Preparation method and application of platelet lysate rich in extracellular vesicles

Abstract

The present invention provides a method for preparing a platelet lysate rich in extracellular vesicles and its application. The platelet lysate prepared by intermittent ultrasonic lysis does not require the exogenous addition of biochemical components such as thrombin, collagen, or calcium ions, making it simpler and safer. By setting reasonable ultrasonic process parameters, the prepared platelet lysate is not only rich in various factors (such as bFGF, VEGF, PDGF-BB, and IL-6, IL-15, IL-17, and INF-γ) but also has a high content of bioactive proteins. Furthermore, the extracellular vesicles are not only high in content but also small in diameter, with an average diameter of as low as 79.9 nm. The proportion of exosomes is relatively large, and the extracellular vesicles have high expression of the transmembrane protein CD9 and low expression of CD81.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and medical technology, and particularly relates to a preparation method of a platelet lysate rich in extracellular vesicles and an application thereof. Background Art

[0002] Platelets contain three main types of granules: α-granules, dense granules, and lytic granules. α-granules are the largest and most abundant secretory granules within platelets and contain growth factors important for angiogenesis and tissue formation. Upon activation, platelets release various platelet-derived cytokines, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). These growth factors stimulate the proliferation of fibroblasts, smooth muscle cells, and osteoblasts, and attract a variety of cellular components involved in the repair of damaged tissues. Platelet lysate (PL) is a platelet-derived product obtained by artificially releasing the abundant active factors within platelets. Platelet lysate is currently prepared primarily through repeated freeze-thaw cycles, followed by the addition of thrombin or calcium ions. Thrombin and calcium ions are physiological platelet activators and are effective activators. However, thrombin is primarily derived from bovine sources, and due to safety concerns, researchers are constantly searching for better activation methods. Repeated freeze-thaw cycles disrupt cell membranes, releasing active products but also causing varying degrees of damage to these substances, depending on the number of freeze-thaw cycles. Extracellular vesicles (EVs) are membrane vesicles released from the plasma membrane (microcapsules or microparticles) or intracellular bodies (exosomes). They are produced upon platelet activation and play important roles in inflammation, cancer progression, innate defense, tissue repair, thrombosis, and neurobiology. Platelet-derived EVs (PEVs) transport diverse cargoes (such as RNA, lipids, and proteins) that can be transferred to recipient cells. The therapeutic potential of PEVs is being explored, as they can promote coagulation and angiogenesis in various animal models of bleeding and trauma. Furthermore, PEVs have been found to be beneficial in the treatment of chronic injuries and trauma. Current processes for preparing platelet lysates rarely consider simultaneously increasing the content of extracellular vesicles (EVs). Summary of the Invention

[0003] Therefore, based on the above background, the present invention provides a method for preparing a platelet lysate rich in extracellular vesicles and its application. The platelet lysate prepared by the present invention has a high content of extracellular vesicles, which can provide direction for the further development and application of platelet lysate.

[0004] The technical solution of the present invention is:

[0005] A method for preparing a platelet lysate rich in extracellular vesicles comprises the following steps:

[0006] S1: Collect whole blood, add it to anticoagulant tube, remove red blood cells, and prepare platelet concentrate;

[0007] S2: Washing and resuspending platelets in HEPES buffer containing an ACD anticoagulant, wherein the mass concentration of the ACD is 0.3%;

[0008] S3: intermittently ultrasonically lyse the platelet resuspension in step S2 under an ice-water bath;

[0009] S4: The lysate from step S3 is centrifuged at 4° C., and the supernatant is collected as the platelet lysate rich in extracellular vesicles.

[0010] Furthermore, the conditions for the intermittent ultrasonic lysis in step S3 are: ultrasonic treatment for 5 seconds, rest for 5 seconds, 30 cycles, and ultrasonic power of 100W.

[0011] Furthermore, the operation of removing red blood cells and preparing concentrated platelets in step S1 is as follows:

[0012] 1) Centrifuge the plasma at room temperature and collect the plasma and buffy coat;

[0013] 2) Centrifuge the plasma and buffy coat from step 1) at room temperature and collect the precipitate.

[0014] Furthermore, the concentration of platelets in the resuspension prepared in step S2 is 1000×10 9 / L.

[0015] Based on the same inventive concept, the present invention provides a platelet lysate rich in extracellular vesicles prepared by the above-mentioned preparation method.

[0016] Based on the same inventive concept, the present invention applies the platelet lysate rich in extracellular vesicles prepared by the above preparation method to cell culture.

[0017] Furthermore, the cells include human umbilical vein endothelial cells.

[0018] Based on the same inventive concept, the present invention applies the platelet lysate rich in extracellular vesicles prepared by the above preparation method to prepare extracellular vesicles.

[0019] Furthermore, the preparation of the extracellular vesicles is performed as follows:

[0020] ① Take the platelet lysate rich in extracellular vesicles, centrifuge it, and take the supernatant;

[0021] ② Centrifuge the supernatant obtained in step 1) again and take the precipitate.

