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Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate

A platelet lysate, umbilical cord stem cell technology, applied in the field of regenerative medicine and biology, can solve pollution, allergic pollution and other problems

Active Publication Date: 2013-10-16
武汉天晴干细胞有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are two risks in the use of animal serum. The first is the allergic contamination reaction of the treatment body caused by heterologous animal-derived protein residues, and the second is the potential contamination of viruses, mycoplasma and other pathogens of known or unknown animal origin.

Method used

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  • Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate
  • Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate
  • Method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate

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Embodiment 1

[0038] A method for culturing autologous umbilical cord mesenchymal stem cells with human umbilical cord blood platelet rich lysate. The steps are: preparation of umbilical cord blood platelet rich serum, preparation of platelet lysis solution, preparation of platelet-free serum, and cytokine PDGF in platelet lysis solution -Determination of AB, FGF2, TGF-β, VEGF content, isolation and primary culture of umbilical cord stem cells, culture and passage of umbilical cord stem cells, cryopreservation of umbilical cord stem cells, karyotype analysis, surface antigen of umbilical cord mesenchymal stem cells after culture analysis.

Embodiment 2

[0040] According to the method for culturing autologous umbilical cord mesenchymal stem cells with human cord blood platelet-rich lysate described in Example 1, the preparation of cord blood platelet-rich serum: 50-120 ml of collected cord blood is aliquoted into 50 ml sterile centrifuge tubes Centrifuge at 150-220g for 5-25 minutes at a temperature of 4-30 degrees. The upper layer is platelet-rich serum. The blood collection bag or blood collection tube uses 2000-5000UI heparin as the anticoagulant. The upper serum accounts for 40% of the total volume. %.

Embodiment 3

[0042] According to the method for culturing autologous umbilical cord mesenchymal stem cells with the human umbilical cord blood platelet-rich lysate described in embodiment 1 or 2, the preparation of the platelet lysate: transfer the platelet-rich serum to a new sterile centrifuge tube, using Sysmex The hematology analyzer (KX-21) counts the number of red blood cells, white blood cells, and platelets contained in whole blood and serum. The ideal number of platelets in the serum for umbilical cord mesenchymal stem cell culture is 0.5~1×10 9 / ml; put the serum in the refrigerator at -80 degrees for 2-24 hours; after the frozen platelet-containing serum is rapidly recovered at 37 degrees, centrifuge at 800-1200g for 5-30 minutes to remove cell debris and supernatant The solution is the platelet lysate, which is proportioned at a certain concentration and used for the cultivation of autologous umbilical cord stem cells.

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Abstract

The invention relates to a method for cultivating autologous umbilical cord mesenchymal stem cells by adopting human umbilical cord blood rich platelet lysate. The conventional cell bank preparing processes adopt blood serum of calves or fetal calves to cultivate, digest, cryopreserve and the like, so that the risk of heterogeneous protein residue certainly exists in the application of clinic umbilical cord stem cells in the future. The method comprises the following steps of umbilical cord blood rich platelet serum preparation, platelet lysate preparation, preparation of serous without platelet, the confirmation of the content of cell factors, namely PDGF-AB, FGF2, TGF-Beta and VFGF in the platelet lysate, the separation and the primary culture of umbilical cord stem cells, the culture and passage of umbilical cord stem cells, the cryopreservation of umbilical cord stem cells, and the karyotype analysis. The method is used for cultivating stem cells.

Description

Technical field: [0001] The invention relates to the fields of regenerative medicine and biotechnology, and in particular to a comprehensive utilization of platelet-rich serum in umbilical cord blood and a production system of autologous mesenchymal stem cells without animal origin. Background technique: [0002] In China, the development of stem cell life bank is increasingly concerned by families of childbearing age. While most pregnant women choose to store cord blood for their children, they also choose to cultivate and preserve cord stem cells. The current cell bank preparation process uses calf or fetal calf serum for cultivation, digestion, and freezing. Therefore, there is indeed a risk of foreign protein residues in the future clinical application of umbilical cord stem cells. [0003] Mesenchymal stem cells are a type of stem cells with the potential for self-renewal, proliferation and multidirectional differentiation. They adhere to the wall and present fibroblast-like c...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/0789
Inventor 张怡陈小威宋晓乐
Owner 武汉天晴干细胞有限公司
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