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Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue

A technology of mesenchymal stem cells and outer amniotic membrane, which is applied in the field of isolation and culture of mesenchymal stem cells, can solve the problems of limited growth space of mesenchymal stem cells, influence on the separation, extraction, development and utilization of umbilical cord mesenchymal stem cells, and influence on the vitality of stem cells, etc.

Inactive Publication Date: 2016-03-23
郭镭 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, umbilical cord mesenchymal stem cells are mostly prepared by enzymatic digestion and tissue adhesion. However, in the process of separation and extraction, the interference of red blood cells is an important factor affecting the quality and quantity of umbilical cord mesenchymal stem cells. The existence of a large number of red blood cells makes mesenchymal stem cells The growth space of mesenchymal stem cells is limited, and at the same time, it affects the vitality of stem cells, which has a great impact on the isolation, extraction, development and utilization of umbilical cord mesenchymal stem cells

Method used

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  • Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue
  • Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue
  • Method for culturing umbilical cord mesenchymal stem cells in separated mode from umbilical cord outer layer amnion tissue

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Selection of serum-free medium for mesenchymal stem cells

[0087] (1) Content screening of serum substitutes

[0088] Test medium: 0.1 volume part of β-mercaptoethanol, 10ng / ml recombinant human basic fibroblast growth factor (b-FGF, Peprotech company), 1 volume part of non-essential amino acid aqueous solution (11140, Gibco company), 1, 2, 5, 8, 10, 12, 15 or 20 parts by volume of KnockoutFBS serum substitute (10828-028, Gibco), 89 parts by volume of a-MEM.

[0089] In the biological safety cabinet, take the third-generation hUC-MSC isolated from the umbilical cord Huatong glue tissue of the natural delivery neonates, and use them as 2×10 4 Cells / cm 2 The density is inoculated in T75 cell culture flask, and 12-15ml of conventional commercially available medium is added to culture cells. After culturing and observing that the cells have fully adhered to the wall, 15 mL of test medium was replaced. Observe the cell growth.

[0090] Results: In the three concentrati...

Embodiment 2

[0095] Example 2 Method for assisting extraction of umbilical cord mesenchymal stem cells with red blood cell lysate

[0096] Collection and transportation of samples: The umbilical cord samples of newborns delivered by cesarean section are collected under aseptic conditions and placed in the umbilical cord preservation and transport solution (D-Hank's solution) containing penicillin sodium, streptomycin sulfate, gentamicin and amphotericin B The final concentration of penicillin sodium, streptomycin sulfate and gentamicin in the protection solution is 150U / mL, and the final concentration of amphotericin B is 300U / ml), transported to the clean GMP cell laboratory on ice within 2 hours;

[0097] Cleaning and disinfection of samples: Put fresh umbilical cord samples in a 50ml sterile centrifuge tube in a biological safety cabinet, rinse with 75% alcohol twice, and rinse with sterile normal saline three times;

[0098] Pretreatment of umbilical cord tissue: Use ophthalmological scissor...

Embodiment 3

[0102] Example 3 Method for assisting extraction of umbilical cord mesenchymal stem cells with red blood cell lysate

[0103] The red blood cell lysate used contains 10g / L of NH 4 Cl and 0.1mMNa 2 -EDTA, pH is 7.2-7.4.

[0104] Refer to the method in Example 2. After culturing to the 5th day, the mesenchymal stem cells crawled out from the bottom of the tissue block. Around the 7th day, the mesenchymal stem cells formed a swirling cell cluster. After removing the tissue block and replacing the fresh medium, About 12 days, the cells reached 60% confluence and were passaged after trypsin digestion; after the third passage, the purity of the cells was greater than 99%, and the viability was greater than 95%.

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Abstract

The invention provides a method for culturing umbilical cord mesenchymal stem cells in a separated mode from umbilical cord outer layer amnion tissue. The method includes the steps that umbilical cord tissue of a healthy newborn is used, washed, disinfected and mechanically cut into pieces to separate an outer layer amnion, and the outer layer amnion is cultured in a suspension mode with a serum-free culture medium after being treated with erythrocyte lysate; medium change is carried out every three to five days, after the attachment rate in a plate reaches 30-70%, trypsinization is used, cells are collected centrifugally for passage amplification, and confluence is carried out when the cell confluence rate reaches 80-90% to obtain the high-purity umbilical cord mesenchymal stem cells.

Description

Technical field [0001] The invention relates to a method for stably separating and extracting umbilical cord mesenchymal stem cells, in particular to a method for separating and culturing mesenchymal stem cells from amniotic membrane tissue outside the umbilical cord. Background technique [0002] Mesenchymal stem cells (mesenchymalstemcells, MSC) are important members of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development, and exist in the connective tissue and interstitium of organs throughout the body. MSC was first discovered in bone marrow in the late 1970s. However, it was not until the 1990s when the multi-directional differentiation potential of MSC was recognized that this kind of stem cells received more and more attention. [0003] Mesenchymal stem cells are pluripotent stem cells. Current studies have shown that MSC can be induced to differentiate into a variety of tissue cells and cell-like cells such as fat, bone, bone ligam...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 郭镭
Owner 郭镭
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