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Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells

A platelet-rich plasma and platelet technology, applied in animal cells, vertebrate cells, bone/connective tissue cells, etc., can solve the problems of allogeneic contamination immune response, blood source tension, difficult to obtain, etc., to achieve rich source of raw materials, reduce The effect of wasted, fully utilized

Active Publication Date: 2012-03-07
SICHUAN NEO LIFE STEM CELL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, cell products produced using animal serum often have many problems: large differences between FBS batches reduce the repeatability and stability of cell culture; there are risks of heterogeneous contamination and immune reactions, such as Heiskanen et al. and Sundin et al. After transplantation of cultured cells with FBS, antibodies against FBS proteins have been found (Heiskanen A et al., Stem Cells. 2007; 25; 197-202; Sundin M et al., Haematologica. 2007; 92:1208-15)
Currently, platelet-rich plasma obtained by these methods is difficult to separate platelets and leukocytes, and it is difficult to realize the separate utilization of platelets and leukocytes, and the high concentration of leukocytes mixed in platelet-rich plasma may have side effects on tissue repair, etc.
On the one hand, it is difficult to obtain a large amount of peripheral blood due to the shortage of blood sources, and the collection of peripheral blood will also bring pain to the donor. polluted

Method used

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  • Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells
  • Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells
  • Preparation of human platelet-rich plasma and application of same in isolation and culture of human mesenchymal stem cells

Examples

Experimental program
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Effect test

Embodiment 1

[0021] In a conventional manner, the collected human adult blood sample was added with 6% hydroxyethyl starch to a content of ~1% (w / v) in the system and mixed thoroughly. Centrifuge at 70g for 6 minutes to remove the lower red blood cells. The remaining part was centrifuged at 420g for 11 minutes to separate the "platelet + white blood cell" mixture layer, which can be used for the storage of hematopoietic stem cells. The remaining platelet-poor plasma layer was centrifuged at 1000g for 10 minutes, the platelets were aggregated to the bottom of the tube, and most of the supernatant was sucked off to increase the concentration of platelets. After resuspension, the concentration of platelets was measured using a Backman Coulter Ac.T Diff2 hematology analyzer , and then take them out after freezing at -20°C, and immediately place them in a water bath at 37°C, shake them continuously to mix them and melt them quickly. After repeated freezing-thawing twice, cell debris was re...

Embodiment 2

[0023]The corresponding cbPRP was obtained by using the umbilical cord blood of a healthy puerpera with full-term normal delivery or caesarean section as the raw material and operating in the same manner as in Example 1.

Embodiment 3

[0025] The MSCs culture experiment was carried out with the cbPRP obtained in Example 2.

[0026] Culture of umbilical cord MSCs:

[0027] The umbilical cords were collected from healthy puerperas with full-term vaginal delivery or cesarean section. After collection, they were stored in PBS solution containing double antibodies at 4°C and processed within 48 hours. Umbilical cord to remove blood vessels, cut to 1mm 3 about. Every 1.5cm umbilical cord tissue piece was inoculated into a 90mm cell culture dish, and was mixed with DMEM-LG / F12 (1 : 1, v / v) Add 10(v)% FBS and add 5(v)% cbPRP (platelet concentration 1000×10 9 cells / L) at 37°C with 5% CO 2 cultured in a saturated humidity incubator. According to the growth of the cells, change the medium every 3-5 days. The result is as figure 1 As shown, cells crawl out from around the tissue block. When the cells reached 70% confluence, they were digested with 0.05 (w / v)% trypsin / EDTA, and then pressed at 10,000 cells / cm ...

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Abstract

The invention provides preparation of human platelet-rich plasma and application of the same in isolation and culture of human mesenchymal stem cells. According to the invention, erythrocytes and a mixture of leucocytes and platelets are separated from human whole blood through centrifugation, then centrifugation with a centrifugal force greater than a centrifugal force used in above-mentioned centrifugation is carried out on residual blood plasma, obtained sediment is collected and is subjected to freezing-thawing treatment respectively at a temperature no more than -20 DEG C and a temperature no less than normal temperature, and the sediment is removed through separation so as to obtain human platelet-rich plasma. The invention has the following beneficial effects: raw materials used inthe method are widely available; the blood resource is fully utilized; the obtained product of platelets is purer, contains more abundant nutritional components including growth factors and has a more ideal effect when used for isolation and culture of human mesenchymal stem cells.

Description

technical field [0001] The present invention relates to the preparation of human platelet-rich plasma in the field of biomedicine and its application in the separation and cultivation of human mesenchymal stem cells, especially the waste plasma that has been separated from the mixture of red blood cells and white blood cells and platelets by conventional methods. Human umbilical cord blood / plasma that has been fully utilized and discarded is used as a raw material to further prepare, including but not limited to, human platelet-rich plasma for the isolation and culture of human mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are multipotent adult stem cells with self-renewal and multiple differentiation potentials. MSCs were first isolated from the bone marrow and grew adherently in vitro in a fusiform shape. In addition to the osteogenic, adipogenic, and chondrogenic differentiation of mesoderm, MSCs have been shown to differentiate acr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775
Inventor 赖真阳陈强叶飞钟立武冉丹王锐锋
Owner SICHUAN NEO LIFE STEM CELL BIOTECH
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