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Amniotic mesenchymal stem cell culture medium and method for culturing amniotic mesenchymal stem cells

An amniotic mesenchymal and stem cell technology, applied in the field of stem cells, can solve the problems of slow proliferation of mesenchymal stem cells, limited number of proliferating cells, low expression, etc., to meet research and development needs, improve expression rate and cell purity, and promote effect of proliferation

Active Publication Date: 2017-07-18
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In view of this, the present invention discloses a medium for amniotic mesenchymal stem cells, which is used to solve technical problems such as slow proliferation of mesenchymal stem cells, low expression, limited number of proliferated cells, etc.

Method used

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  • Amniotic mesenchymal stem cell culture medium and method for culturing amniotic mesenchymal stem cells
  • Amniotic mesenchymal stem cell culture medium and method for culturing amniotic mesenchymal stem cells
  • Amniotic mesenchymal stem cell culture medium and method for culturing amniotic mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0024] Example 1 Primary isolation of amniotic mesenchymal stem cells

[0025] Under sterile conditions, take the fresh amniotic membrane discarded after delivery, peel off the amniotic membrane, wash it with PBS buffer solution, and cut the amniotic membrane tissue into 5×5cm 2 For tissue fragments of different sizes, add 3-5 times the volume of 0.25% trypsin, place on a 200r / min 37°C constant temperature shaker, digest for 30min, and shake vigorously several times every 10min during this period to remove epithelial cells.

[0026] After the digestion is complete, transfer it to a storage bottle, add 150 mL of PBS buffer solution, shake vigorously, and repeat this operation 2 times. Transfer the amnion tissue to a small beaker and chop it to 1mm 3 . Then transfer to a storage bottle, add 20 mL of 0.5% type I collagenase and complete medium (high glucose DMEM+10% FBS), so that the final concentration of collagenase is 0.1-0.2%. Place in a constant temperature shaker at 37°C...

Embodiment 2

[0028] (1) Add the following components to the DMEM / F12 medium, and mix well to obtain a culture medium for amniotic mesenchymal stem cells. Wherein, the culture medium comprises the following components:

[0029] Fetal bovine serum: 7.5% vol;

[0030] Double antibody: 1% vol;

[0031] Basic fibroblast growth factor (bFGF): 1.25ng / mL;

[0032] Epidermal growth factor (EGF): 1.25ng / mL;

[0033] Platelet-derived cell growth factor (PDGF): 0.3ng / mL;

[0034] Ginseng saponin: 1g / mL;

[0035] Lentinan: 1g / mL;

[0036] Basal medium: DMEM / F12 medium.

[0037] (2) The primary amniotic mesenchymal stem cells of Example 1 were resuspended using the medium of step (1), and then 5×10 4 ~8×10 4 Cells / mL were seeded in a 15cm culture dish, and placed in CO 2 The culture was carried out in an incubator, and the medium was changed after 48 hours of cultivation, and the medium was changed every 2 days thereafter.

[0038] When the primary amniotic mesenchymal stem cells grew to 80% c...

Embodiment 3

[0045] (1) Add the following components to the DMEM / F12 medium, and mix well to obtain a culture medium for amniotic mesenchymal stem cells. Wherein, the culture medium comprises the following components:

[0046] Fetal bovine serum: 7.5% vol;

[0047] Double antibody: 1% vol;

[0048] Basic fibroblast growth factor (bFGF): 1ng / mL;

[0049] Epidermal growth factor (EGF): 1ng / mL;

[0050] Platelet-derived cell growth factor (PDGF): 0.3ng / mL;

[0051] Ginseng saponin: 1g / mL;

[0052] Lentinan: 1g / mL;

[0053] Basal medium: DMEM / F12 medium.

[0054] (2) The P6 generation amniotic mesenchymal stem cells in Example 2 are subcultured with the medium of step (1), and the culture medium is 5×10 4 Cells / mL were inoculated into 15 cm culture dishes, five dishes were inoculated, and a total of 5 × 10 cells were inoculated. 6 , then placed in CO 2 The culture was carried out in an incubator, and the medium was changed after 48 hours of culture, and then the medium was changed every...

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Abstract

The invention belongs to the technical field of stem cells, and particularly relates to an amniotic mesenchymal stem cell culture medium and a method for culturing amniotic mesenchymal stem cells. The amniotic mesenchymal stem cell culture medium provided by the invention comprises a basic fibroblast growth factor, a platelet derived cell growth factor, panax japlcus var saponin and lentinan. Through reasonable compatibility of the components, cell proliferation can be promoted, the expression rate and the cell purity of the amniotic mesenchymal stem cells are improved, and a sufficient number of stem cells are obtained within a relatively short time; the amniotic mesenchymal stem cell culture medium is applicable to two-dimensional or three-dimensional culture of the amniotic mesenchymal stem cells, so that research and development needs are met.

Description

technical field [0001] The invention belongs to the technical field of stem cells, and in particular relates to a medium for amniotic mesenchymal stem cells and a method for culturing amniotic mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are an important member of the stem cell family, derived from the mesoderm and ectoderm in the early stages of development, belonging to pluripotent stem cells, which can differentiate into pancreatic islets, neural , vascular endothelium, bone, cartilage, muscle, liver, cardiac muscle and other tissue cells. MSCs have become the seed cells for cell and gene therapy research because of their relative lack of immunogenicity, simple culture technology, and cryopreservation. They have potential clinical application value and have become a hot spot in stem cell research. However, the culture effect of the existing mesenchymal stem cell culture medium is not ideal, and there are technical defects such as sl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/0775
CPCC12N5/0605C12N5/0668C12N2501/11C12N2501/115C12N2501/135C12N2501/90C12N2501/999
Inventor 王一飞陈海佳葛啸虎李丽娟王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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