A preparation method and kit for clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells

A technology of pluripotent stem cells and mesenchymal stem cells, which is applied in the field of preparation of clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells, can solve the problems that mesenchymal stem cells cannot be used, and achieve the effect of unified source

Active Publication Date: 2020-10-20
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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Problems solved by technology

However, this culture method requires the replacement of two different differentiation media, and the differentiated mesenchymal stem cells cannot be used clinically.

Method used

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  • A preparation method and kit for clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells
  • A preparation method and kit for clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells
  • A preparation method and kit for clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells

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preparation example Construction

[0034] The invention provides a method for preparing clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells, comprising the following steps:

[0035]1) digesting human induced pluripotent stem cells to make a single cell suspension, and inoculating it on a pretreated culture plate;

[0036] 2) When the cells reach 80% confluence, use the induction medium for induction culture, and change the medium every day;

[0037] 3) When the cells reach 100% confluence, they are digested to make a single cell suspension, inoculated on the expansion medium for expansion culture, and the cells obtained after 3 to 4 generations of expansion culture are clinical-grade Mesenchymal stem cells.

[0038] In the present invention, human induced pluripotent stem cells are digested to make a single cell suspension, which is seeded on a pretreated culture plate.

[0039] In the present invention, the preparation method of human induced pluripotent stem cells is to separat...

Embodiment 1

[0049] The formula of MSC induction medium (referred to as M+S+C2):

[0050] 1L α-MEM basal medium contains 10% human platelet extract, 10 μmol / L SB431542 and 2 μmol / L CHIR99021. MSC induction medium was obtained after conventional sterilization.

Embodiment 2

[0052] Recipe of MSC Induction Medium:

[0053] 1L α-MEM basal medium contains 10% human platelet extract, 5 μmol / L SB431542 and 5 μmol / L CHIR99021. MSC induction medium was obtained after conventional sterilization.

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Abstract

The invention provides a preparation method and kit of clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells, belonging to the technical field of cell culture. The induction medium is based on α-MEM and also includes platelet extract, SB431542 and CHIR99021. The preparation method of clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells comprises the following steps: digesting human induced pluripotent stem cells to make a single cell suspension, and inoculating them on a pretreated culture plate; waiting for the cells to reach 80% confluence The induction medium was used for induction culture, and the medium was changed every day; when the cells reached 100% confluence, they were digested and made into a single cell suspension, and then inoculated on the expansion medium for expansion and culture for 3 to 4 generations to obtain clinical-grade mesenchyme stem cells. The mesenchymal stem cells prepared by the method provided by the invention provide a source of mesenchymal stem cells with uniform source, stable supply and consistent characteristics for clinical application.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a preparation method and a kit of clinical-grade human induced pluripotent stem cell-derived mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSC, mesenchymal stem cells) are a cell group derived from the mesoderm and ectoderm in the early embryonic development. They have multi-directional differentiation potential and low immunogenicity. and other fields are widely used. At present, mesenchymal stem cells are mainly derived from umbilical cord, fat, bone marrow and other tissues. The number of passages is limited, and there are individual differences in mesenchymal stem cells from different donor sources. It is difficult to meet the needs of clinical applications for stable and consistent cell sources. In addition, mesenchymal stem cells transplanted into the body can differentiate into a variety of terminal cell types. During the d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775
CPCC12N5/0662C12N2500/84C12N2500/02C12N5/0668C12N2501/15C12N2506/45C12N2501/415
Inventor 刘中民贾文文鲁济真汤红明
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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