Umbilical cord tissue freeze preservation and unfreezing method

A technology for umbilical cord tissue and cryopreservation, which is applied in tissue culture, bone/connective tissue cells, biochemical equipment and methods, etc., can solve problems such as cell chemical damage, reduce the probability of cell damage and apoptosis, and improve freezing. storage effect, the effect of promoting mutual adhesion

Inactive Publication Date: 2017-12-15
北京唐颐惠康生物医学技术有限公司
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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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  • Umbilical cord tissue freeze preservation and unfreezing method
  • Umbilical cord tissue freeze preservation and unfreezing method
  • Umbilical cord tissue freeze preservation and unfreezing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] A method for freezing and resuscitating umbilical cord tissue includes the following steps:

[0059] S1: Wash the fresh umbilical cord with normal saline and cut into small pieces, adhere to the wall for 36 hours, and subculture the obtained cells for 6 days;

[0060] S2: Place the passaged cells obtained in step S1 in DMEM medium, add the cryopreservation protection solution that has been sonicated in advance at 2°C, centrifuge at low speed and gently shake and mix, the cryopreservation protection solution is added with At least one cell adhesion molecule and at least one cell activity inhibitor;

[0061] S3: Put the cell mixture obtained in step S2 in liquid nitrogen, and cool to -196°C for storage;

[0062] S4: Take out the frozen cells, immediately transfer them to a 37°C water bath for 1 min, wash them twice with PBS buffer after taking them out, centrifuge and discard the washing solution;

[0063] S5: Add the resuscitation preservation solution that has been ultrasonicall...

Embodiment 2

[0065] A method for freezing and resuscitating umbilical cord tissue includes the following steps:

[0066] S1: Wash the fresh umbilical cord with normal saline, cut into small pieces, adhere to the wall for 48 hours, and subculture the obtained cells for 8 days;

[0067] S2: Place the passaged cells obtained in step S1 in DMEM medium, add cryopreservation protection solution that has been sonicated in advance at 5°C, centrifuge at low speed and gently shake and mix, the cryopreservation solution is added with At least one cell adhesion molecule and at least one cell activity inhibitor;

[0068] S3: Put the cell mixture obtained in step S2 in liquid nitrogen, and cool to -196°C for storage;

[0069] S4: Take out the frozen cells, immediately transfer them to a 37°C water bath for 2 minutes, wash them with PBS buffer 4 times after taking them out, centrifuge and discard the washing solution;

[0070] S5: Add the resuscitation preservation solution that has been ultrasonically treated in...

Embodiment 3

[0072] A method for freezing and resuscitating umbilical cord tissue includes the steps described in Example 1, wherein the specific method of step S1 is as follows:

[0073] S1.1: Take a fresh umbilical cord, wash it with normal saline, collect Wharton’s glue and cut it into 1~2mm pieces, spread it in a cell culture flask, add 5mL primary culture medium, and place it in 5% CO 2 , Culture in a 37°C incubator, and change the medium every 3 to 4 days. The primary medium is a DMEM medium supplemented with 8% fetal bovine serum and 100 mg / L streptomycin;

[0074] S1.2: When the cell confluence reaches 80-90%, collect adherent cells, add 5mL subculture medium after digestion, and place in 5% CO 2 , Cultivate in an incubator at 37°C, and cultivate until the cell fusion degree reaches 80-90%. The subculture medium is supplemented with 12% fetal bovine serum and 2-5 mg / L citrus DMEM medium.

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Abstract

The invention provides an umbilical cord tissue freeze preservation and unfreezing method. According to the umbilical cord tissue freeze preservation and unfreezing method, cell aggregate freeze preservation is adopted instead of single cell freeze preservation, so that cell damage apoptosis rate is reduced, and survival rate is increased; cell adhesion molecules are added to promote bonding of cells, reduce the amount of single cells, and increase cell survival rate; ultrasonic treatment is adopted so as to ensure full penetration of a refrigeration protective solution and an unfreezing protective solution into intercellular spaces, solutions are dispersed into small liquid drops, and the permeability is improved; a cell activity inhibitor is adopted for reversible inhibition of physiological activities in cells, and improving of freeze preservation effect without influencing cell activity. The umbilical cord tissue freeze preservation and unfreezing method is capable of reducing physical and chemical damages on cells in freezing and unfreezing process, and increasing cell survival rate and unfreezing rate, so it is convenient for subsequent using.

Description

Technical field [0001] The invention belongs to the technical field of cryopreservation and resuscitation of human tissue, and particularly relates to a method for cryopreservation and resuscitation of umbilical cord tissue. Background technique [0002] The umbilical cord is a tubular structure connecting the fetus and the placenta in mammals. Umbilical cord blood is rich in mesenchymal stem cells. Mesenchymal stem cells are an important member of the stem cell family. They are primitive cells with self-replication and multi-differentiation potential. They are the original 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 for seed cells for tissue and organ damage repair caused by aging and disease. Therefore, storing mesenchymal stem cells can provide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0775
CPCA01N1/0221A01N1/0226C12N5/0665C12N2501/01C12N2501/06C12N2501/58C12N2501/999
Inventor 曹毓琳刘俊江时兆田白志惠王俊王沾
Owner 北京唐颐惠康生物医学技术有限公司
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