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Separate culture method for human amniotic mesenchymal stem cells

A technique for the separation and culture of stromal cells, applied in the field of separation and culturing of amniotic mesenchymal stem cells, can solve problems such as transmission and virus infection, and achieve the effects of high cell purity, promotion of proliferation, and convenience in preparation and purchase.

Pending Publication Date: 2017-05-17
北京天晟宇生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the application of animal serum such as FBS, after stem cell transplantation, the infusion of heterologous proteins poses a potential threat to patients, which may produce anti-FBS antibodies and other unknown adverse reactions, and may cause the spread of viral infections between humans and other species

Method used

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  • Separate culture method for human amniotic mesenchymal stem cells
  • Separate culture method for human amniotic mesenchymal stem cells
  • Separate culture method for human amniotic mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Isolation and culture of human amniotic mesenchymal stem cells

[0040] 1. Cell separation

[0041] (1) The amniotic membrane was taken from healthy puerpera who had cesarean section at term. deal with.

[0042] (2) Set the diameter to The amniotic membrane was washed with 0.9% sodium chloride solution, cut to about 1mm×1mm×1mm, treated with 0.1% collagenase and % trypsin digested at 37°C Dilute to 40ml with α-MEM (Gibco, USA).

[0043] (3) Filter the above liquid with 100-mesh and 200-mesh filters successively to remove undigested tissues.

[0044] (4) The filtered cell-containing liquid is placed in Place in a centrifuge tube after balancing and place it in a low-temperature centrifuge, and centrifuge for 8 minutes at a centrifugal force of 300Xg and a temperature of 4±2°C.

[0045] After shutdown, gently take out the centrifuge tube and place it on the test tube rack. The content of the centrifuge tube is divided into two parts, the upper laye...

Embodiment 2

[0053] Example 2: Flow Cytometry

[0054] 1. Method

[0055] For the human amniotic mesenchymal stem cells obtained in Example 1, the expression of the human leukocyte differentiation antigen CD73 was detected by flow cytometry with an anti-human CD73 monoclonal antibody (BioLegend, USA), and the expression of the human leukocyte differentiation antigen CD73 was detected with an anti-human CD90 monoclonal antibody (BioLegend, USA). ) by flow cytometry to detect the expression of human leukocyte differentiation antigen CD90, with anti-human Monoclonal antibody (BioLegend, USA) was used to detect human leukocyte differentiation antigen by flow cytometry The expression of the situation, with anti-human Monoclonal antibody (BioLegend, USA) was used to detect human leukocyte differentiation antigen by flow cytometry The expression of HLA-DR monoclonal antibody (BioLegend, USA) by flow cytometry (model FACSCalibur BD Company) to detect the expression of human leukocyte anti...

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Abstract

The invention discloses a separate culture method for human amniotic mesenchymal stem cells, and belongs to the technical field of biology. The separate culture method comprises the following steps: (I) cell separation; (II) cell culture: inoculating a single separated human amniotic karyocyte into a plastic culture bottle of 75cm<2>, culturing in a 5 percent CO2 incubator at the temperature 37 DEG C, wherein a culture solution is a culture medium special for the human amniotic mesenchymal stem cell; replacing the culture solution 24 hours later, discarding non-adherent cells, and replacing the culture solution every 2 to 3 days; fusing the cells after the human amniotic mesenchymal stem cells grow to 70 to 80 percent, digesting with pancreatin, and performing passage; performing passage once in the culture medium special for the human amniotic mesenchymal stem cells every 2 to 3 days at the temperature of 37 DEG C and in 5 percent CO2. The obtained human amniotic mesenchymal stem cells express by the following three kinds of cell membrane molecules, namely, a human leukocyte differentiation antigen CD73, a human leukocyte differentiation antigen CD90 and a human leukocyte differentiation antigen CD105, and do not express a human leukocyte differentiation antigen CD45 and a human leucocyte antigen HLA-DR.

Description

technical field [0001] The invention relates to a method for separating and culturing amniotic mesenchymal stem cells, belonging to the field of biotechnology. Background technique [0002] Human amniotic stem cells have high self-renewal, proliferation, implantability and multilineage differentiation capabilities, and there is no risk of immune rejection and tumorigenicity after transplantation in allogeneic individuals. Moreover, human amniotic stem cells are derived from the discarded placenta after childbirth, and their clinical application will not bring about medical ethics controversy. Amniotic stem cells have excellent biological characteristics such as colony formation ability, proliferation ability, embryonic stemness, immune regulation ability, and no allogeneic transplantation rejection and tumorigenicity. They can be used for cell therapy of many diseases and repair of tissue and organ damage. It is an ideal seed cell resource in the field of regenerative medic...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/073
Inventor 刘英谷涌泉
Owner 北京天晟宇生物科技有限公司
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