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Method for preparing umbilical cord mesenchymal stem cells

A technology of mesenchymal stem cells and umbilical cords, applied in the field of preparation of umbilical cord mesenchymal stem cells, can solve the problems of stem cell damage, poor activity, and difficulty in expanding mesenchymal stem cells

Inactive Publication Date: 2015-10-21
SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The trypsin in the digestive juice affects the cytoskeleton by dissociating the intercellular mucin and glycoprotein to separate the cells, and has a strong destructive effect on the protein components of the intercellular substance and cell membrane proteins, thus causing the isolated stem cells Injury leads to poor activity, and it is difficult to expand the ideal number and activity of mesenchymal stem cells

Method used

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Examples

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Comparison scheme
Effect test

preparation example Construction

[0018] On the basis of the above-mentioned Wharton's gum digestive fluid of the present invention, the present invention further proposes a method for preparing umbilical cord mesenchymal stem cells, comprising the following steps:

[0019] S10, obtaining the broken tissue of umbilical cord Wharton jelly;

[0020] S20, digesting the umbilical cord Wharton's gum tissue with Wharton's gum digestive juice, and collecting the digestive juice;

[0021] S30, extracting umbilical cord mesenchymal stem cells from the digestive juice.

[0022] Wherein, step S10 obtains the broken tissue of the raw material Wharton gum, and the following steps can be adopted in specific implementation:

[0023] S11, obtaining the umbilical cord;

[0024] The umbilical cord obtained in step S11 can be the umbilical cord cut during the production operation, or the umbilical cord that can be directly used in other ways; after the umbilical cord is obtained, in order to maintain the shape of its cell tiss...

Embodiment 1

[0040] (1) Digestion solution: 1g / L hyaluronidase (Sigma company), 100U / ml ABC enzyme (Beijing Siqingyuan Biotechnology Co., Ltd.)

[0041] Dissolve hyaluronidase in PBS with pH 7.2 to obtain 50ml of hyaluronidase solution with a mass concentration of 1g / L, and set aside;

[0042] Dilute 100U / mL chondroitin sulfate ABC enzyme to 10U / mL with PBS of pH 7.2, and prepare 50ml for later use;

[0043] Finally, mix the hyaluronidase solution with the chondroitin sulfate ABC enzyme at a volume ratio of 1:1.

[0044] Then the umbilical cord mesenchymal stem cells were prepared by enzymatic digestion using the digestive juice configured above, specifically:

[0045] S11, the 15-20cm umbilical cord cut during the production operation is placed in sterile saline for injection containing anticoagulant (preferably with 1% double antibody), and transported to the laboratory at 4°C at low temperature to ensure that the cells Freshness, to avoid the quality of subsequent stem cell isolation ...

Embodiment 2

[0066] This embodiment 2 is a comparative example, the processing process of the umbilical cord adopted in this embodiment 2 is the same as that of Example 1, and the difference as a comparison is that the collagenase containing 0.1% (mass percentage) is used in this embodiment 2 The PBS solution of type I and 0.25% (mass percent) trypsin (both purchased from American AMRESCO Company) was used as a digestive solution, and the umbilical cord mesenchymal stem cells were isolated and prepared according to the same steps as in Example 1.

[0067] Similarly, in this embodiment 2, the mesenchymal stem cells will be harvested according to the test method of the above-mentioned embodiment 1 for various data and performance tests, and the results are as follows:

[0068] (1) Detection results of the number of cell bodies: the collected cells were taken as a sample with a standard volume, the cells were counted by the trypan blue method, and repeated three times, the average number of ce...

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Abstract

The invention provides a method for preparing umbilical cord mesenchymal stem cells and aims to obtain umbilical cord tissues. The method comprises the steps of conducting digestive treatment on the umbilical cord tissues with Whartons jelly digestive juice, and collecting the digestive juice, wherein the Whartons jelly digestive juice comprises hyaluronidase and a chondroitin sulfate ABC enzyme which separate and extract the umbilical cord mesenchymal stem cells from the digestive juice. By means of the method for preparing the umbilical cord mesenchymal stem cells, the hyaluronidase and the chondroitin sulfate ABC enzyme are adopted for digesting Whartons jelly in a joint mode in the enzymic digestion process, the umbilical cord mesenchymal stem cells with the best cell quality can be obtained on the whole, the influence of long-time digestion on cell bodies by a pancreatic enzyme and collagenase is avoided, and the obtaining rate of the umbilical cord mesenchymal stem cells which are finally obtained through the preparation is higher; the obtained stem cells have higher cell viability and differentiation potential.

Description

technical field [0001] The invention belongs to the technical field of stem cell preparation, and in particular relates to a preparation method of umbilical cord mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) can differentiate into fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and other tissue cells under specific induction conditions in vivo or in vitro, and After continuous subculture and cryopreservation, it still has multi-directional differentiation potential, and can be used as an ideal seed cell for the repair of tissue and organ damage caused by aging and disease. Mesenchymal stem cells mainly include bone marrow mesenchymal stem cells, fat mesenchymal stem cells and umbilical cord mesenchymal stem cells according to the tissue barrier. Among them, umbilical cord mesenchymal stem cells are collected from medical waste neonatal umbilical cords. Compared with bone marrow mesenchymal stem ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 曾宪卓鲁菲
Owner SHEN ZHEN ISTEM REGENERATIVE MEDICINE SCI TECH CO LTD
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