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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.

Pending Publication Date: 2017-12-08
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is a lack of effective means for platelet cytokine extraction in the biomedical field, and the only published PL (platelet lysate) preparation method has many shortcomings: ① Insufficient platelet lysis, low yield of cytokines; ② Lack of concentration and enrichment steps, Low concentration of cytokines; ③ Lack of quality detection methods

Method used

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  • Enrichment method of platelet cell factor concentrates
  • Enrichment method of platelet cell factor concentrates
  • Enrichment method of platelet cell factor concentrates

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses an enrichment method of platelet cell factor concentrates. The method comprises the steps as follows: whole blood is taken, platelets are collected, a cell suspension is prepared with normal saline, subjected to ultrasonic crushing multiple times under the ultrasonic power of 100-200 W and crushed for 1-60 s each time, a crushed mixed solution is filtered, filtrate is taken, and the platelet cell factor concentrates are obtained. The obtained platelet ultrasonic lysates (cell factor concentrates) comprise TGF-beta, PDGF, IGFs, FGFs, EGF, VEGF and the like, have multiple bioactivities, can increase cartilage cell activity of organisms and promote stem cells to proliferate and differentiate and contributes to regeneration and repair of cartilages and bone tissue. The composition can be prepared in the form of injection, powder and the like and has excellent application prospects in the medical bone tissue engineering field.

Description

(1) Technical field [0001] The invention relates to a method for enriching platelet cytokine concentrate which can be used for repairing cartilage and bone tissue damage. (2) Background technology [0002] Platelets are anucleated cells in mammalian blood, which can 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 (transforming growth factor-β, TGF-β), insulin-like growth factor-1 (insulin-like growth factor, IGF-1), fibroblast growth factor (fibroblast growth factor, FGF), epidermal growth factor (epidermal growth factor, EGF), vascular endothelial growth factor (vascular endothelial growth factor, VEGF), etc. (Sanchez et al., Int J Oral Maxillofac Implants, 2003, 18 (1): 93-103). Cell growth factors released by platelets have strong biological activity, can induce the division and proliferation of bone cells and synthesis of collagen, and promo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/52C07K14/485C07K14/49C07K14/495C07K14/50C07K14/515C07K14/65C07K1/34C12N1/06
CPCC07K14/485C07K14/49C07K14/495C07K14/50C07K14/515C07K14/52C07K14/65C12N1/06
Inventor 单乐天童培建杜文喜尹利明周莉
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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