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Method for rapidly cryopreserving and resuscitating endometrial stem cells

An endometrial, stem cell technology, applied in non-embryonic pluripotent stem cells, animal cells, reproductive tract cells, etc., can solve problems such as cell chemical damage

Inactive Publication Date: 2019-03-26
CENTURY BIOSTRENGTH BEIJING PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, vitrification requires the use of cytotoxic dimethyl sulfoxide, which is prone to chemical damage to cells

Method used

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  • Method for rapidly cryopreserving and resuscitating endometrial stem cells

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Effect test

Embodiment 1

[0067] A method for rapidly freezing and recovering endometrial stem cells, comprising the steps of:

[0068] S1: Extract endometrial tissue, adhere to culture for 48 hours, and subculture the obtained cells for 6 days;

[0069] S2: Place the subcultured cells obtained in step S1 in DMEM medium, add cryopreservation solution pre-cooled to 2-5°C, shake and mix gently, and ultrasonicate at low frequency for a short time, add to the cryopreservation solution There is at least one cell adhesion molecule and at least one inhibitor of cell viability;

[0070] S3: Place the cell mixture obtained in step S2 in liquid nitrogen, and cool down to -196°C for storage;

[0071] S4: Take out the frozen cells, first rapidly raise the temperature to 30-40°C, then quickly cool down to 2-10°C, repeat the above process 3 times; wash 2 times with PBS buffer after taking out, centrifuge, and discard the washing solution;

[0072] S5: To the cells obtained in step S4, add a resuscitation preservat...

Embodiment 2

[0074] A method for rapidly freezing and recovering endometrial stem cells, comprising the steps of:

[0075] S1: Extract endometrial tissue, adhere to culture for 36 hours, and subculture the obtained cells for 8 days;

[0076] S2: Place the subcultured cells obtained in step S1 in DMEM medium, add cryopreservation solution pre-cooled to 2-5°C, shake and mix gently, and ultrasonicate at low frequency for a short time, add to the cryopreservation solution There is at least one cell adhesion molecule and at least one inhibitor of cell viability;

[0077] S3: Place the cell mixture obtained in step S2 in liquid nitrogen, and cool down to -196°C for storage;

[0078] S4: Take out the frozen cells, first rapidly raise the temperature to 30-40°C, then quickly cool down to 2-10°C, repeat the above process 3 times; wash 4 times with PBS buffer after taking out, centrifuge, and discard the washing solution;

[0079] S5: To the cells obtained in step S4, add a resuscitation preservat...

Embodiment 3

[0081] A method for rapidly freezing and resuscitating endometrial stem cells, comprising the steps described in Example 1, wherein the specific method of step S1 is as follows:

[0082] S1.1: Collect endometrial decidua tissue samples, wash them with normal saline, cut them into pieces of 1-2mm, spread them in cell culture flasks, add type I collagenase, shake and digest for 60 minutes, then add primary The medium was digested, and the supernatant was removed after centrifugation, and the primary medium was added and placed in 5% CO 2 , Cultivate in an incubator at 37°C, replace the medium every 3 to 4 days, and the primary medium is a DMEM culture solution supplemented with 10% fetal bovine serum and 30mg / L chlorogenic acid;

[0083] S1.2: When the cell confluency reaches 80-90%, collect adherent cells, add subculture medium after digestion, and place in 5% CO 2 , in an incubator at 37°C until the cell confluency reaches 80-90%, the subculture medium is supplemented with 15...

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Abstract

The invention provides a method for rapidly cryopreserving and resuscitating endometrial stem cells. The method is capable of reducing the probability of cell damage and apoptosis and improving the survival rate. According to the method, cryopreservation fluid or resuscitation fluid is added into the cells to perform short-term low-frequency ultrasonic treatment, so that mutually adhesive cells can produce slight loosening and gaps on premise of not damaging the cell structure, and the fluid can be dispersed into droplets and conveniently infiltrates into the cells; cell adhesion molecules arecapable of promoting mutual adhesion of the cells, so that individual cells are further reduced, and the cell survival rate is improved; and a cell activity inhibitor is capable of performing reversible inhibition on some physiological activities in the cells, and the cryopreservation effect is improved on premise of not influencing the cell activity. According to the means, physical and chemicaldamage to the cells in the cryopreservation and resuscitation process can be reduced, so that the cell survival rate and cryopreservation and resuscitation rate of the cells are improved.

Description

technical field [0001] The invention belongs to the technical field of human tissue cryopreservation and resuscitation, in particular to a method for rapidly freezing and resuscitating endometrial stem cells. Background technique [0002] The endometrium is the layer that makes up the lining of the uterus in mammals. Current studies have confirmed that there are a certain number of stem cells in the endometrial tissue. Stem cells are primitive cells with self-replication and multi-directional differentiation potential. They are the origin cells of the body and the primitive cells that form various tissues and organs of the human body. Under certain conditions, it can differentiate into a variety of functional cells or tissues and organs, which are called "universal cells" in the medical field, and are suitable as seed cells for the repair of tissue and organ damage caused by aging and disease. In fertile women, the endometrium will undergo periodic regeneration, differentia...

Claims

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Application Information

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IPC IPC(8): C12N5/074A01N1/02
CPCA01N1/0221C12N5/0682
Inventor 董明珠宋莉莉潘新刘世红卢家堃
Owner CENTURY BIOSTRENGTH BEIJING PTY LTD
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