[0022] Based on the same inventive concept, the present invention provides extracellular vesicles prepared using the platelet lysate super-enriched in extracellular vesicles prepared above.

[0023] Furthermore, the extracellular vesicles can be used to prepare preparations that can promote wound healing.

[0024] The above technical solution has the following beneficial effects:

[0025] 1) In the process of preparing platelet lysate by intermittent ultrasonic lysis, no exogenous addition of biochemical components such as thrombin, collagen or Ca ions is required, which is simpler and safer.

[0026] 2) By setting reasonable ultrasonic process parameters, the platelet lysate prepared in the present invention is not only rich in various factors (such as bFGF, VEGF, PDGF-BB, etc., as well as IL-6, IL-15, IL-17 and INF-γ) but also has a high content of bioactive proteins.

[0027] The extracellular vesicles in the platelet lysate prepared by the present invention not only have a high content but also a small diameter. The average diameter of the extracellular vesicles can be as low as 79.9 nm. The proportion of exosomes is relatively large, and the transmembrane protein CD9 of the extracellular vesicles is highly expressed, while CD81 is lowly expressed.

[0028] 3) The platelet lysate prepared by the present invention can be concentrated and purified to prepare extracellular vesicle products, which can provide new ideas and directions for the preparation of platelet extracellular vesicle-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a bar graph showing the contents of bFGF, VEGF, and PDGF-BB in platelet lysates prepared using three different methods according to the embodiments of the present invention.

[0030] Figure 2 This is a bar graph showing the contents of IL-6, IL-15, IL-17, and INF-γ in platelet lysates prepared using three different methods according to the embodiments of the present invention.

[0031] Figure 3 The live cell densities at 24 h and 48 h after cell culture using platelet lysates prepared by three different methods in the examples of the present invention are shown.

[0032] Figure 4 Diameter distribution diagram of extracellular vesicles in platelet lysates prepared by three different methods in the examples of the present invention.

[0033] Figure 5This is a graph showing the relative content of protein factors in the extracellular vesicles prepared according to an embodiment of the present invention.

[0034] Figure 6 This is an expression diagram of the transmembrane protein content in the lysate prepared in the examples of the present invention. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The equipment used in the following examples: JY98-IID ultrasonic cell disruptor; Mindray fully automatic blood cell analyzer, BC-20.

[0039] Example 1: A method for preparing a platelet lysate rich in extracellular vesicles, comprising the following steps:

[0040] S1: Collect whole blood from a healthy individual without any disease into an anticoagulant tube, remove red blood cells, and prepare concentrated platelets;

[0041] The specific operations of this step are:

[0042] 1) Centrifuge the plasma in the anticoagulant tube at room temperature: 300g*20min, and collect the plasma and buffy coat;

[0043] 2) Centrifuge the plasma and buffy coat from step 1) at room temperature at 900 g for 15 minutes, and collect the precipitate.

[0044] S2: Wash and resuspend platelets in HEPES buffer containing ACD anticoagulant. The platelet concentration in the resuspension prepared in this step is 1000×10 9 / L; the mass concentration of the ACD is about 0.3%.

[0045] S3: The platelet resuspension in step S2 was subjected to intermittent ultrasonic lysis in an ice-water bath; the conditions for the intermittent ultrasonic lysis in this step were: ultrasonic treatment for 5 seconds, rest for 5 seconds, 30 cycles, and ultrasonic power of 100W.

[0046] S4: Centrifuge the lysate from step S3 at 4°C at 400g for 20 min, and collect the supernatant, which is the platelet lysate rich in extracellular vesicles.

[0047] Example 2: In this example, the preparation method of Example 1, the repeated freeze-thaw method, and the external thrombin method are used to prepare platelet lysate, wherein the platelet lysate prepared in Example 1 is referred to as SPL, the platelet lysate prepared by the repeated freeze-thaw method is referred to as FTPL, and the platelet lysate prepared by the external thrombin method is referred to as TPL.

[0048] The operation of the repeated freeze-thaw method is as follows:

[0049] Collect blood from healthy individuals without any disease into anticoagulant tubes, remove red blood cells and prepare concentrated platelets;

[0050] Platelets were washed with HEPES buffer containing ACD anticoagulant, placed at -80°C for 12 hours and incubated in a water bath at 37°C for 30 minutes, repeated three times, and centrifuged at 4000g for 20 minutes at 4°C. The supernatant was collected as the platelet lysate prepared by repeated freeze-thaw cycles; the mass concentration of ACD was approximately 0.3%;

[0051] The operation of the thrombin addition method is as follows:

[0052] Collect blood from healthy individuals without any disease into anticoagulant tubes, remove red blood cells and prepare concentrated platelets;

[0053] Platelets were washed with HEPES buffer containing ACD anticoagulant, and a mixture of thrombin and calcium ions was added (final concentration: 100 u / ml thrombin and 22.8 mM calcium ions). The platelets were incubated in a 37°C water bath for 30 minutes, centrifuged at 4000 g for 20 minutes at 4°C, and the supernatant was collected as the platelet activation solution prepared with thrombin. The mass concentration of ACD was approximately 0.3%.

