Method of preparing mesenchymal-like stem cells and mesenchymal-like stem cells prepared thereby

A technology of mesenchymal stem cells and human pluripotent stem cells, which is applied in the field of similar mesenchymal stem cells prepared by packaging, can solve the problems of increasing the culture cycle, high treatment cost, genetic stability, and difficulty in effectively controlling the pluripotency of embryonic stem cells.

Active Publication Date: 2022-02-25
ミラセルバイオ カンパニー リミテッド
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, since the method using a flow cytometer requires the use of laser light, there are problems in that the cell survival rate is reduced and the culture period is increased because the number of cells obtained after separation is small.
Treatment with cytokines and chemicals is problematic in terms of continued high treatment costs and genetic stability after differentiation and proliferation
In addition, it is difficult to effectively control the pluripotency of embryonic stem cells

Method used

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  • Method of preparing mesenchymal-like stem cells and mesenchymal-like stem cells prepared thereby
  • Method of preparing mesenchymal-like stem cells and mesenchymal-like stem cells prepared thereby
  • Method of preparing mesenchymal-like stem cells and mesenchymal-like stem cells prepared thereby

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preparation example Construction

[0053] According to an embodiment of the present invention, a method for preparing similar mesenchymal stem cells is provided, the method comprising the following steps: (a) preparing human pluripotent stem cells that are cultured for less than 70 passages after the establishment of the cell line; (b) from selecting cystic embryoid bodies from among the embryoid bodies induced to differentiate said human pluripotent stem cells; (c) isolating similar mesenchymal stem cells by loading said cystic embryoid bodies onto a cell-permeable three-dimensional culture insert; (d) separating only a cell population in the form of a monolayer among cells passing through the cell-permeable three-dimensional culture insert; and (e) by homogenizing the cell population in the form of a monolayer horizontally and vertically to The size of 100 μm to 500 μm is used to cultivate similar mesenchymal stem cells, and the similar mesenchymal stem cells have anti-inflammatory effect and immunosuppressive...

Embodiment 1

[0117] Example 1: Isolation and culture of similar mesenchymal stem cells with enhanced anti-inflammatory efficacy and immunosuppressive effect

[0118] figure 1 It is a schematic diagram illustrating the process of isolating and culturing similar mesenchymal stem cells from human pluripotent stem cells according to an embodiment of the present invention. Human pluripotent stem cells that were subcultured less than 70 times after the establishment of the cell line were used. And under the conditions of 37° C. and 5% CO2, the human pluripotent stem cells that have been subcultured for less than 70 times after the establishment of the cell line are maintained in the cell culture medium. Human pluripotent stem cells maintained in culture were isolated from culture plates by simple enzymatic treatment. Afterwards, only cystic (Cystic) embryoid bodies were selected by the formation of embryoid bodies. Only the first 2 ml of culture medium was added to the new culture plate dur...

Embodiment 2

[0119] Example 2: Isolation and proliferation of similar mesenchymal stem cells with enhanced anti-inflammatory efficacy and immunosuppressive effect

[0120] Among similar mesenchymal stem cells isolated in Example 1 above, cells in the form of a population that passed through the cell-permeable three-dimensional culture insert and moved below the cell-permeable three-dimensional culture insert were isolated. And mechanically remove the multilayer (Multilayer) population in the separated population. Of the isolated populations, only the monolayer (Monolayer) population was cut into a size of 500 μm or less in width and 500 μm or less in length using a micropipette, homogenized and selectively cultured. Preferably, the similar mesenchymal stem cells are cultured by homogenizing the word-form population to a size of 100 μm to 100 μm in width and length. After culturing for 5 to 7 days, only similar mesenchymal stem cells protruding from the population in the form of single c...

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Abstract

The invention relates to a method for effectively preparing mesenchymal-like stem cells having anti-inflammatory efficacy and immunosuppression, the method including: inducing differentiation of the human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines to select cystic embryoid bodies; loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells; isolating only monolayer-shaped cell clusters from the cells passing through the cell-permeable 3D culture unit, and uniformizing the same. Further, the invention relates to mesenchymal-like stem cells prepared by the preparation method, a transporter or a therapeutic composition including the mesenchymal-like stem cells, and a therapeutic composition for prevention or treatment of diseases that includes an active ingredient or exosomes secreted from the mesenchymal-like stem cells.

Description

technical field [0001] The present invention relates to a method for preparing similar mesenchymal stem cells from human pluripotent stem cells and similar mesenchymal stem cells prepared by the preparation method. Specifically, human pluripotent stem cells (Human pluripotent stem cells) cultured for less than 70 passages after the establishment of pluripotent cell lines were used to select Cystic embryoid bodies among various forms of embryoid bodies induced by their differentiation , and by loading cystic embryoid bodies onto a cell-permeable three-dimensional culture insert (3Dculture unit) to isolate mesenchymal stem-like cells in which only the monolayer form was isolated and homogenize the cell population in the form of a monolayer, thereby preparing mesenchymal stem cells-like. The high-purity similar mesenchymal stem cells prepared by the above method have anti-inflammatory effects and immunosuppressive properties, and the expression of CD90 and SOX2 is above 95%. In...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61K45/06A61P29/00A61P37/06A61P25/00A61P37/02A61P9/10A61P11/00A61P1/00A61P9/14A61P17/02A61P19/02A61P13/12A61P13/10A61P21/00A61P19/08A61P27/16A61P17/00A61P17/06A61P17/16A61P27/02A61P25/28A61P25/16A61K8/99A61Q19/00
CPCC12N5/0662A61K35/28A61K45/06A61P29/00A61P37/06A61P25/00A61P37/02A61P9/10A61P11/00A61P1/00A61P9/14A61P17/02A61P19/02A61P13/12A61P13/10A61P21/00A61P19/08A61P27/16A61P17/00A61P17/06A61P17/16A61P27/02A61P25/28A61P25/16A61K8/99A61Q19/00C12N2513/00C12N2506/03A61K8/98A61P35/00C12N2500/10C12N2506/45C12N2533/30C12N2535/00C12N2506/02
Inventor 洪起成郑炯敏金恩永申姃民A.R.姜
Owner ミラセルバイオ カンパニー リミテッド
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