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In vitro induction amplification, freeze preservation and anabiosis method of immune cells

An immune cell and cryopreservation technology, applied in the biological field, can solve the problems of natural killer cell culture, cryopreservation and recovery methods that need to be improved, expansion multiples, poor tumor killing effect, complicated culture system, etc., to achieve good cell characteristics , low cost and high induction efficiency

Active Publication Date: 2017-05-24
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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  • In vitro induction amplification, freeze preservation and anabiosis method of immune cells
  • In vitro induction amplification, freeze preservation and anabiosis method of immune cells
  • In vitro induction amplification, freeze preservation and anabiosis method of immune cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Using the method for inducing and expanding immune cells of 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 T75 bottles coated with anti-human CD16

[0090] 1.1 Add 5 mL of 2.5 μg / mL anti-human CD16 monoclonal antibody dissolved in medical physiological saline to a sterile culture flask, gently shake the culture flask to spread the antibody over the culture surface, and avoid light overnight at 4°C.

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

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

[0093] 2.1 Use a sterile blood collection bag with anticoagulant to collect about 100ml of human peripheral blood. R...

Embodiment 2

[0136] In this example, six cryopreservation solutions containing plasma and six cryopreservation solutions containing plasma were used to cryopreserve the NK cells induced and expanded in vitro in Example 1, and compare the effects of cryopreservation to observe the effect of plasma on The effect of cryopreservation of NK cells, and reducing the DMSO concentration to reduce the possibility of cytotoxicity. details as follows:

[0137] 1. Freeze NK cells with cryopreservation liquid without plasma

[0138] 1.1. Methods of cryopreservation and recovery of NK cells:

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

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

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

[0142] Cryopreservation solution 3: 10% by volume DMSO+10% by volume...

Embodiment 3

[0184] The NK cells cryopreserved in the cryopreservation solution 9 of Example 2 were recovered, and the specific method was as follows

[0185] 1. The recovery process

[0186] (1) Take out the NK cells cryopreserved in the cryopreservation solution 7 of Example 2 in liquid nitrogen, and quickly melt them in a 37-42°C water bath.

[0187] (2) The cells were treated with an equal volume of X-VIVO15 medium (denoted as group P) and an equal volume of resuscitation solution containing 2.5% HSA and 5% Dextran 40 (denoted as F1 group).

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

[0189] 2. Training after resuscitation

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

[0191] 3. Calculation

[0192] S...

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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 present invention relates to the field of biotechnology, in particular to methods for in vitro induction, expansion, cryopreservation and recovery of immune cells. Background technique [0002] In recent years, biological therapy has become the fifth major treatment model after surgery, radiotherapy, chemotherapy and endocrine therapy, and has gradually received attention. Adoptive Cellular Immunotherapy (ACI) is one of the cell biological treatment methods. It is directed to infusion of immune cells with anti-tumor activity to tumor patients, which directly kills tumor cells or stimulates the body's immune response to kill tumor cells to achieve tumor treatment. purpose. At present, adoptive immune cells in clinical applications include DC-CIK cells, TIL cells, LAK cells and NK cells. Among them, 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, also called NK cell...

Claims

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Application Information

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IPC IPC(8): C12N5/0784C12N5/0783A01N1/02
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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