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Preparation and application of mesenchymal stem cell secretion factors

A technology of mesenchymal stem cells and secreting factors, which is applied in the application of promoting angiogenesis and skin anti-aging and repairing, and the preparation of mesenchymal stem cells secreting factors, achieving the effects of high safety, high content, and strong ability to promote skin defect repair.

Active Publication Date: 2020-03-24
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no beauty product about iMSC promoting angiogenesis and skin anti-aging repair

Method used

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  • Preparation and application of mesenchymal stem cell secretion factors
  • Preparation and application of mesenchymal stem cell secretion factors
  • Preparation and application of mesenchymal stem cell secretion factors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Induction, expansion and identification of iMSCs

[0046] Select iPSCs in good condition, and replace the iPSC medium with complete MSC medium. Medium was changed every 2-3 days for approximately 14 days. Differentiated cells were flow-sorted using the MSC marker CD105 to obtain CD105-positive cells. Use MSC complete medium to continue the expansion culture. MSC complete medium components are: low-sugar DMEM, 10% fetal bovine serum, non-essential amino acids, double antibodies (containing 100units / mL penicillin and 100μg / mL streptomycin), 2mM GlutaMAX, 2ng / mL EGF, 2ng / mLFGF, 55 μM β-mercaptoethanol. Differentiation process see figure 1 .

[0047] After digestion of P4 generation cells with a confluence of 90% (use Tryple E for cell digestion), count and prepare a density of 1 × 10 6 Each cell / mL cell suspension was filtered with a 40 μm cell mesh, 1 mL of the filtrate was taken, and 10 μL of fluorescently labeled cell surface marker antibodies were added...

Embodiment 2

[0051] Example 2 Preparation and storage of iMSC-CdM

[0052] Select P5-P8 generations of iMSCs with strong proliferation ability to prepare the cytokine supernatant; the preparation steps of iMSC-CdM are as follows: P5-P8 generations of iMSCs, when the cell confluence is 60%-70%, aspirate to completely culture After rinsing three times with 0.9% normal saline, add an appropriate amount of phenol red-free low-sugar DMEM (Gibco, #11054) according to the area of ​​the culture dish (see Table 2 for details); after culturing for 24 hours, collect the cell culture supernatant and centrifuge at 1200rpm Cell debris and impurities were removed for 5 minutes, filtered with a 0.22 μm filter membrane, and subpackaged; the iMSC-CdM prepared through the above steps were tested for mycoplasma and endotoxin (third-party testing company), and the results were negative.

[0053] The iMSC-CdM prepared by the above steps were aliquoted and stored at -20°C, thawed at 4°C before use, and stored at...

Embodiment 3

[0058] Example 3 Effect of Different Cell Confluency on Total Protein and Key Components of iMSC-CdM

[0059] The preparation of iMSC-CdM was carried out when the cell confluency was 60-70% and 80-90%, respectively, and the preparation steps were as described in Example 2.

[0060] The total protein concentration in iMSC-CdM was determined by BCA method.

[0061] The concentration of key components in iMSC-CdM including angiogenin, bFGF, HGF, and VEGF was determined by protein chip or Elisa method.

[0062] Such as Figure 4As shown in (A, B), the preparation of iMSC-CdM is carried out when the cell confluence is 60%-70%, the total protein content in iMSC-CdM is about 1.2 times of that of 80%-90% confluence, and the angiogenesis The content of production factors, including angiogenin, bFGF, HGF, and VEGF, was higher than that at 80%-90% confluence. A cell confluence of 60%-70% is more conducive to enriching the protein components in iMSC-CdM than 80%-90%.

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Abstract

The invention provides a preparation method of mesenchymal stem cell secretion factors. Compared with mesenchymal stem cells from other sources, such as umbilical cord mesenchymal stem cells, the mesenchymal stem cells from pluripotent induced stem cells used in the invention are more stable in source and not easy to age after in vitro passage. The preparation method is simple in preparation process, the component of the obtained mesenchymal stem cells secrete factors is a human-derived stem cell secretion factor; the potential tumorigenic hidden danger of the stem cells is avoided; no exogenous additive or preservative is contained; no mycoplasma or endotoxin is contained; irritability is not easily caused; storage is convenient; better angiogenesis promoting capacity and skin defect healing promoting capacity are achieved; obvious promoting effect on vascular cell proliferation and angiogenesis is achieved; skin repair can be promoted, and skin aging can be delayed through external application; and a promising application prospect in skin injury treatment and development of beauty and skin care products is achieved. .

Description

technical field [0001] The invention belongs to the field of medical care and health, and specifically relates to the preparation of a mesenchymal stem cell secreted factor derived from pluripotent induced stem cells and its application in the direction of promoting angiogenesis and skin anti-aging repair. Background technique [0002] Aging is an inevitable stage in the development of body life activities, and skin aging is the most external feature of body aging. Studies have shown that skin aging is a complex physiological process, which is the result of multiple mechanisms. In addition to the reduction of collagen fiber synthesis and elastic fiber degeneration in a broad sense, vascular aging is also considered to be one of the important signs of skin aging. Slowing down vascular aging and promoting angiogenesis will be a new direction for skin rejuvenation treatment. [0003] Mesenchymal stem cells (MSC) have the characteristics of strong tissue repair ability, strong ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61P17/02A61P17/00A61K8/99A61Q19/00A61Q19/08C12N5/0775
CPCA61K35/28A61P17/02A61P17/00A61K8/99A61Q19/00A61Q19/08C12N5/0662C12N2509/00C12N2500/32C12N2500/44
Inventor 梁小婷林芳刘中民
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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