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Cell cryopreservation liquid, preparation method and application thereof, and cryopreservation method and transfusion method of mononuclear cells

A technology for cryopreservation and nuclear cells, which can be applied in applications, pharmaceutical formulations, and preservation of human or animal bodies. Low cost and cost-effective effect

Inactive Publication Date: 2021-07-27
北京益华生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] When the cell cryopreservation solution in the prior art is used to freeze mononuclear cells, the viability of the recovered cells can basically meet the requirements. However, since the cell cryopreservation solution generally contains dimethyl sulfoxide (DMSO) components, and some cell cryopreservation fluids also contain exogenous / animal-derived serum components, or some potentially risky components are added
[0005] Studies have shown that DMSO has certain toxic and side effects, and its interaction with protein hydrophobic functional groups will cause protein hydrolysis, which has toxic and side effects on blood vessels and kidneys, and even has carcinogenic effects; the existence of exogenous / animal-derived serum will also cause Leading to hidden dangers such as rejection / infectious disease / allergies, the safety of returning frozen cells to the human body cannot be guaranteed
Long-term cryopreservation of cells in the above-mentioned cell cryopreservation solution will inevitably affect the cell fluid composition of the frozen cells, and there will be certain safety risks after the cells are returned to the human body
[0006] Furthermore, when the cells cryopreserved by the above-mentioned cell cryopreservation solution are reinfused, because the cell cryopreservation solution has safety risks, the cells need to be centrifuged and washed repeatedly. On the one hand, the damage to the cells due to centrifugation will affect its effect. On the other hand, centrifugation and repeated washing of cells requires the clinical application of ultra-clean benches, centrifuges and other equipment in hospitals, which is not economical, but also inconvenient and inefficient to operate.

Method used

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  • Cell cryopreservation liquid, preparation method and application thereof, and cryopreservation method and transfusion method of mononuclear cells
  • Cell cryopreservation liquid, preparation method and application thereof, and cryopreservation method and transfusion method of mononuclear cells
  • Cell cryopreservation liquid, preparation method and application thereof, and cryopreservation method and transfusion method of mononuclear cells

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preparation example Construction

[0061] The present invention also proposes a method for preparing a cell cryopreservation solution. First, mix human autologous serum and hydroxyethyl starch 130 / 0.4 sodium chloride injection; then add trehalose, BDNF and GDNF to it, and mix evenly to obtain cell Freezing solution.

[0062]The present invention also proposes the aforementioned cell cryopreservation solution for cryopreserving mononuclear cells.

[0063] The cell cryopreservation solution is suitable for the cryopreservation of various types of cells, especially for the cryopreservation of mononuclear cells such as peripheral blood or umbilical cord blood, and the viability of the cells is better.

[0064] The present invention also proposes a mononuclear cell cryopreservation method, using the aforementioned cell cryopreservation solution to freeze mononuclear cells.

[0065] Preferably, it comprises the following steps:

[0066] Prepare cell cryopreservation solution according to the aforementioned preparat...

Embodiment 1

[0073] 1) Preparation of cell freezing solution:

[0074] Mix 30 mL of human autologous serum with 70 mL of hydroxyethyl starch 130 / 0.4 sodium chloride injection, add 0.35 g of trehalose, 1 μg of BDNF and 3 μg of GDNF, and mix well to obtain a cell cryopreservation solution.

[0075] 2) Extraction of mononuclear cells:

[0076] The peripheral blood or umbilical cord blood of the human body is collected, and the mononuclear cells are extracted; the peripheral blood or umbilical cord blood and the human autologous serum are collected from the same human body. The extraction steps of the mononuclear cells are as follows:

[0077] Put the collected peripheral blood or umbilical cord blood into a 50mL centrifuge tube, and centrifuge at 3000rpm for 30min; after centrifugation, take the white layer, add Hanks solution with a volume ratio of 1:1, and mix well.

[0078] Pour 25mL of lymphatic separation solution into another 50mL centrifuge tube, and slowly spread the mixed periphera...

Embodiment 2

[0092] 1) Preparation of cell freezing solution:

[0093] Mix 50 mL of human autologous serum with 50 mL of hydroxyethyl starch 130 / 0.4 sodium chloride injection, add 1 g of trehalose, 5 μg of BDNF and 3 μg of GDNF, and mix well to obtain a cell cryopreservation solution.

[0094] 2) Extraction of mononuclear cells: same as in Example 1.

[0095] 3) Cryopreservation test: the same as in Example 1.

[0096] After testing, after the mononuclear cells were frozen by the cell freezing solution of the embodiment of the present invention, the results are shown in Table 2 below:

[0097] Table 2

[0098]

[0099] Remarks: h in the table means hour, and m means month.

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Abstract

The invention relates to a cell cryopreservation solution, a preparation method and application thereof, and a cryopreservation method and a reinfusion method of mononuclear cells. The cell cryopreservation liquid is used for cryopreserving mononuclear cells, and is composed of human autoserum, hydroxyethyl starch 130 / 0.4 sodium chloride injection, trehalose, BDNF and GDNF. The preparation method comprises the following steps: mixing the human autoserum and the hydroxyethyl starch 130 / 0.4 sodium chloride injection; adding trehalose, BDNF and GDNF into the mixture, and uniformly mixing; freezing the mononuclear cells by using the cell freezing medium; and taking out the cryopreserved mononuclear cells from the liquid nitrogen, thawing in a water bath at 37 DEG C, and directly inputting into the human body. The technical problem to be solved is how to provide a cryopreservation solution which enables cells cryopreserved by the cryopreservation solution to be directly transfused back to a human body after being thawed, and the cells cryopreserved by the cryopreservation solution not only have higher cell viability, but also are economical in cost, convenient, rapid and efficient, so that the cryopreservation solution is more practical.

Description

technical field [0001] The invention belongs to the technical field of cell cryopreservation, and in particular relates to a cell cryopreservation solution, a preparation method and application thereof, a mononuclear cell cryopreservation method and a reinfusion method. Background technique [0002] Mononuclear cells mainly include immune cells such as lymphocytes, monocytes, dendritic cells (dc) and NK cells. Among them, lymphocytes have the characteristics of immune regulation and self-replication, and can play a key role in the treatment of viral, bacterial, and fungal infections. Immune cells have great clinical application value in cancer treatment. [0003] As the age increases, the immune function of the human body will also decline. The peripheral blood or cord blood mononuclear cells are frozen and stored when they are young, and the frozen mononuclear cells are reinfused into the human body when they are old. It can effectively solve the problem of ineffective pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02A61K35/16A61K47/26A61K47/36A61K47/42
CPCA01N1/0226A01N1/0221A61K35/16A61K47/26A61K47/36A61K47/42
Inventor 周慧杨铭斌姜粉军姜宁健袁春梅刘杨吴威
Owner 北京益华生物科技有限公司