twice with PBS buffer. Centrifuge at a speed of 1000rpm for 10min each time, resuspend the washed cells in 500μl of PBS buffer at a density of 10 8 each / 500μl.

[0045] 2. Fix the separation column in the MACS magnetic field, slowly pass the labeled cells through the MiniMACS separation column, and elute to remove CD34 - cells, remove the separation column from the magnetic field, pressurize the elution column with 1mL buffer, and collect CD34 + cells and counted. Analysis of CD34 by flow cytometry + Cell Purity. The results showed that the CD34 + The purity of hematopoietic stem cells is above 96%.

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Abstract

The invention relates to a preparation method of NK cells suitable for immunotherapy, and particularly provides an in-vitro preparation method of NK cells. The NK cells are formed by in-vitro induced differentiation of embryo stem cells. The preparation method comprises the following steps: carrying out amplification culture on embryo stem cells, carrying out induced differentiation on the embryo stem cells to obtain hematopoietic stem cells, purifying and amplifying the CD34+ hematopoietic cells, and carrying out induced differentiation on the hematopoietic cells to obtain the NK cells. The invention also provides an induced culture system for NK cell in-vitro preparation. The preparation method is simple; and the obtained NK cells have obvious killing effects on tumor target cells.

Description

technical field [0001] The invention relates to the field of biotechnology, and relates to an in vitro preparation method of NK cells, in particular to a method for preparing NK cells from embryonic stem cells induced differentiation. Background technique [0002] NK cells (natural killer cells), also known as natural killer cells, are a type of CD56 + , CD3 - Lymphocytes are the effector cells of the natural immune system in the body, and they can recognize tumor and virus-infected cells without antigen pre-sensitization. It is located in the first line of defense of the body's defense system. At the same time, it can regulate the acquired immune response by secreting a variety of cytokines and chemokines in the early stage, so it is also a bridge connecting innate immunity and acquired immunity. Non-self cells play an important role. [0003] At present, NK cell therapy is obtained clinically by culturing in vitro, expanding the NK cells in the patient's own peripheral ...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 周萱
Owner 奥思达干细胞有限公司
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