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Method for preparing efficient clinical-level CD 56<+> group immune cell

A technology of immune cells and cells, applied in the biological field, can solve the problems of limited expansion and difficult to achieve clinical reinfusion, and achieve the effects of promoting differentiation and maturation, simple and easy technical operation, and reduced culture cost.

Inactive Publication Date: 2017-04-05
JILIN TUO HUA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Clinically use CD56 such as NK, CIK or NKT + When group immune cells are used to treat tumors, a high number of cells is required, and the expansion of ordinary cell induction culture methods is limited, and it is difficult to meet the requirements of clinical reinfusion. Therefore, it is necessary to find another method that can enhance CD56 at the same time. + The proliferative activity of group immune cells, and the immune cell culture method that can enhance the killing ability of cells at the same time

Method used

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  • Method for preparing efficient clinical-level CD 56&lt;+&gt; group immune cell
  • Method for preparing efficient clinical-level CD 56&lt;+&gt; group immune cell
  • Method for preparing efficient clinical-level CD 56&lt;+&gt; group immune cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Isolation of mononuclear cells from peripheral blood or cord blood

[0038] 1. Aseptically collected 50ml of anticoagulated peripheral blood from the patient (male, 17 years old, acute myeloid leukemia, collected from the Department of Hematology, Siping Hospital, China Medical University with the consent of the patient);

[0039] 2. 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;

[0040] 3. Absorb the upper layer of plasma for later use during cultivation; dilute the blood at a ratio of 0.9% normal saline: remaining blood = 1:1, take two 50ml centrifuge tubes and add 15ml of lymphocyte separation solution (Tianjin Haoyang Biological Products Technology Co., Ltd. Company, article number: LTS1077006), slowly press the diluted blood: lymphocyte separation solution = 2:1 on the lymphocyte separation sol...

Embodiment 2

[0044] Embodiment 2: Anti-CD16 antibody coating culture vessel

[0045] Cell culture flasks (Corning, 75cm 2 , Cat. No.: 3275) was added to the coating solution containing anti-CD16 antibody (Biolegend, Cat. No.: 302013) (obtained by adding 5 μg antibody to 5 ml PBS buffer solution), incubated at room temperature for 4 h, and the final concentration of antibody was 1 μg / ml.

Embodiment 3

[0046] Example 3: Inoculation of mononuclear cells

[0047] Slowly rinse the culture flask coated with the antibody in Example 2 twice with 0.9% normal saline, resuspend the cells obtained in Example 1 with 45 ml of AIM-V serum-free medium (GIBCO, product number: 0870112-DK), and adjust the cell Density to 1×10 6 cells / ml and inoculated into culture flasks.

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Abstract

The invention provides a method for preparing efficient clinical-level CD 56<+> group immune cell. The method comprises the following steps: a) separating single karyocyte from peripheral blood or umbilical cord blood; b) inoculating the single karyocyte in the step a) to an anti-CD16 antibody-coated culture dish; c) adding rabbit-anti-human thymic cell immune globulin, Lifein and animal origin-free cytokine in the culture dish for inducing and stimulating the single karyocyte inoculated in the step b); d) supplementing the culture dish and the animal origin-free cytokine according to the cells counting results every 2-3 days; and e) obtaining the CD 56<+> group immune cell. The method can reduce the cost, accords with the requirement of clinical-level preparation, and has good killing activity in vitro and vivo.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a highly efficient clinical grade CD56 + Method for preparing population immune cells. 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 cancer cells in the body after surgery. Cell therapy mainly includes natural killer cell (natural killer cell, NK) therapy, NKT cell therapy, CIK cell therapy, etc. CD8 + CD56 + or CD8 － CD56 + as the main effector cells ( figure 1 ). [0003] NK cells are a special lymphocyte population that can directly kill target cells without major histocompatibility complex (MHC) restriction, and are considered to be important effector cells for tumor immunotherapy. CD56 + The killing of tumor cells by lymphocytes has the characteristics of no n...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783A61K35/17A61P35/00A61P37/02
CPCA61K35/17C12N5/0646C12N2501/04C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/599C12N2501/998
Inventor 姜丽君
Owner JILIN TUO HUA BIOTECH
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