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Method for culturing autologous umbilical cord mesenchymal stem cells with platelet-rich lysate from human umbilical cord blood

A platelet lysate and mesenchymal stem cell technology, which is applied in the field of autologous mesenchymal stem cell-free production system, can solve the problems of pollution, allergic pollution, etc.

Active Publication Date: 2016-08-10
武汉天晴干细胞有限公司
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  • 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 culturing autologous umbilical cord mesenchymal stem cells with platelet-rich lysate from human umbilical cord blood
  • Method for culturing autologous umbilical cord mesenchymal stem cells with platelet-rich lysate from human umbilical cord blood
  • Method for culturing autologous umbilical cord mesenchymal stem cells with platelet-rich lysate from human umbilical cord blood

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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 of which are: preparation of umbilical cord blood platelet-rich serum, preparation of platelet lysate, preparation of platelet-free serum, and cytokine PDGF in the platelet lysate - 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, chromosome karyotype analysis, surface antigen of umbilical cord mesenchymal stem cells after culture analyze.

Embodiment 2

[0040] According to the method for culturing autologous umbilical cord mesenchymal stem cells with human umbilical cord blood platelet-rich lysate described in Example 1, the preparation of the described umbilical cord blood platelet-rich serum: 50-120 ml of the collected umbilical cord blood are divided 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, use 2000-5000UI heparin as anticoagulant in the blood collection bag or blood collection tube, the upper serum accounts for 40% of the total volume %.

Embodiment 3

[0042] According to the human umbilical cord blood platelet-rich lysate described in embodiment 1 or 2, the method for culturing autologous umbilical cord mesenchymal stem cells, the preparation of the platelet lysate: transfer the platelet-rich serum to a new sterile centrifuge tube, and use Sysmexhematology The 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 serum for umbilical cord mesenchymal stem cell culture is 0.5~1×10 9 Between / ml; place the serum in a -80 degree refrigerator for 2 to 24 hours; after the frozen platelet-containing serum is rapidly recovered at 37 degrees, it is centrifuged at 800~1200g for 5~30 minutes to remove cell debris and the supernatant The platelet lysate is the platelet lysate, which is used for the cultivation of autologous umbilical cord stem cells after proportioning at a certain concentration.

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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, in particular to the comprehensive utilization of platelet-rich serum in umbilical cord blood and an animal-source-free production system of autologous mesenchymal stem cells. Background technique: [0002] In China, the development of stem cell life bank has attracted increasing attention from 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 bovine serum for culture, digestion, and cryopreservation. Therefore, there will indeed be residual risks of foreign proteins in the future clinical application of umbilical cord stem cells. [0003] Mesenchymal stem cells are a type of stem cells with self-renewal, proliferation and multi-directional differentiation potential. They adhere to the wall in vitro an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775C12N5/0789
Inventor 张怡刘艳青
Owner 武汉天晴干细胞有限公司
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