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A kind of mesenchymal stem cell and its preparation method and application

A technology for stem cells and human pluripotent stem cells, which can be used in biochemical equipment and methods, animal cells, vertebrate cells, etc., and can solve the problems of limited number of MSCs, unstable MSC effects, and large demand for clinical cells.

Active Publication Date: 2020-08-21
安徽中盛溯源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present invention solves the following problems: 1) The MSC induced by the traditional serum-containing culture system is unstable and the purity is not high; 2) The number of MSC obtained by the traditional 2D culture method is limited, and the demand for clinical cells is large

Method used

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  • A kind of mesenchymal stem cell and its preparation method and application
  • A kind of mesenchymal stem cell and its preparation method and application
  • A kind of mesenchymal stem cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] A method for preparing mesenchymal stem cells, comprising the steps of:

[0088] S1: Human pluripotent stem cells form embryoid bodies;

[0089] S2: Differentiation of embryoid bodies to mesoderm cells;

[0090] S3: Differentiation of mesoderm cells into mesenchymal stem cells.

[0091] Specifically, the flow chart is as figure 1 As shown, the steps are as follows:

[0092] (1) Human pluripotent stem cells form embryoid bodies (D-1~D0)

[0093] The hPSCs used in the experiment have been strictly verified for pluripotency (expressing various pluripotency markers, and can form teratomas including inner, middle and outer germ layers in immunodeficient mice). hPSCs are normally cultured in hPSC maintenance medium, the medium used is E8 or TeSR or other similar medium.

[0094] In this example, human pluripotent stem cells were prepared by the method disclosed in patent CN 108085299A.

[0095] After human pluripotent stem cells were cultured to a confluence of 70-90%,...

Embodiment 2

[0114] This example studies the effects of 2D culture and 3D culture on the number of iMSCs obtained. The only difference between the 2D culture in this example and Example 1 is that 2D culture is used in the whole induction process.

[0115] The experimental results are shown in Table 3:

[0116] table 3

[0117]

[0118] It can be concluded from Table 3 that with the 3D differentiation method, the cells can grow out of the EBs and get iMSCs with a cell volume about 10 to 15 times that of the initial cells. Twice as many cells were obtained and 1 / 4 the amount of medium was used. Therefore, 3D culture is preferred for large-scale production of iMSCs.

Embodiment 3

[0120] This example studies the influence of different Rock inhibitor concentrations on the experimental results. The ROCK inhibitor concentrations were set at 2.5, 5 and 10 μM, respectively. The result is as figure 2 As shown, when the concentration of ROCK inhibitor was 2.5 and 5 μM, the EBs formed by pluripotent stem cells were small and heterogeneous in shape; when the concentration of ROCK inhibitor was 10 μM, the EBs formed were very round and uniform in shape.

[0121]Thus, the concentration of ROCK inhibitor plays a large role in the formation of EBs during the first 8–32 hours of our differentiation method.

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Abstract

The present invention belongs to the field of stem cell biology, relates to lineage-specific differentiation of human multipotential or pluripotent stem cells, and particularly relates to a mesenchymal stem cell, and a preparation method and an application thereof. The method for preparing the mesenchymal stem cell comprises the following steps: S1, human multipotential stem cells form embryoid bodies; S2, the embryoid bodies differentiate into mesoderm cells; and S3, the mesodermal cells differentiate into the mesenchymal stem cells. The preparation method has a clear cell differentiation path, high differentiation efficiency, and stable differentiation effect and does not use serum-containing culture system or trophoblast cells, and the obtained cell population has high purity and largenumber, and thus is suitable for subsequent production and application of clinical-grade cell preparations.

Description

technical field [0001] The invention belongs to the field of stem cell biology, and relates to the lineage-specific differentiation of human multipotent or multipotential stem cells, in particular to a mesenchymal stem cell and its preparation method and application. Background technique [0002] Mesenchymal stem cells (MSCs) are mostly derived from the mesoderm in the early stage of development. They have the ability of self-renewal and the potential to differentiate into functional cells such as bone, cartilage, and fat. They are multipotent stem cells. MSC was originally found in bone marrow. In addition, MSC exists in amniotic membrane, umbilical cord, umbilical cord blood, placenta, fat, dental pulp and other tissues in human and animal bodies. Among them, the MSC content in umbilical cord tissue is the most abundant, from adipose tissue A large number of MSCs can also be obtained, and the content of MSCs in bone marrow tissue is relatively small, accounting for 1 / 10 of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/28
CPCA61K35/28C12N5/0662C12N2500/30C12N2501/15C12N2501/155
Inventor 李海兰俞君英张颖
Owner 安徽中盛溯源生物科技有限公司
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