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Method of freezing Schwann cells by compound low-temperature cryopreserving system

A technology of Schwann cells and cryopreservation, which is applied in the preservation, application, animal husbandry and other directions of human or animal body to achieve the effect of increasing osmotic pressure, reducing ice crystal damage and slowing down drastic changes.

Active Publication Date: 2017-07-28
WUHAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on the use of supramolecular gel systems for cryopreservation of cells

Method used

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  • Method of freezing Schwann cells by compound low-temperature cryopreserving system
  • Method of freezing Schwann cells by compound low-temperature cryopreserving system
  • Method of freezing Schwann cells by compound low-temperature cryopreserving system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation and properties of supramolecular gels:

[0041] 1. Preparation of gelling factor BDT:

[0042] (1) Synthesis of dodecyl-Boc-L-tyrosine methyl ester (BDTE) (Boc-L-tyrosine methyl ester alkylation):

[0043] Weigh 3.998g (0.0135mol) of Boc-L-tyrosine methyl ester into a 25mL round bottom flask, add 10ml of DMF, and after the raw material is completely dissolved, add 3.7113g of K 2 CO 3 (0.027mol) was used as an acid-binding agent, and then 4 mL of bromododecane (0.0167mol) was added to react at room temperature for 12 hours. After the reaction was completed, add 20 mL of water to wash, suction filter, wash the filter cake, and vacuum dry for 24 hours to obtain a white solid. Recrystallize once from absolute ethanol (20ml) to obtain a pure white solid.

[0044] (2) Synthesis of BDT (hydrolysis reaction of BDTE):

[0045] Dissolve 0.5007g (0.0011mol) BDTE in a 25mL flask with 10mL ethanol, add 1.7mL (0.0017mol) of 1mol / L NaOH solution dropwise in an ice-wat...

Embodiment 2

[0050] Preparation and properties of composite cryopreservation system:

[0051] Take the BDT obtained in Example 1 and add it to 1ml RPMI1640 medium, put it in a 60°C water bath to dissolve, and after it is completely dissolved, add a certain amount of RPMI1640 medium solution containing a composite cryoprotectant dropwise to prepare a composite low-temperature cryopreservation system. use. The final concentration of the composite system configured is 1.5g / LBDT, 8vol% DMSO, 0.05mol / L trehalose, and its microscopic appearance is observed with an optical microscope in an ice-water bath (0-4°C), as shown in image 3 shown. The microstructure of the supramolecular gel formed by the gel factor in the RPMI1640 medium solution containing the compound cryoprotectant is different, but it still presents a three-dimensional network structure. When the gel is wrapped, the penetration rate of the osmoprotectant will be greatly reduced, thereby reducing the damage to the cells caused by ...

Embodiment 3

[0055] Freezing process of Schwann cells:

[0056] 1. The configuration of the cell culture medium system: the configuration of the cell culture medium system: add 10 vol% fetal bovine serum and 1 vol% double antibody to the RPMI1640 medium to prepare a full culture medium for cells. The whole culture medium is divided into two parts, one part is added with the BDT gel factor obtained in Example 1 (concentration is 1.5g / L) and dissolved at 60°C, after it is completely dissolved, it is filtered and sterilized, and the other part does not do any After processing, aliquot them separately and place them at 4°C for later use.

[0057] 2. Configuration of the cryoprotectant system: add 16vol% DMSO, 32vol% DMSO, 0.2mol / L trehalose to the whole culture medium, filter and sterilize with a 0.22μm filter membrane, divide and store at 4°C for later use.

[0058] 3. The freezing process of Schwann cells: Digest the cells in the logarithmic phase with 0.25% trypsin, divide them into three ...

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Abstract

The invention discloses a method of freezing Schwann cells by a compound low-temperature cryopreserving system. The method comprises the following steps of: heating amino acid gel factors and dissolving the same in a cell culture medium, filtering and degerming the cell culture medium, and preparing a cell suspension liquid with the cell culture medium containing the gel factors; dropwise adding a cell culture medium containing a compound freezing protecting agent into the cell suspension liquid at 0-4 DEG C and uniformly mixing the mixture, wherein the compound freezing protecting agent is prepared by mixing a permeable freezing protecting agent and a non-permeable freezing protecting agent; and putting a cryopreserving tube in a programmed cryopreserving box to be frozen for 24h at 80 DEG C below and then transferring the cryopreserving tube to liquid nitrogen to be cryopreserved. The supermolecular gel formed by self-assembling the amino acid gel factors has a unique three-dimensional netty structure. When the freezing protecting agent is dropwise added, cells are coated by the supermolecular gel, so that the permeating damage of the freezing protecting agent is reduced; in temperature-reducing and restoring processes, damage of ice crystals is reduced as the three-dimensional netty structure of the supermolecular gel limits growth and recrystallization of the ice crystals.

Description

technical field [0001] The invention belongs to the technical field of cryopreservation of cells, and in particular relates to a method for freezing Schwann cells. Background technique [0002] Nerve repair and regeneration has always been a research hotspot in biology and tissue engineering, and Schwann cells play an important role in nerve regeneration. Therefore, many related studies need to use Schwann cells, so how to store a large number of Schwann cells in vitro has become a big problem, and cryopreservation is currently the most common and effective preservation method. [0003] Cryopreservation is to place cells in an ultra-low temperature (usually -80°C or lower) environment to inhibit cell metabolism for long-term storage. At present, cryopreservation technology has been successfully applied to the preservation of stem cells, red blood cells, liver cells, sperm, oocytes, bone cells, skin, cornea and other cells and tissues. There have been relevant literature re...

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 陈万煜陈熹
Owner WUHAN UNIV OF TECH
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