Human umbilical mesenchymal stem cell and preparation method thereof

A technology of mesenchymal stem cells and human umbilical cord, which is applied in the field of preparation of original mesenchymal stem cells, can solve the problems of MSC quantity disputes, etc., and achieve the effect of simple operation, low cost, high purity and cell activity

Active Publication Date: 2011-07-20
UNION STEMCELL & GENE ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Other methods such as culturing MSCs from umbilical cord blood and peripheral blood are somewhat difficult, and t

Method used

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  • Human umbilical mesenchymal stem cell and preparation method thereof
  • Human umbilical mesenchymal stem cell and preparation method thereof
  • Human umbilical mesenchymal stem cell and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1: Preparation of human umbilical cord placental mesenchymal stem cells

[0025] With the informed consent of the puerpera, the umbilical cord or placenta of healthy fetuses delivered at term were collected. Before the umbilical cord or placenta is collected, the puerpera needs to be tested for HIV antibody, hepatitis B virus antibody, hepatitis C virus antibody, treponemal pallidum antibody, alanine aminotransferase, mycoplasma, etc., all of which are qualified to ensure safety before collection.

[0026] 1. After the umbilical cord or placenta is removed from the operating table, immerse in 0.9% saline containing antibiotics and store at 4°C;

[0027] 2. Take out the umbilical cord or placenta in the clean bench, and rinse the residual blood on the surface with D-PBS;

[0028] 3. Cut the umbilical cord or placenta into 1-3mm 3 Put the tissue pieces of different sizes into a 200mL blue cap reagent bottle, add digestion solution with a mass volume percentage c...

Embodiment 2

[0031] Example 2: Cell characteristics of human umbilical cord placental mesenchymal stem cells

[0032] 1. Immunophenotypic detection of CD29, CD44, CD73, CD90, CD105, CD31, CD34, HLA-DR (see Table 1). Inoculate the mesenchymal stem cells derived from human umbilical cord placenta at passage 4-7 in a roller bottle, digest with 10ml trypsin when the cells reach 80-90% confluence, transfer the cell suspension into a 50ml centrifuge tube and centrifuge at 1000rpm 3 minutes. After centrifugation, the supernatant was discarded, washed twice with PBS, and divided into 1×10 tubes. 6Cells, the first tube is a blank control (only containing mesenchymal stem cells), which is used to adjust the voltage of the flow cytometer, and the second tube is added with an isotype control antibody (PE-MOUSE IgGl / FITC-MOUSE IgG1 / APC-Mouse IgG1κ) 10 μl, add 10 μl of other corresponding antibodies to the other tubes, mix well, incubate at 4°C in the dark for 30 minutes, wash once with PBS, discard t...

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Abstract

The invention discloses a human umbilical mesenchymal stem cell and a preparation method thereof. The human umbilical mesenchymal stem cell is prepared by carrying out aseptic treatment, mechanical shearing, Wharton jelly exposure, collagenase digestion and isolated culture on an umbilical stalk of a healthy fetus by a term abdominal delivery. After freezing and thawing, the cell activity does not decline dramatically, thereby ensuring that the human umbilical mesenchymal stem cell is an ideal seed cell for cellular therapy. The invention also relates to a primitive mesenchymal stem cell prepared by the method, and the primitive mesenchymal stem cell is confirmed as the mesenchymal stem cell by the identification of morphology, immunophenotype and invitro differentiation experiments in accordance with the mesenchymal stem cell standards established by International Society for Cellular Therapy (ISCT). The cell count, cell activity, purity, doubling time and multiplication capacity of the human umbilical mesenchymal stem cell are apparently higher than those of bone marrow-derived mesenchymal stem cell.

Description

technical field [0001] The invention relates to a preparation method of primitive mesenchymal stem cells, in particular to the preparation of human umbilical cord mesenchymal stem cells by using human umbilical cord Wharton's jelly as a material and the preparation method thereof. technical background [0002] Mesenchymal stem cells (MSCs) are a type of tissue stem cells with multi-directional differentiation potential, which can be obtained from fetal blood, liver, bone marrow, fat and other tissues. Bone marrow-derived MSCs may be highly contaminated with viruses, and their cell number, expansion, and differentiation capabilities tend to decline significantly with age. Other methods, such as culturing MSCs from umbilical cord blood and peripheral blood, are difficult, and the amount of MSCs in full-term delivery umbilical cord blood or mobilized peripheral blood is still controversial. Recently, some scholars have isolated and cultured MSCs from postpartum waste material ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 洪敬欣韩俊领刘剑
Owner UNION STEMCELL & GENE ENG
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