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NK cell amplification method capable of remarkably enhancing killing activity

A technology of NK cells and killing activity, which is applied in the field of cell biology, can solve the problems of limited expansion times and low killing activity, and achieve the effect of enhancing activity

Pending Publication Date: 2021-12-31
上海劲泉医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] 1. Adding soluble recombinant growth factors, IL-2, IL-15 and other conventional NK cell culture methods has the disadvantages of limited expansion and low killing activity
[0007] 2. Using K562 as trophoblast cells (K562IL15-4-IBBligand) for NK cell expansion and culture after gamma radiation does not meet GMP standards including safety concerns

Method used

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  • NK cell amplification method capable of remarkably enhancing killing activity
  • NK cell amplification method capable of remarkably enhancing killing activity
  • NK cell amplification method capable of remarkably enhancing killing activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Construction of recombinant baculovirus

[0065] 1. Construction of pFB-HU-IL2, pFB-HU-IL15, pFB-HU-IL21 and pFB-HU-IFN-γ plasmid vectors

[0066] 1) Query the human IL-2 gene sequence, human IL-15 gene sequence, human IL-21 gene sequence and human IFN-γ gene sequence respectively on the NCBI website;

[0067] 2) using the PCR amplification method to obtain the human IL-2 gene sequence, human IL-15 gene sequence, human IL-21 gene sequence and human IFN-γ gene sequence for future use;

[0068] 3) The pFBGFPR plasmid with CMV promoter (such as Figure 1a shown) to carry out AgeI-AccI double enzyme digestion, and then insert IL2, IL21, IL15 and IFN-γ genes respectively to obtain the following recombinant plasmid: pFB-HU-IL2 (such as Figure 1b shown), pFB-HU-IL15 (as shown Figure 1c shown), pFB-HU-IL21 (such as Figure 1d shown) and pFB-HU-IFN-γ (as Figure 1e shown),

[0069] 2. Preparation of pFB-HU-IL2, pFB-HU-IL15, pFB-HU-IL21 and pFB-HU-IFN-γ recom...

Embodiment 2

[0077] Example 2: Preparation of mesenchymal stem cells

[0078] Prepare reagents:

[0079]a, Mesenchymal stem cell serum-free medium 500ml / bottle at 2-8°C, store in the dark (YDcon)

[0080] b, Mesenchymal stem cell serum-free medium supplement 5ml / bottle -20°C dark storage (YDcon)

[0081] c, 0.05% trypsin-EDTA (sigma)

[0082] d, Pancreatin inhibitor 500ml / bottle stored at 2-8°C in the dark (YDcon)

[0083] e, PBS x 20

[0084] The preparation steps of mesenchymal stem cells (MSC cells) are as follows:

[0085] 1. Collect healthy umbilical cords from the obstetrics and gynecology department, put them in a 50ml sterile centrifuge tube containing 25ml (150u Qingda / PBS), and cold-chain them to the laboratory at 4°C.

[0086] (Requirement: When taking the umbilical cord, take the umbilical cord blood at the same time, and send the umbilical cord blood to Adikon for testing, or the hospital provides the maternal blood report.)

[0087] 2. A small sample of umbilical cord t...

Embodiment 3

[0098] Example 3: Preparation of activated NK cells

[0099] In the preparation procedure of this example, the culture medium does not contain animal protein components, but contains recombinant or medical-grade human protein serum-free cell culture medium, which has high proliferative properties for human peripheral blood T lymphocytes, and is suitable for the activation and expansion of NK cells. It is the best medium for cell immunotherapy.

[0100] 1. Preparation of 24-well culture plate

[0101] (1) In a 24-well culture plate, add Anti-CD16 MAb stock solution.

[0102] (2) Gently shake the culture flask to allow the solution to spread on the surface of the culture flask.

[0103] (3) Incubate for 1 hour at room temperature or store at 4°C until removed before use. Coating solution (MAb) was removed.

[0104] (4) Wash the culture flask twice with physiological saline. Washed culture flasks should be used immediately.

[0105] 2. Blood separation

[0106] (1) Collect...

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Abstract

The invention provides an NK cell amplification method capable of remarkably enhancing killing activity. The method comprises the following steps: (1) constructing a recombinant baculovirus containing a cytokine gene required by NK amplification and a mammalian promoter; (2) infecting MSC cells by using the recombinant baculovirus prepared in the step (1); and (3) adding the infected MSC cells in the step (2) to activated and cultured NK cells for co-culture, and continuing to perform passage multiplication culture on the NK cells after co-culture for 24-60 hours. According to the amplification method, the killing activity of the NK cells can be remarkably enhanced and the amplification factor can be greatly improved. Meanwhile, the amplification method meets the GMP requirements and clinical use standards in the whole process.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a NK cell expansion method capable of significantly enhancing killing activity. Background technique [0002] NK cells are called natural killer cells (Natural kill cells), which are an important part of the human innate immune system. The phenotype of the cells is CD3-CD56+. When NK cells play the role of killing target cells, they do not require pre-sensitization of antigens and are not restricted by MHC. Moreover, the difference in the density of NK cell CD56 molecule expression divides NK into two subgroups, CD56dim and CD56bright. CD56dim accounts for more than 90% of NK cells, mainly due to cytotoxicity and strong killing activity; CD56bright can produce a large number of cells Factors that mainly play an immunomodulatory role. [0003] Unlike T cells and B cells, NK lacks gene rearrangement, and unlike T cells and B cells, it does not recognize target cells through specific TC...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/10C12N15/866C12N15/24C12N15/26C12N15/23C12N7/01
CPCC12N5/0646C12N15/86C12N5/0665C12N7/00C07K14/55C07K14/5443C07K14/54C07K14/57C12N2502/137C12N2510/00C12N2710/14021C12N2710/14043
Inventor 张尚权
Owner 上海劲泉医疗科技有限公司