Method for separating and cultivating porcine marrow endothelial progenitor cell

A technology of endothelial progenitor cells and culture methods, which is applied in the field of isolation and culture of porcine bone marrow endothelial progenitor cells, can solve the problems of high cost of immunomagnetic bead separation methods, inconsistent phenotypic expression rates, and reduced cell acquisition rate, etc. The effect of high efficiency, convenient material collection and wide source

Inactive Publication Date: 2008-09-17
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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Problems solved by technology

[0012] The traditional culture method is more cumbersome, in the use of CD34 + MNC CD34 is unavoidable in the process of sorting MNC by antibody-coated immunomagnetic beads - The loss of the magnetic beads and the damage to the cells by the magnetic beads themselves will greatly reduce the acquisition rate of the c...

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  • Method for separating and cultivating porcine marrow endothelial progenitor cell
  • Method for separating and cultivating porcine marrow endothelial progenitor cell
  • Method for separating and cultivating porcine marrow endothelial progenitor cell

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Embodiment 1

[0075]1) Isolation of mini-pig bone marrow mononuclear cells: Take 5-10 ml of anti-coagulated bone marrow from the ilium of mini-pig under sterile conditions, transfer the bone marrow cells into a test tube and mix them with sterile PBS solution 1:1 and centrifuge (20°C, 400g, 10 minute). After centrifugation, remove the supernatant, and then mix with sterile PBS solution 1:1. Take 10ml of Ficoll solution (Histopaque-10771, 1.077g / ml, Sigma company) and add it into a blank test tube in two parts; slowly drop the mixed bone marrow cell liquid into the test tube containing Ficoll solution (this step must be slow, be careful not to Break the plane between Ficoll and bone marrow cell fluid) and then centrifuge (20°C, 900g, 30 minutes). After centrifugation, first remove the uppermost supernatant (be careful not to take away the mononuclear cell layer under the supernatant), take out the mononuclear cell layer and add it to a blank test tube, then add 1:1 sterile PBS solution and ...

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Abstract

The invention relates to the biological cell technology field, particularly a method for separating and culturing miniature swine bone marrow-derived endothelial progenitor cells. The conventional EPCs culture method has the disadvantages of complexity and greatly reduced cell harvest rate by using immunomagnetic bead separation. The method in the invention comprises the steps of directly using myelomonocyte to conduct culture in vitro to obtain EPCs, adding growth factors in culture solution, planting in a definite density in a culture flask/ vessel or a glass sheet enveloped by fibronectin, and conducing appropriate culture to obtain EPC. The method with high repeatability simplifies the steps of the conventional culture method, effectively avoids unnecessary cell loss caused by the conventional separation and culture method, increases the pick-up rate of EPCs, and saves a large amount of funds. The method of the invention also prepares for clinical application in future, and can reduce the usage of bone marrow and provide a technology platform for curing traumatic or ischemic diseases by autotransfusion.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for isolating and culturing porcine bone marrow endothelial progenitor cells. Background technique [0002] With the rapid development of stem cell technology and theory since the end of the 20th century, the research and clinical treatment of trauma-induced ischemic diseases and trauma repair mechanisms are facing new opportunities. Studies have shown that after body injury, stem / progenitor cells with multidirectional differentiation potential in the blood circulation can mobilize, proliferate, and migrate into injured organs under the action of cytokines / growth factors produced by inflammatory cells, and differentiate into corresponding tissue cells for further development. Injury repair, on the other hand, stem cells migrating to the injury site can also respond physiologically to different local stimuli (including hypoxia or ischemia), and sequentially release vasoactive...

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Application Information

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IPC IPC(8): C12N5/06C12N5/071
Inventor 方国恩周虹罗天航吴建国卢正茂毛岸荣
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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