Method for enriching and purifying blood platelets
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A platelet enrichment technology, applied in the field of biomedicine, can solve the problems of low PRP concentration or purity, white blood cell and red blood cell contamination, plasma residual quality, etc., to avoid potential adverse reactions, reduce pollution, and improve the effect of enrichment purity.
Active Publication Date: 2015-01-28
杭州三江上御生物科技有限公司
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Through the application of the present invention, the enrichment purity of platelets can be effectively improved, the contamination of wh
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Embodiment 1
[0036] (1) Collect human whole blood into vacuum blood collection tubes containing sodium citrate, the mass concentration of sodium citrate being 3.2% to 3.8%. The volume ratio of anticoagulant to whole blood is 1:9. 3 mL whole blood was collected.
[0037] (2) Fully mix the blood and anticoagulant to avoid coagulation.
[0038] (3) Distribute 3 mL of blood containing anticoagulants into centrifuge tubes and centrifuge at 1000 rpm for 10 minutes. After centrifugation, the blood should be divided into three layers: the bottom layer is red blood cells, the top layer is platelet-poor layer, and the middle layer is platelet-rich layer. Contains platelets and white blood cells.
[0039] (4) Use a pipette to extract the uppermost layer and the middle layer and transfer them to a new centrifuge tube without intentionally avoiding the extraction of red blood cells to obtain enrichment A.
[0040] (5) Add 1 mL of normal saline to the original centrifuge tube containing the remaining...
Embodiment 2
[0052] Example 2: Preparation of concentrated platelets
[0053] The operation steps are the same as in Example 1, except that in step (6), the erythrocyte lysate is a lymphocyte separation liquid (P8610-200, Shanghai Suo Laibao Biotechnology Co., Ltd.), and the lymphocyte separation liquid and enrichment C The volume ratio is 1:1.
[0054] The prepared human, rat and mouse platelets have a purity of more than 99%.
Embodiment 3
[0055] Example 3: Preparation of concentrated platelets
[0056] The operation steps are the same as in Example 1, except that in step (1), heparin (15 IU / mL) is used as the anticoagulant, and the volume ratio of anticoagulant to whole blood is 1:9.
[0057] The prepared human, rat and mouse platelets have a purity of more than 99%.
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Abstract
The invention discloses a method for enriching and purifying blood platelets. The method comprises the following steps: adding an anticoagulant into whole blood and centrifuging at the speed of 1000rpm for 10 minutes; absorbing one part above a red blood cell layer and adding normal saline into the residual red blood cell layer; centrifuging at the speed of 1000rpm for 10 minutes; taking one part above the red blood cell layer and mixing to obtain an enrichment C; adding a red blood cell lysis solution into the enrichment C; uniformly mixing and standing for half an hour; centrifuging at the speed of 1500rpm for 20 minutes; collecting liquid supernatant A and a middle white flocculent layer A; centrifuging the liquid supernatant A at the speed of 1500rpm for 10 minutes; collecting a bottom white layer B and mixing the bottom white layer B with the middle white flocculent layer A; adding the normal saline and centrifuging at the speed of 1500rpm for 10 minutes; and collecting a middle white layer C, namely the concentrated and enriched blood platelets. According to the method for enriching and purifying the blood platelets, a purification method for removing blood serum and breaking red blood cells on the basis of centrifuging and enriching is added, so that the red blood cells can be completely removed; and the enriching purity of the red blood cells is effectively improved and the pollution of white blood cells and the red blood cells is alleviated.
Description
Technical field: [0001] The invention belongs to the technical field of biomedicine and relates to a centrifugal preparation method suitable for high-purity platelets of different species including humans and experimental animals. Background technique: [0002] Platelets are anucleated cells in mammalian blood, which are smaller than red blood cells and white blood cells. In addition to providing aggregation during hemostasis, platelets can also release a large number of cell growth factors through the degranulation of intracellular α granules, including platelet-derived Growth factor (PDGF), transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), etc., can induce The division and proliferation of bone cells and the synthesis of collagen promote the repair and reconstruction of bone tissue (Marx RE, Implant Dent, 2001, 10(4):225-228). Among them, PDGF can promote the chemotaxis an...
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