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Culturing method for NKT cells

A technology of NKT cells and culture methods, applied in the field of NKT cell culture and three-dimensional culture of NKT cells, can solve the problem of not being able to contact the culture medium well, and achieve the effect of sufficient area, high multiplication factor and high killing effect.

Inactive Publication Date: 2016-05-18
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The culture method of the present invention utilizes hydroxypropyl methylcellulose to establish a three-dimensional structure of the culture medium in the early stage of NKT cell culture, so as to solve the problem that NKT cells form agglomerated cells and accumulate at the bottom during the proliferation process and cannot contact the culture medium well

Method used

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  • Culturing method for NKT cells
  • Culturing method for NKT cells
  • Culturing method for NKT cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Isolation of mononuclear cells

[0036]Collect 20mL of peripheral blood, add 10mL of normal saline to dilute, and slowly add to a centrifuge tube containing 15mL of lymphocyte separation solution, so that the diluted blood and lymphocyte separation solution are separated clearly. -30min, extract the middle buffy coat cells to obtain mononuclear cells, add normal saline to wash twice, count, and the cell volume is 2×10 7 pcs - 4×10 7 indivual.

Embodiment 2

[0037] Embodiment 2, the preparation of hydroxypropyl methylcellulose solution

[0038] Take a certain amount of deionized water and heat it to 70°C-75°C, add 10g-20g hydroxypropyl methylcellulose to 100mL deionized water to prepare a hydroxypropylmethylcellulose solution with a mass concentration of 8%-20%. Dissolve on a magnetic stirrer, stirrer speed 300r / min, dissolve for 10min, let stand to eliminate air bubbles, cool to room temperature, the viscosity of the obtained hydroxypropyl methylcellulose solution is controlled at room temperature 40Mpa.s-100Mpa.s, 0.1mol / mL HCl or NaOH solution to adjust the pH to 6.8-7.5, set aside.

Embodiment 3

[0039] Embodiment 3, the culture method of NKT cell described in the present invention

[0040] 1. Obtain mononuclear cells (PBMC) 2-4×10 7 pcs, according to 5-15×10 6 Inoculate X-VIVO15 serum-free medium per mL, and add hydroxypropyl methylcellulose solution, IL-2, IL-15, IL-21 and CD3 monoclonal antibody to cultivate NKT cells; among them, add HPMC to 100mL culture medium Solution 10mL, maintain HPMC mass percentage at 1.5-2%, solution viscosity 20Mpa.s; IL-2 final concentration is 200U / mL, IL-15 final concentration is 200U / mL, IL-21 final concentration is 200U / mL, CD3 The final concentration of monoclonal antibody is 100U / mL;

[0041] 2. Replenish once every 2-3 days, add X-VIVO15 serum-free medium, hydroxypropyl methylcellulose solution, IL-2, IL-15, IL-21 and CD3 monoclonal antibody for each rehydration; 10mL of HPMC solution was added to 100mL of culture solution to maintain the mass percentage of HPMC at 1.5-2%, and the solution viscosity was 20Mpa.s; the final conce...

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Abstract

The invention belongs to the field of cytobiology, and discloses a culturing method for NKT cells. The culturing method for the NKT cells comprises the steps that mononuclear cells are inoculated into an X-VIVO15 serum-free culture medium, a hydroxypropyl methyl cellulose solution, 1L-2, 1L-15, 1L-21 and CD3 monoclonal antibodies are added, and then the NKT cells are cultured. According to the culturing method, the normal-temperature gelling effect of hydroxypropyl methyl cellulose is utilized, the hydroxypropyl methyl cellulose solution is added, a three-dimensional structure of a culture solution is built in the culturing initial stage of the NKT cells, therefore, the NKT cells proliferate in the three-dimensional space, the space is bigger, and the area of the contacted culture solution is sufficient. Experimental results show that by culturing the NKT cells through the culturing method for the NKT cells, the cells are good in state and high in proliferation speed and proliferation multiple, the NKT effect cells with the higher purity can be obtained, and the killing effect of the obtained NKT cells is obviously higher than that of NKT cells obtained through a conventional culturing method.

Description

technical field [0001] The invention belongs to the field of cell biology, and in particular relates to a method for culturing NKT cells, in particular to a three-dimensional culture method for NKT cells. Background technique [0002] Tumor refers to the new organism formed by the proliferation of local tissue cells under the action of various tumorigenic factors, because this new organism is mostly in the form of space-occupying blocky protrusions, also known as neoplasm. According to the cell characteristics of new organisms and the degree of harm to the body, tumors are divided into two categories: benign tumors and malignant tumors, and cancer is the general term for malignant tumors. [0003] Traditional tumor treatment methods include surgery, radiotherapy and chemotherapy. However, surgery cannot eliminate scattered and invisible cancer cells, so there is a risk of recurrence and metastasis. Chemotherapy and radiotherapy can eliminate tumor cells and alleviate the c...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/515C12N2501/90
Inventor 陈海佳王一飞葛啸虎应杰
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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