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Preparation method and application of treatment-grade mesenchymal stem cells based on induced pluripotent stem cells

A stem cell and cell technology, applied in the fields of biomedicine and biology, can solve the problems of complicated operation, unstable quality, small output, etc., and achieve the effects of high efficiency, simple operation and short culture period.

Pending Publication Date: 2019-12-31
NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the known methods of using iPSCs to prepare MSCs mostly require adherent culture, digestion, and repeated passage, and involve co-cultivation of mouse-derived cells, coating of heterogeneous animal-derived substances, flow sorting, or virus transfection, etc., and the operation is very complicated and cumbersome. , is not suitable for large-scale production, the yield is small and the quality is unstable, and foreign substances are introduced, and the clinical application value is limited

Method used

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  • Preparation method and application of treatment-grade mesenchymal stem cells based on induced pluripotent stem cells
  • Preparation method and application of treatment-grade mesenchymal stem cells based on induced pluripotent stem cells
  • Preparation method and application of treatment-grade mesenchymal stem cells based on induced pluripotent stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Establishment of the method for preparing MSCs derived from human iPSCs

[0042] 1. Culture of human-derived iPSCs

[0043] We use TeSR that is xeno-free from animal source components TM -E8 TM (STEMCELL Technologies, Catalog#5990) human ESC / iPSC medium, cultured human iPSCs in a feeder-free culture mode (take normal human skin fibroblasts, see references Warren L, Ni Y, Wang J, Guo X. Feeder-free derivation of human induced pluripotent stem cells with messenger RNA. Sci Rep.2012; 2: 657. doi: 10.1038 / srep00657. Epub 2012 Sep 14. Acquired by the method of cultivation), the culture conditions are 37 ° C, 5% -10% o 2 and 10%-15% CO 2 , 95% humidity. Change fluid daily.

[0044] For the typical morphology of iPSCs maintained in culture, see figure 1 . The typical morphology of iPSCs, the clonal cells are aggregated in a round shape with uniform shape and neat edges. Photographed under a microscope at 100×.

[0045] 2. Induction medium combination A susp...

Embodiment 2

[0051] 1. Culture of human-derived iPSCs

[0052] With embodiment 1.

[0053] 2.1. Induction medium combination A Suspension culture induces iPSCs-EB differentiation

[0054] Induction medium combination A is: ①TeSR TM -E8 TM Human ESC / iPSC medium; ② CHIR-99021 (CT99021), the final concentrations are 1 μM, 25 μM and 50 μM; ③ human serum (allogeneic source), the final concentrations are set to 1%, 10% and 20% (volume percentage); ④ Fatty acid mixture (volume ratio 1:1000, Sigma, L0288), 1× non-essential amino acid solution (NEAA, Gibco, 11140-050), human fibroblast growth factor (bFGF, R&D, 233-FB-01M, 1-50ng / mL can be used, 20ng / mL is used in this implementation case), vitamin C (Vc, Sigma, A4403, 1-10 μM is all available, 2 μM is used in this implementation case). All the other are with embodiment 1.

[0055] 2.2. Receive MSCs

[0056] With embodiment 1.

Embodiment 3

[0058] 1. Culture of human-derived iPSCs

[0059] With embodiment 1.

[0060] 2.1. Induction medium combination A Suspension culture induces iPSCs-EB differentiation

[0061] Induction medium combination A is: ①TeSR TM -E8 TM Human ESC / iPSC medium; ② CHIR-99021 (CT99021), the final concentrations were 1 μM, 25 μM and 50 μM; ③ Fetal bovine serum, the final concentrations were set to 1%, 10% and 20% respectively; ④ Fatty acid mixture (vol. Ratio 1:1000, Sigma, L0288), 1× non-essential amino acid solution (NEAA, Gibco, 11140-050), human fibroblast growth factor (bFGF, R&D, 233-FB-01M, 1-50ng / mL can be , this implementation case selects 20ng / mL), vitamin C (Vc, Sigma, A4403, 1-10 μ M can be selected, and this implementation case selects 2 μ M). All the other are with embodiment 1.

[0062] 2.2. Receive MSCs

[0063] With embodiment 1.

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Abstract

The invention discloses a preparation method of iPSCs-derived MSCs. The preparation method comprises the following steps of culturing iPSCs; digesting the iPSCs, and performing suspension culture on digested iPSCs cells so as to form an embryoid body; adding an induced culture medium combination A in the suspension culture process of the embryoid body for induction and disintegration; and after the embryoid body is disintegrated, performing direct adherence culture, or after the embryoid body is digested, performing adherence culture, or after the embryoid body is digested, performing sortingculture to obtain the MSCs. The invention also discloses an application of the iPSCs-derived MSCs. The novel method for quickly preparing the iPSCs-derived MSCs disclosed by the invention has the characteristics that xenogeneic exogenous substances do not exist, the suspension culture is performed, the culture period is shorter, the operation is simple, infinite amplification is achieved, the efficiency is high, and the preparation method is more suitable for large-scale production. The obtained iPSCs-derived MSCs conforms to essential features of the MSCs through identification, besides, hasfunctions being similar to those of bone marrow derived MSCs, and can be used for tissue repair and treatment of immunization relevant diseases.

Description

technical field [0001] The invention belongs to the fields of biomedicine and biotechnology, and in particular relates to a preparation method and application of therapeutic grade mesenchymal stem cells based on induced pluripotent stem cells. Background technique [0002] Based on the immunomodulatory effect and multilineage differentiation potential of mesenchymal stem cells (MSCs), the application research of MSCs in the fields of immune-related diseases and regenerative medicine has attracted extensive attention. However, the rarity of MSCs, heterogeneity in different tissues and organs, and the need to obtain them through invasive means (except for umbilical cord sources) limit the potential of MSCs for clinical translation. In addition, MSCs have limited ability to expand during culture, and usually begin to age after 8-10 passages, so they cannot produce enough cells for clinical treatment. Therefore, finding simple, reliable, and sufficient cell sources is crucial f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/077A61K35/28A61P11/00A61P39/06A61P37/00
CPCC12N5/0668C12N5/0653C12N5/0654C12N5/0655A61K35/28A61P11/00A61P39/06A61P37/00C12N2500/02C12N2506/1392
Inventor 季晨博尤梁惠曹彦
Owner NANJING MATERNITY & CHILD HEALTH CARE HOSPITAL
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