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Construction method of CD3<->CD56<+>NK cell high-efficient multiplication culture system

A technology of CD3-CD56 and NK cells, which is applied in the field of expansion of biological products, can solve the problems of cumbersome methods, unfavorable promotion, restrictions, etc., and achieve the effect of high killing efficiency, easy promotion and low cost

Inactive Publication Date: 2009-03-18
JIANGSU PROVINCE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First of all, the mode of action of CD137 and IL-15 needs to be improved. Currently, retroviruses are used to simultaneously transfect 4-1BBL and IL-15 into tumor cell lines. Although the amplification efficiency can be improved, the method is cumbersome and not conducive to popularization. The use of retroviruses has certain risks and the introduction of transgenic cells in the culture system also restricts CD3 - CD56 + Isolation and purification of NK cells

Method used

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  • Construction method of CD3&lt;-&gt;CD56&lt;+&gt;NK cell high-efficient multiplication culture system
  • Construction method of CD3&lt;-&gt;CD56&lt;+&gt;NK cell high-efficient multiplication culture system
  • Construction method of CD3&lt;-&gt;CD56&lt;+&gt;NK cell high-efficient multiplication culture system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1: the preparation of the plastic culture plate of high adhesion

[0031] 1. Poly-D-Lysine or Poly-L-Lysine solution using sterile distilled water ddH 2 O 1:10 dilute the poly-lysine solution.

[0032] 2. Add Poly-D-Lysine or Poly-L-Lysine dilution (300 μl) to a plastic culture plate, culture dish or culture bottle, and place at room temperature for 5-10 minutes.

[0033] 3. Suck off the Poly-D-Lysine or Poly-L-Lysine dilution in the plastic plate, petri dish or culture bottle, and dry overnight at room temperature for use.

Embodiment 2

[0034] Example 2: Coating of anti-CD137 mAb and IL-15

[0035] 1. Anti-CD137 mAb and IL-15 were added to phosphate buffered solution (PBS) at the same time, and diluted to concentrations of 1-10 μg / ml and 10-100 ng / ml respectively.

[0036] 2. Add the above anti-CD137 mAb and IL-15 mixed dilution (300 μl) into the plastic culture plate, culture dish or culture bottle prepared in Example 1, and overnight at 4°C.

[0037] 3. Suck off the diluent from the plastic plate or culture dish or culture bottle, and set aside.

Embodiment 3

[0038] Embodiment 3: the culture of NK cell

[0039] 1. Peripheral blood mononuclear cells (PBMC), CD3 - CD56 + Preparation of NK cells: Take 100 ml of peripheral blood from normal volunteers (obtained from Nanjing Red Cross Blood Station), separate (Ficoll) to obtain PBMC. After washing the cells, dilute to 3×10 with RPMI1640 medium containing 10% calf serum 6 / ml. Placed in a 6-well culture plate (2ml / well) for 2 hours (5% CO 2 , 37° C.) gently suck out the suspended cells to obtain PBMCs. CD3 - CD56 + NK cells were obtained from PBMCs (purity >95%) using magnetic bead negative selection or flow cytometric sorting.

[0040] 2. Expansion and enrichment of NK cells

[0041] PBMC was adjusted to a concentration of 1×10 with RPMI1640 containing 10% calf serum. 5 / ml, and add 500IU / ml IL-2, then add it to the culture plate prepared in Example 2, culture for 3-6 days, and collect the cells to be the amplified CD3 - CD56 + NK cells. Collect cell counts and use flow cyto...

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Abstract

The invention discloses a method for constructing CD3<->CD56<+>NK cell efficient culture system, and mainly provides a culture system for adhering invitro mass amplified CD3<->CD56<+>NK cells which consist of anti-CD137mAb and IL-15 by using Poly-D-Lysine or Poly-L-Lysine as adhesion agent. Compared with the prior other methods, the method obviously has the advantages of simple and reasonable method, easy promotion, less required raw materials, low cost, shorter preparation cycle, high amplification efficiency, high killing efficiency to tumor cells and so on. Experiments prove that the capability of killing A549 of the CD3<->CD56<+>NK cells which are amplified by the method is increased by one time compared with CD3<->CD56<+>NK cells which are amplified by other known cytokines or cytokine combination in the same cell concentration.

Description

technical field [0001] The present invention relates to a poly-D-Lysine adherent mouse anti-human CD137 monoclonal antibody anti-CD137 mAb and recombinant human cytokine IL-15, which can be used to treat tumor diseases CD3 - CD56 + The invention relates to a construction method of an NK cell expansion culture system, which belongs to a biological product expansion method. Background technique [0002] NK cells were discovered in 1975. They are large granular lymphocytes morphologically, derived from bone marrow, and account for 5%-10% of the total number of peripheral blood lymphocytes. It has a broad-spectrum anti-tumor cell effect, especially on lymphoma and leukemia cells. It is the first line of defense against tumor immunity, targeting CD3 - CD56 + The killing effect of NK sensitive target cells (such as K562 cells), the killing function is not affected by whether the target cells express MHC; CD3 - CD56 + NK cells are also an important link connecting innate immun...

Claims

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Application Information

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IPC IPC(8): C12N5/08C12N5/0783
Inventor 束永前居颂文
Owner JIANGSU PROVINCE HOSPITAL
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