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Permeable protection agent, serum-free frozen stock solution, and preparation and application methods of serum-free frozen stock solution

A protective agent and permeability technology, applied in the field of cell culture, can solve the problems of increasing animal pathogen pollution, cell body and human injury, inability to clinical infusion, etc., and achieves low cost, good application prospects, and good effect of adherent growth.

Pending Publication Date: 2019-06-14
北京美迪阿姆科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Generally, the medium, serum and dimethyl sulfoxide (DMSO) are mixed according to a certain ratio to cryopreserve the cells. The dosage of DMSO is 10%, but high concentration of DMSO is highly toxic and will cause damage to the cell body and the human body; In addition, the cost of serum used in the traditional cryopreservation solution formula is high, which increases the possibility of animal pathogen contamination
In addition, studies have shown that cells that have been in contact with fetal bovine serum for a long time will endocytose fetal bovine serum in the solution medium, and mesenchymal stem cells after endocytosis of fetal bovine serum may undergo some protein expression changes. Immunological reaction caused by xenogeneic animal protein can occur after the injection, so it cannot be directly used for clinical infusion
Therefore, the use of serum-containing cryopreservation medium to freeze cells will bring certain risks to clinical use.

Method used

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  • Permeable protection agent, serum-free frozen stock solution, and preparation and application methods of serum-free frozen stock solution
  • Permeable protection agent, serum-free frozen stock solution, and preparation and application methods of serum-free frozen stock solution
  • Permeable protection agent, serum-free frozen stock solution, and preparation and application methods of serum-free frozen stock solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] 1) Proportion by volume of serum-free freezing solution: 88% serum-free medium (serum-free medium adopts the scheme in the invention patent application with opening number CN108707579A), 12% fulvic acid;

[0020] 2) Install the sterilizing filter containing the serum-free cryopreservation solution in an ultra-clean workbench or a safety cabinet, pump out the mixed liquid with a pressure pump, and pack it into sterile containers; Seal the container and store it in a refrigerator at 4 to 10°C, protected from light;

[0021] 3) Cell cryopreservation: resuspend the cells in the serum-free cryopreservation solution, add the cell suspension to the cryopreservation tube, place the cryopreservation tube in a programmed cooling box that is already at normal temperature, and then place the programmed cooling box Place in a -80°C ultra-low temperature refrigerator overnight, and transfer to a liquid nitrogen tank freezer for further preservation the next day;

[0022] Cryopreserv...

Embodiment 2

[0024] 1) Proportion by volume of serum-free freezing solution: 92% serum-free medium (serum-free medium adopts the scheme in the invention patent application with opening number CN108707579A), 8% fulvic acid;

[0025] 2) Install the sterilizing filter containing the serum-free cryopreservation solution in an ultra-clean workbench or a safety cabinet, pump out the mixed liquid with a pressure pump, and pack it into sterile containers; Seal the container and store it in a refrigerator at 4 to 10°C, protected from light;

[0026] 3) Cell cryopreservation: resuspend the cells in the serum-free cryopreservation solution, add the cell suspension to the cryopreservation tube, place the cryopreservation tube in a programmed cooling box that is already at normal temperature, and then place the programmed cooling box Place in a -80°C ultra-low temperature refrigerator overnight, and transfer to a liquid nitrogen tank freezer for further preservation the next day;

[0027] Cryopreserva...

Embodiment 3

[0029] 1) Proportion by volume of serum-free freezing solution: 94% serum-free medium (serum-free medium adopts the scheme in the invention patent application with opening number CN108707579A), 6% fulvic acid (Fulvic Acid), CAS Accession No. 479-66-3;

[0030] 2) Install the sterilizing filter containing the serum-free cryopreservation solution in an ultra-clean workbench or a safety cabinet, pump out the mixed liquid with a pressure pump, and pack it into sterile containers; Seal the container and store it in a refrigerator at 4 to 10°C, protected from light;

[0031] 3) Cell cryopreservation: resuspend the cells in the serum-free cryopreservation solution, add the cell suspension to the cryopreservation tube, place the cryopreservation tube in a programmed cooling box that is already at normal temperature, and then place the programmed cooling box Place in a -80°C ultra-low temperature refrigerator overnight, and transfer to a liquid nitrogen tank freezer for further preser...

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Abstract

The invention relates to a permeable protection agent, a serum-free frozen stock solution, and preparation and application methods of the serum-free frozen stock solution. The permeable protection agent is fulvic acid. The technical scheme of the invention has the technical effects that the cost is low; the toxicity is low; and the survival rate of cells is high after resuscitation.

Description

technical field [0001] The present invention relates to the field of cell culture, in particular to an osmotic protectant for cryopreservation of lymphocytes derived from human blood, granulocytes and pluripotent stem cells derived from umbilical cord blood, a serum-free cryopreservation solution and the serum-free cryopreservation solution Methods of preparation and use. Background technique [0002] Cell cryopreservation and recovery are important techniques in cell culture technology, and cell cryopreservation is an important means for long-term preservation of cells. Cell cryopreservation solution, as a solution that must be used for cell cryopreservation, its function is to suspend the cells that need to be frozen in the cryopreservation solution, supply the nutrients necessary for cell life and metabolism, and prevent or reduce the impact of frozen ice crystals on the cells. cell damage. [0003] In the prior art, osmotic protectants are often used for cell cryoprese...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 张风春施喆陈虎张淑芸杨玉惠
Owner 北京美迪阿姆科技发展有限公司
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