In vitro expansion method of NK cells and kit for method

A technology of NK cells and cells, which is applied in the direction of cell culture active agents, animal cells, vertebrate cells, etc., can solve the problems of troublesome safety, high cost and insufficient amplification methods, and achieve the effect of simple use and meeting clinical needs

Active Publication Date: 2018-06-22
JILIN TUO HUA BIOTECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In order to solve the problems of insufficient NK cell purity, low amplification factor, high cost and cumbersome amplification method and insufficient safety in the prior a

Method used

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  • In vitro expansion method of NK cells and kit for method
  • In vitro expansion method of NK cells and kit for method
  • In vitro expansion method of NK cells and kit for method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: In vitro expansion of NK cells in umbilical cord blood samples

[0029] 1. Melt 1.5 μg of HER-2 antibody (Tongli Haiyuan, product number: TL-503) as an antibody for inducing differentiation of NK cells, add it to 15ml of normal saline, mix well, and add it to the 175cm 2 The bottom area of ​​the cell culture bottle, and the liquid is fully dispersed at the bottom of the bottle, and placed in a refrigerator at 4°C overnight.

[0030] 2. Aseptically collect 50ml of umbilical cord blood from healthy puerperas (collected from the Department of Obstetrics and Gynecology, Siping Hospital, China Medical University with the consent of the patient);

[0031] 3. Transfer the collected blood sample to a 50ml centrifuge tube (Corning, product number: 430828), and use a Thermo 4KR centrifuge at room temperature to adjust the speed to 800g, and centrifuge for 10 minutes;

[0032] 4. Absorb the upper layer of plasma for later use during cultivation; dilute the blood accord...

Embodiment 2

[0040] Example 2: Cell mass, amplification factor and viability after cell expansion

[0041] Total cell expansion factor: the cells obtained on the 7th, 14th, and 21st days of culture in the above examples were stained with trypan blue and then counted with a hemocytometer, and the current total cells were divided by the total number of mononuclear cells before culture , the value is the amplification factor of the cells, cell viability (%)=number of unstained cells / total number of observed cells×100. The results are shown in Table 1 and Figure 1A and Figure 1B , Figure 1A In the picture under the inverted microscope on the 7th day of culture, it can be seen that the cells are bright and in good condition, the proliferation spheres are scattered, and the cells are in the shape of irregular long sweet potatoes; Figure 1B The picture under the inverted microscope on the 14th day of culture shows that the cell density is significantly higher than that of the 7th day, and ...

Embodiment 3

[0044] Example 3: Detection of CD3 before and after cell expansion by flow cytometry - CD16 + CD56 + NK cell ratio

[0045] Flow cytometry detection method: Take the cell samples obtained after culturing the 7th day, 14th day and 21st day in Example 1 5×10 5 Cells / tube, wash 2 times with PBS. Add flow detection antibodies respectively for double-labeled flow phenotype detection: FITC-labeled mouse anti-human CD3 antibody (BioLegend, Cat. No. 300306) and APC-labeled mouse anti-human CD16 antibody (BioLegend, Cat. Product No. 304610); incubate at room temperature for 20 min, wash with PBS twice, and analyze the cells by flow cytometry.

[0046] The result is as Figure 2A to Figure 2C As shown, after 14 days of induction culture CD3 - CD16 + CD56 + About 18.4% of NK cells (Q1 area) on day 7 ( Figure 2A ) increased to about 87.8% ( Figure 2B ), CD3 was detected by flow cytometry after 21 days of culture - CD16 + CD56 + NK cells (Q1 area) increased to 96.8% ( Figu...

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Abstract

The invention provides an in vitro expansion method of NK cells and a kit for the method. According to the method, the NK cells obtained by carrying out in vitro induction and amplification on mononuclear cells which are separated from umbilical cord blood or peripheral blood are subjected to flow cytometry, which shows that the proportion of CD3<->CD16<+>CD56<+> cells reaches 90 percent or above;in addition, the culture better meets the requirements of clinical preparation; the NK cells show good killing activity in vitro and in vivo. On the other hand, the invention provides application ofan HER-antibody and alpha-mannatide for induced differentiation of the NK cells, as well as rabbit anti-human thymocyle immunoglobulin, 1L-15 and IL-2 for amplifying the NK cells in preparing a kit for obtaining high-purity NK cells in vitro.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a high-purity clinical-grade NK cell in vitro amplification method and kit. Background technique [0002] Cell therapy is another tumor treatment method after surgery, radiotherapy and chemotherapy. It is generally used as an auxiliary means of surgery, radiotherapy and chemotherapy to prolong the life of patients and remove residual tumor cells in the body after surgery. NK cells are a special group of lymphocytes that are not restricted by the major histocompatibility complex (MHC) and can directly kill target cells. They are considered to be important effector cells for tumor immunotherapy. Associated with immune regulation and, in some cases, involved in hypersensitivity reactions and autoimmune diseases. The number of NK cells in peripheral blood is very limited, so the clinical application of NK cells is limited. The current method of obtaining clinical-grade NK cells is in v...

Claims

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Application Information

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IPC IPC(8): C12N5/0783A61K35/17A61P35/00
CPCA61K35/17C12N5/0646C12N2500/90C12N2501/04C12N2501/2302C12N2501/2315C12N2501/998
Inventor 姜丽君毕薇薇李超邢亚丽
Owner JILIN TUO HUA BIOTECH
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