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Immune cell freezing medium and preparation method and application thereof

A technology of immune cells and cryopreservation, applied in the field of cell biology, can solve the problems of cytotoxic side effects, protein denaturation, and inability to use cryopreservation.

Pending Publication Date: 2021-05-18
圣至润合(北京)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

Using DMSO, glycerin, etc., although the cost is low and the effect is good, but it can interact with the hydrophobic groups of the protein, resulting in protein denaturation, which has certain toxic and side effects on the cells, and cannot be used for cryopreservation of therapeutic cells
Using fetal bovine serum, although no foreign protein will be introduced, which reduces the possibility of animal pathogen contamination, and will not affect human adoptive immunotherapy, but the survival rate of immune cells in this frozen solution is low
The use of human donor plasma / serum is safe and reliable, but due to individual differences, the quality of the cryopreservation solution is inconsistent, and the effect of cryopreservation / resuscitation is also different, which is not conducive to large-scale cryopreservation of immune cells

Method used

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  • Immune cell freezing medium and preparation method and application thereof
  • Immune cell freezing medium and preparation method and application thereof
  • Immune cell freezing medium and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Preparation of 1ml Immune Cell Cryopreservation Solution

[0027] (1) Take vitamin C, vitamin E, insulin-transferrin-selenium, reduced glutathione, recombinant human interleukin 2, recombinant human interleukin 4, hyaluronic acid, trehalose, dextran, glucose, hydroxyethyl starch , Classify and dissolve according to their respective solubility characteristics, and filter through 0.22μm;

[0028] (2) Mix with 0.6ml compound amino acid injection, and adjust the pH to 7.4;

[0029] (3) Add 0.1ml of sodium lactate Ringer's solution and 0.3ml of human serum albumin and mix well to obtain 1ml of immune cell cryopreservation solution, and the final concentration of each component in the cryopreservation solution is as follows: 100μg / ml vitamin C, 100μg Vitamin E / ml, 50μl / ml insulin-transferrin-selenium, 100U / ml recombinant human interleukin 2, 100U / ml recombinant human interleukin 4, 1mg / ml reduced glutathione, 10mg / ml Hyaluronic acid, 0.8mg / ml trehalose, 5mg / ml de...

Embodiment 2

[0030] Example 2: Acquisition of immune cells

[0031] (1) Take a 50ml centrifuge tube and add 15ml of Ficoll separation solution;

[0032] (2) Slowly add 30ml of well-mixed peripheral blood suspension along the tube wall (the peripheral blood is donated by volunteers, it is only used for research and development needs, and an informed consent has been signed);

[0033] (3) Slowly move to the centrifuge, 650g, rise 1 drop 1, centrifuge for 30min;

[0034] (4) After centrifugation, each tube is divided into 4 layers, from bottom to top, followed by red blood cell layer, Ficoll layer, mononuclear cell layer and plasma layer, absorb the plasma layer and transfer it to a 50ml sterile centrifuge tube for later use until it is separated from the buffy membrane. Layer 5mm;

[0035] (5) Carefully transfer the mononuclear cell layer to a 50ml sterile centrifuge tube;

[0036] (6) Add PBS to 50ml and mix well;

[0037] (7) Centrifuge at 650 g for 10 minutes to collect the cell pelle...

Embodiment 3

[0039] Example 3: Cryopreservation and recovery of immune cells

[0040] (1) Take 5*10 immune cells collected from the new culture 6 , add 1ml of the immune cell cryopreservation solution prepared in Example 1 and mix well, and place it in a 1ml cryopreservation tube; at the same time, use the traditional cell cryopreservation solution 1 (formulation: 10% DMSO + 90% fetal bovine serum) as Control 1; traditional cell cryopreservation solution 2 (formulation: 10% DMSO + 90% autologous plasma) was used as control 2; the dosage was the same.

[0041](2) Part of the immune cells were frozen and stored according to the method of programmed cooling, that is, preset at 4°C for 20 minutes;

[0042] (3) The other part of the immune cells was directly stored at -80°C, and stored overnight in liquid nitrogen.

[0043] (4) After all the immune cells were stored in liquid nitrogen for more than 14 days, they were taken out and recovered, and the recovery rate of cryopreservation was teste...

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Abstract

The invention provides an immune cell cryopreservation solution and a preparation method and application thereof. The immune cell cryopreservation solution is specifically prepared from human serum albumin, a cell balance solution, a cell stabilizer, a cell protective agent and an amino acid solution. The immune cell cryopreservation solution contains various cell protection components, so that internal and external injuries caused by ice crystals during cryopreservation of immune cells are avoided, and the recovery rate of the unfrozen immune cells is increased. The immune cell cryopreservation solution does not contain serum components, DMSO and the like, introduction of heterologous exogenous components and harmful components is avoided, and the immune cell cryopreservation solution is safer and more reliable in clinical application.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and relates to an immune cell cryopreservation solution and a preparation method and application thereof. Background technique [0002] At present, immune cell therapy can be widely used in the treatment of viral diseases, malignant blood system diseases, liver cancer, lung cancer and other solid tumors, and the curative effect is very significant. Compared with traditional treatment, immune cell therapy has very significant advantages. Immune cell therapy refers to the collection of the body's own immune cells, which are cultured in vitro to increase the number by thousands of times and enhance the targeted killing function, and then infuse them back into the human body to kill pathogens and cancer cells in the blood and tissues. , Mutated cells, breaking immune tolerance, activating and enhancing the immune ability of the body, taking into account the dual effects of treatment and health c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0284
Inventor 张旭辉胡向军李胜华修冰水徐绍梅
Owner 圣至润合(北京)生物科技有限公司
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