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Cell freezing medium as well as preparation method and application thereof

A cryopreservation, cell technology, applied in the field of cell culture, can solve problems such as protein denaturation, achieve good differentiation ability, high survival rate, maintain cell survival rate and proliferation activity.

Pending Publication Date: 2022-05-10
SHENZHEN WINGOR BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] DMSO is the most commonly used osmotic refrigerant in cell preservation. DMSO can penetrate into the cells, allowing water to seep out and reduce the damage to cells from ice crystals formed during cryopreservation. However, DMSO has certain toxic effects and can be hydrophobic with proteins. The group reacts, leading to protein denaturation
Therefore, the existing cryopreservation solution (complete medium containing 10% DMSO) still has significant deficiencies in maintaining cell viability and proliferative activity after recovery

Method used

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  • Cell freezing medium as well as preparation method and application thereof
  • Cell freezing medium as well as preparation method and application thereof
  • Cell freezing medium as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1 A kind of cryopreservation solution of stem cells

[0049] According to the composition and ingredients of the final concentration, all the components of the frozen storage solution shown in Table 1 below were dissolved with IMDM basal medium, and finally DMSO (dimethyl sulfoxide) with a fraction of 10% of the frozen storage solution was added to obtain final product.

[0050] Table 1 Example 1 Mesenchymal stem cell cryopreservation solution component table

[0051]

[0052]

[0053]

Embodiment 2

[0054] Example 2 Cell Cryopreservation

[0055] Cell cryopreservation involves the following steps:

[0056] 1) The P2 generation cells were cultured for about 72 hours, and harvested when the cell confluence reached 85% or more under a microscope;

[0057] 2) The culture solution in the culture bottle was harvested into a T175 culture bottle as a digestion stop solution, and 10 mL of normal saline washing solution was added to each culture bottle. Add 5 mL of 0.125% trypsin to each of the washed T175 culture flasks, shake the culture flask gently, so that the trypsin in the culture flask can quickly infiltrate the bottom of the culture flask, and immediately add 15 mL of stop solution to the culture flask after digestion for 1.5 minutes to stop Digestion;

[0058] 3) Harvest the digested cell suspension into a 225mL centrifuge tube, wash the culture flask with 10-15mL of normal saline in sequence, wash the residual cells attached to the culture flask, and harvest the washed...

Embodiment 3

[0062] Example 3 Cell recovery

[0063] The cryopreserved human umbilical cord mesenchymal stem cells were resuscitated, including the following steps:

[0064] Take out the cryopreservation tube described in Example 2 from the liquid nitrogen, put it into a water bath at 37 degrees Celsius, and shake gently to melt the frozen cell suspension. The operation time should not exceed 1 minute, and add the cell suspension in the cryopreservation tube to In 10mL of complete medium, mix well, centrifuge at 300g for 10min, remove the supernatant, resuspend the cell pellet with complete medium, inoculate 1×10 6 Cells were cultured in T175 flasks.

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Abstract

The invention discloses a cell freezing medium as well as a preparation method and application thereof. Comprising the following components: an IMDM basal culture medium, DMSO, 4-hydroxyethyl piperazine ethanesulfonic acid, pyridoxal hydrochloride, inositol, putrescine, serotonin, mercaptoethanol, arachidonic acid, cholesterol, catechin, bile alkali chloride, calcium pantothenate, nicotinamide, trehalose, polyoxypropylene polyoxyethylene, Tween 80, albumin, transferrin, fibronectin, fisetin, piperlongumine, tocopheryl acetate and linoleic acid. Linolenic acid, myristic acid, oleic acid, palmitic acid, palmitoleic acid and stearic acid; the composition further comprises amino acids, hormones, inhibitors, cell factors, inorganic compounds and vitamins. The cell cryopreservation liquid provided by the invention is used for cryopreserving mesenchymal stem cells, so that the human umbilical cord mesenchymal stem cells have high survival rate after cryopreservation and resuscitation, and meanwhile, a better differentiation capability is kept; and the cell survival rate and proliferation activity can be better maintained.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a cell cryopreservation solution and its preparation method and application. Background technique [0002] Stem cell therapy is a very promising means of treating a variety of diseases that is currently developing rapidly, and the development of this technology requires the construction of a stem cell bank as a support, and cryopreservation of cells is one of the key technologies. When freezing cells, the water in the internal and external environment of the cells will form ice crystals, which will lead to a series of physical and chemical changes in the cells, such as mechanical damage, increased electrolyte concentration, osmotic pressure changes, dehydration, pH changes, and protein denaturation. even lead to cell death. Therefore, protective agents such as glycerin and DMSO need to be added to the cell cryopreservation solution to lower the freezing point. U...

Claims

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Application Information

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IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 任浩姜舒张芸刘赢滢谢亮赵传鑫李欣纪惜銮翁东旭黄伟李结明
Owner SHENZHEN WINGOR BIO TECH
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