Enrichment method of platelet cell factor concentrates
A cytokine and platelet technology, applied in platelet-derived growth factor, fibroblast growth factor, cytokine/lymphokine/interferon, etc., can solve the lack of platelet cytokines, lack of concentration and enrichment steps, and insufficient platelet lysis and other problems, to increase the vitality of chondrocytes, help regeneration and repair, and avoid the interference of miscellaneous cells and plasma.
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Embodiment 1
[0033] (1) Collect 9ml of human whole blood into a container containing 1ml of anticoagulant (3.2% sodium citrate aqueous solution), the volume ratio of anticoagulant to whole blood is 1:9, mix well to avoid coagulation .
[0034] (2) Distribute the blood mixed in step (1) into centrifuge tube a and centrifuge at 1000rpm for 10 minutes. After centrifugation, the blood should be divided into three layers: the lower layer is red blood cells, the upper layer is platelet-poor layer, and the middle layer is platelet-rich layer (contains platelets and white blood cells).
[0035] (3) Use a pipette to extract the upper layer and the middle layer of the centrifuge tube a in step (2) and transfer them to a new centrifuge tube b, without deliberately avoiding the extraction of red blood cells.
[0036] (4) Add an appropriate amount of normal saline to the centrifuge tube a of the upper layer and the middle layer taken out in step (3), centrifuge at a speed of 1000rpm for 10 minutes, an...
Embodiment 2
[0049] The operating steps are the same as in Example 1, except that the rat sample is used. In step (6), the concentration of the cell suspension is 10 6 / ml, the size of the load probe of the ultrasonic breaker is 4mm, the ultrasonic power is 150W, the time of each crushing is 30s, and the interval is 10s, and the crushing is 3 times.
[0050] The contents of TGF-β, PDGF, IGF-1 and VEGF in the prepared cytokine concentrate were higher than 100pg / ml, 50pg / ml, 300pg / ml and 70pg / ml respectively, as shown in Table 3.
[0051] Same as step (7) of Example 1, the platelet cytokine concentrate collected in Example 2 was set to high concentration (stock solution), medium concentration (1 / 10 stock solution), low concentration (1 / 50 stock solution), and added to culture In the cell plate with mesenchymal stem cells, culture them at 37°C for 24h, 48h, and 72h respectively. At each time point, the cell viability (MTT method) and ELISA detection were performed, and compared with the PL me...
Embodiment 3
[0055] The operating steps are the same as in Example 1, except that the mouse sample is used. In step (6), the concentration of the cell suspension is 10 8 / ml, the size of the load probe of the ultrasonic breaker is 6mm, the ultrasonic power is 100W, the time of each crushing is 10s, and the interval is 10s, and the crushing time is 6 times.
[0056] The contents of TGF-β, PDGF, IGF-1 and VEGF in the prepared cytokine concentrate are respectively higher than 100pg / ml, 50pg / ml, 300pg / ml and 70pg / ml.
[0057] Same as step (7) of Example 1, the platelet cytokine concentrate collected in Example 3 was set to high concentration (stock solution), medium concentration (1 / 10 stock solution), low concentration (1 / 50 stock solution), and added to culture In the cell plate with mesenchymal stem cells, culture them at 37°C for 24h, 48h, and 72h respectively. At each time point, the cell viability (MTT method) and ELISA detection were performed, and compared with the PL method. The resul...
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