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A method for inducing expansion, cryopreservation and recovery of immune cells in vitro

An immune cell, cryopreservation technology, applied in the biological field, can solve the problems of natural killer cell culture, cryopreservation and recovery methods need to be improved, the amplification multiple and tumoricidal effect are not good, and the culture system is complicated, so as to achieve good cell characteristics. , low cost, high induction efficiency

Active Publication Date: 2018-01-09
成都安缇赛尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are already NK cell therapy products on the market, but the recovery effect after cryopreservation is poor, the culture system used is complicated, the amplification multiple and tumor killing effect are not good, and some culture systems have safety risks and other problems.
[0004] Therefore, the methods of culture, cryopreservation and recovery of natural killer cells need to be improved

Method used

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  • A method for inducing expansion, cryopreservation and recovery of immune cells in vitro
  • A method for inducing expansion, cryopreservation and recovery of immune cells in vitro
  • A method for inducing expansion, cryopreservation and recovery of immune cells in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Using the method for inducing expansion of immune cells in the embodiment of the present invention, the isolated mononuclear cells are induced and expanded into immune cells, and the activity of the immune cells is detected.

[0088] 1. Experimental method

[0089] 1. Prepare anti-human CD16 coated T75 vials

[0090] 1.1 Add 5 mL of 2.5 μg / mL anti-human CD16 monoclonal antibody dissolved in medical normal saline into a sterile culture bottle, shake the culture bottle gently to make the antibody cover the culture surface, and overnight at 4°C in the dark.

[0091] 1.2 Recover the antibody coating solution before use, wash the culture bottle once with 5 mL of normal saline, and then use 5 mL of T cell expansion medium (OpTmizer TM CTS TM T-cell expansion SFM) and washed once.

[0092] 2. Collect peripheral blood, separate peripheral blood plasma and mononuclear cells

[0093] 2.1 Collect about 100ml of human peripheral blood with a sterile blood collection bag added ...

Embodiment 2

[0136] In this example, 6 kinds of cryopreservation solutions without plasma and 6 kinds of cryopreservation solutions containing plasma were used respectively to freeze the NK cells induced and expanded in vitro in Example 1, and compare the cryopreservation effects so as to observe the effect of plasma on Effects of cryopreservation of NK cells, and the possibility of reducing DMSO concentrations to reduce cytotoxicity. details as follows:

[0137] 1. Use plasma-free cryopreservation medium to freeze NK cells

[0138] 1.1. NK cell cryopreservation and recovery methods:

[0139] The NK cells obtained on the 10th day of in vitro induction and expansion in Example 1 were cryopreserved in four freezing solutions containing HSA, and the components of the four freezing solutions were as follows:

[0140] Freezing solution 1: 5 vol% DMSO+10 vol% HSA;

[0141] Freezing solution 2: 5 vol% DMSO+15 vol% HSA;

[0142] Freezing solution 3: 10% by volume DMSO + 10% by volume HSA;

[...

Embodiment 3

[0184] The NK cells frozen in the freezing solution 9 of Example 2 were resuscitated, and the specific method was as follows

[0185] 1. Recovery process

[0186] (1) The NK cells cryopreserved in the cryopreservation solution 7 of Example 2 were taken out from the liquid nitrogen, and rapidly thawed in a water bath at 37-42°C.

[0187] (2) The cells were treated with an equal volume of X-VIVO15 medium (referred to as group P) and an equal volume of recovery solution containing 2.5% HSA and 5% Dextran40 (referred to as group F1).

[0188] (3) Centrifuge, take the cells separately, count with a cell viability analyzer, and measure the cell viability.

[0189] 2. Culture after recovery

[0190] Cells in group P and group F1 were cultured in vitro using immune cell scale expansion medium, SuperCulture TML500 human lymphocyte serum-free medium + domestic IL-2 with a final concentration of 1000IU / ml, and the cell technology and Vitality analysis.

[0191] 3. Calculate

[0192]...

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Abstract

The invention discloses an in vitro induction amplification, freeze preservation and anabiosis method of immune cells. The method comprises the following steps: using a CD16 antibody enveloped culture container to obtain an enveloped culture container; using an immune cell activated culture medium to put single karyocytes into the enveloped culture container to carry out first induction and amplification culture so as to obtain primarily induced and amplified immune cells; using an immune cell amplification culture medium to carry out second induction and amplification culture on the primarily induced and amplified immune cells so as to obtain differentiated immune cells; using an immune cell large-scale amplification culture medium to carry out third induction and amplification culture on the differentiated immune cells so as to obtain large-scale function activated immune cells; using an immune cell freeze preservation liquid to freeze and preserve the immune cells so as to obtain frozen and preserved immune cells; water-bathing the frozen and preserved immune cells for melting, mixing with a freezing anabiosis liquid of the immune cells so as to obtain an anabiosis cell mixed liquid; carrying out centrifugal treatment on the anabiosis cell mixed liquid so as to obtain anabiosis immune cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to methods for inducing expansion, cryopreservation and recovery of immune cells in vitro. Background technique [0002] In recent years, biological therapy has become the fifth largest treatment mode after surgery, radiotherapy and chemotherapy, and endocrine therapy, and has gradually received attention. Adoptive cellular immunotherapy (ACI) is one of the methods of cell biological therapy. It refers to the infusion of immune cells with anti-tumor activity to tumor patients, directly killing tumor cells or stimulating the body's immune response to kill tumor cells, so as to achieve the goal of treating tumors. Purpose. Currently, adoptive immune cells in clinical use include DC-CIK cells, TIL cells, LAK cells and NK cells, among which CIK cells, LAK cells and A-NK cells are all anti-cancer systems with natural killer cells as the main body. [0003] Natural killer cells (natural kil...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/00
CPCA01N1/0221C12N5/0639C12N5/0646C12N2500/72C12N2500/84C12N2500/90C12N2501/2302
Inventor 徐智峰张新
Owner 成都安缇赛尔生物科技有限公司
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