Method using umbilical cord tissue cryopreservation to preserve various stem cells, thawing method and application

A cryopreservation method and technology of umbilical cord tissue, applied in the field of life sciences, can solve the problems of affecting the effect of cryopreservation and high tissue water content, and achieve the effects of program-controlled cooling and cryopreservation process fast, improving utilization rate and improving effect

Inactive Publication Date: 2019-01-04
YINFENG BIOLOGICAL GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the pretreatment process for umbilical cord tissue is relatively simple, which causes the water content in the frozen tissue to be too high, which affects the freezing effect
In addition, the program-controlled cooling and cryopreservation methods used in the prior art are all designed based on cell cryopreservation, and need to be redesigned for cryopreservation of umbilical cord Wharton jelly tissue

Method used

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  • Method using umbilical cord tissue cryopreservation to preserve various stem cells, thawing method and application
  • Method using umbilical cord tissue cryopreservation to preserve various stem cells, thawing method and application
  • Method using umbilical cord tissue cryopreservation to preserve various stem cells, thawing method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Umbilical cord tissue cryopreservation method of the present invention is as follows:

[0042] First, separate the frozen umbilical cord tissue into Wharton's jelly, wash the umbilical cord fully with normal saline in a cell culture dish, cut off the two ends of the umbilical cord with tissue scissors, and then rinse; Cut the umbilical cord into small pieces with scissors and clean them; then cut a small opening in each small piece of umbilical cord along the direction parallel to the veins, then tear it longitudinally with tissue forceps and flatten it; then carefully peel off the blood vessels and amniotic membrane from the umbilical cord, The part of Wharton's Jelly was fully washed 3 times with normal saline, and cut to 1-2 cm 3 small pieces.

[0043] One tissue block was reserved for the stripped Wharton's jelly tissue as a control experiment. The control experiment consisted of three parts: 1. Take a piece of fresh tissue and fix it in 10% neutral formalin, send...

Embodiment 2

[0079] The cryopreservation experiment of umbilical cord Wharton jelly tissue was carried out by using the formula of the cryopreservation agent of the No. 5 experimental group in the proportion of DMSO content in the cryopreservation solution added with fetal bovine serum (FBS) in Table 1.

[0080] A section of umbilical cord was selected, and the separation and pretreatment process of the umbilical cord Wharton's jelly tissue was carried out according to the method in Example 1, and one part was reserved as a control test group. The rest were evenly divided into 15 cryopreservation tubes, and the cryopreservation agent of No. 5 experimental group was added, and each 5 cryopreservation tubes were regarded as a group, and the program controlled cooling and freezing were carried out.

[0081] One group adopts a program-controlled cooling and freezing program: keep at 3°C ​​for 4 minutes; drop to -10°C at 2°C / min and keep for 12 minutes; drop to -50°C at 0.5°C / min and keep for 5 ...

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Abstract

The invention relates to a method using umbilical cord tissue cryopreservation to preserve various stem cells, a thawing method and application. The method specifically includes: cleaning umbilical cord tissue, shearing into tissue sections, and stripping mesenchyme Wharton's jelly tissue; using an alpha-MEM protecting agent containing DMSO for preprocessing, performing concentration balance under4-10 DEG C for 10-20 minutes, performing low-temperature centrifuging, and removing supernate; adding dextran or fetal calf serum to allow the dextran or fetal calf serum and the alpha-MEM protectingagent containing DMSO to jointly form cryopreservation liquid, and using a program-controlled temperature lower or graded temperature lowering manner to perform cryopreservation on the tissue. The cryopreservation method has the advantages that the cryopreserved umbilical cord tissue can be effectively protected, subsequent thawing operation is facilitated, and the activity of cells cultured by the thawed tissue is the same as that of cells in fresh tissue.

Description

technical field [0001] The invention relates to the technical field of life sciences, in particular to a method for preserving various stem cells by freezing interstitial tissue of the umbilical cord, a resuscitating method and an application thereof. Background technique [0002] The umbilical cord is a tubular structure connecting the fetus and the placenta in mammals. It consists of two arteries and one vein, and loose, gelatinous mesenchyme can be seen in the gap. Wharton's Jelly tissue in human umbilical cord can be isolated to obtain various kinds of mesenchymal stem cells (Mesenchymol Stem Cells, MSCs), epithelial stem cells (ESCs), endothelial progenitor cells (endothelial progenitor cells, EPCs) and so on. Stem cells, MSCs derived from umbilical cords have the advantages of wide sources, convenient materials, relatively pure, rich content, and low immunogenicity. About 10 7 Primary MSCs. MSCs currently found to positively express CD13, CD29, CD44, CD73, CD90 (Thy-...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/0775
CPCA01N1/021C12N5/0665
Inventor 徐峰波崔光晶金华生德伟王圣川宋现收王肇光
Owner YINFENG BIOLOGICAL GRP
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