A culture method for massively expanding stem cells in vitro from peripheral blood
A culture method and technology of stem cells, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., to achieve the effect of enriching developmental theory
Active Publication Date: 2018-07-17
INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE
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In addition, it has not been clear how endothelial cells as a microenvironment complete the expansion of stem cells? Why are there "hematopoietic endothelial cells"? Why are hematopoietic stem cells derived from endothelial cells? These questions are still a mystery to us. The main reason is the lack of technology to continuously observe the expansion process of hematopoietic stem cells. There have been articles emphasizing the application of real-time video methods. Experiments with this method have demonstrated hematopoietic stem cells adhered to endothelial cells. Capable of expanding non-adherent stem cells grown in suspension
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[0021] Below in conjunction with accompanying drawing and specific embodiment the present invention is described in further detail:
[0022] 1: First prepare the endothelial progenitor cells as the microenvironment
[0023] 1. The commonly used methods for culturing late EPCs (endothelial progenitor cells) are as follows:
[0024] The MNC layer was isolated from peripheral blood by the known Ficoll density gradient method, and then a certain density of mononuclear cells was seeded into a culture dish lined with fibronectin or collagen, and then cultured with EGM2 commercial medium, every other day Replace the medium once, and after 2-3 weeks of culture, there will be late endothelial progenitor cells that grow in the form of colonies and have a cobblestone-like cell shape.
[0025] 2. Currently commonly used umbilical vein endothelial cells isolated from umbilical cord: the method is omitted
[0026] Two: Prepare expanded hematopoietic stem cell seed cells from peripheral bl...
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The invention discloses a cultivation method for mass in-vitro stem cell expansion from peripheral blood. According to the cultivation method, two kinds of endothelial progenitor cells existing outside human bone marrow are taken as microenvironment cells for in-vitro stem cell expansion, and stem cells are expanded in the peripheral blood substantially. The cultivation method has the advantages that the endothelial progenitor cells existing outside the human bone marrow are taken as microenvironments to achieve mass in-vitro peripheral blood stem cell expansion, a real-time monitoring videos are directly applied for visually displaying that the stem cells can be expanded through self-replication when the endothelial progenitor cells serve as the microenvironments, the quantity is increased by 100 times, and the expanded stem cells have asymmetric division capability of the stem cells and the capability of dividing to next level progenitor cells; in other words, the stem cells are increased by 100 times in terms of quantity as compared with cells without microenvironments after the endothelial progenitor cells are taken as the microenvironments, and the expanded stem cells are proved to have two key characteristics of the stem cells, namely the self-replication capability and the capability of dividing to the next level progenitor cells.
Description
technical field [0001] The present invention relates to a kind of stem cell biotechnology, in particular to using two kinds of endothelial progenitor cells that exist outside human bone marrow and can provide a microenvironment for stem cell expansion to co-cultivate with seed stem cells, thereby completing the large-scale expansion of stem cells from peripheral blood in vitro training method. Background technique [0002] Hematopoietic stem cells are a type of stem cells with self-renewal and differentiation potential in hematopoietic tissues. The survival of hematopoietic stem cells is inseparable from the bone marrow microenvironment known as "niche". When co-transplanted with bone marrow stromal cells that can be used as a microenvironment, the transplantation efficiency can be significantly increased, indicating that the bone marrow microenvironment has an important impact on hematopoietic stem cells. These microenvironments include initially discovered osteoblasts in...
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IPC IPC(8): C12N5/0789
Inventor 吴立华
Owner INST OF HEMATOLOGY & BLOOD DISEASES HOSPITAL CHINESE ACADEMY OF MEDICAL SCI & PEKING UNION MEDICAL COLLEGE