[0054] The whole blood for the platelet lysates prepared by the three methods in this example was derived from the same human body.

[0055] Next, the Luminex detection technology was used to detect the content of specific active factors in the lysate prepared above. Figure 1 and Figure 2 As shown, Figure 1 Clearly, SPL contains higher levels of bFGF, as well as relatively high levels of VEGF and PDGF-BB. Basic fibroblast growth factor (bFGF) promotes the proliferation and division of nearly all cells associated with wounds, including fibroblasts, vascular endothelial cells, smooth muscle cells, epithelial cells, chondrocytes, and neurons. This suggests that SPL may have a more significant effect on wound healing.

[0056] Figure 2As shown: SPL contains high levels of IL-6, IL-15, IL-17 and INF-γ, which may indicate that SPL is more effective in improving the body's resistance to infection.

[0057] Example 3: Human umbilical vein endothelial cells (HUVEC) were treated with the lysates prepared by the three preparation methods in Example 2, specifically:

[0058] HUVECs were seeded into 96-well plates, with approximately 3,000 cells per well. When the cell density reached approximately 70%, DMEM culture medium containing the platelet lysate prepared above was added (the added mass concentration of the platelet lysate was 10%). After culturing for 24 and 48 hours, cell viability was detected according to the instructions of the CCK8 kit. The results are shown in the table. Figure 3 .Depend on Figure 3 It is obvious that the cells in the SPL group showed a stronger proliferation-promoting trend after 24 hours of HUVEC treatment, and the cell proliferation-promoting effect of the SPL group was more significant after 48 hours of HUVEC treatment.

[0059] Example 4: This example further analyzes extracellular vesicles.

[0060] The three platelet lysates prepared by the three preparation methods in Example 2 were centrifuged at 10,000 x g, 4° C. for 45 min, and the supernatants were collected. The supernatants were then centrifuged at 100,000 x g, 4° C. for 70 min, and the precipitates were collected. The precipitates were then resuspended in PBS to obtain extracellular vesicle suspensions.

[0061] The diameter and distribution of extracellular vesicles were characterized by nanoparticle size analyzer (NTA), the protein concentration of the extracellular vesicles was measured by BCA method, and the specific markers of the extracellular vesicles were characterized by fluorescent labeling of CD9, CD63, and CD81. Figures 4 to 6 shown.

[0062] Depend on Figure 4 The results showed that the average diameter of extracellular vesicles in the SPL group was the smallest, which was 79.9 nm, while the average diameters of extracellular vesicles in the TPL group and FTPL group were 82.7 nm and 87.4 nm, respectively. The content of extracellular vesicles in the SPL group was relatively richer, with a concentration of up to 3.81×10 9 / ml.

[0063] Considering the important role of protein factors in extracellular vesicles in their biology, the protein / extracellular vesicle content of the three groups of extracellular vesicles was compared. Figure 5It can be seen that the protein / extracellular vesicle ratio of the SPL group was 0.37, while that of the TPL and FTPL groups was 0.23 and 0.11, respectively. This indicates that the extracellular vesicles of the SPL group contain more proteins, that is, there may be more protein active factors, which will have greater potential for the treatment of diseases.

[0064] Depend on Figure 6 It can be seen that after fluorescent labeling of CD9, CD63, and CD81, the SPL group highly expressed CD9 (about 16.3%) and lowly expressed CD81 (about 6.6%), which was similar to the FTPL group, CD9 (about 17.7%) and CD81 (about 6.8%), while the TPL group was the opposite, CD9 (about 5.7%) and CD81 (about 20.6%). This may indicate that the platelet vesicles produced by platelet lysis and the extracellular vesicles produced by thrombin activation have different functional subpopulation classifications.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing a platelet lysate rich in extracellular vesicles, characterized in that: S1: Collect whole blood, add it to anticoagulant tube, remove red blood cells, and prepare platelet concentrate; S2: Washing and resuspending platelets in HEPES buffer containing an ACD anticoagulant, wherein the mass concentration of the ACD is 0.3%; S3: intermittently ultrasonically lyse the platelet resuspension in step S2 under an ice-water bath; The conditions for intermittent ultrasonic lysis were as follows: ultrasonic treatment for 5 s, rest for 5 s, 30 cycles, and ultrasonic power of 100 W. S4: The lysate from step S3 is centrifuged at 4° C., and the supernatant is collected as the platelet lysate rich in extracellular vesicles.

2. The method for preparing a platelet lysate rich in extracellular vesicles according to claim 1, characterized in that: The steps for removing red blood cells and preparing concentrated platelets in step S1 are as follows: 1) Centrifuge the plasma at room temperature and collect the plasma and buffy coat; 2) Centrifuge the plasma and buffy coat from step 1) at room temperature and collect the precipitate.

3. The method for preparing a platelet lysate rich in extracellular vesicles according to claim 2, characterized in that: The platelet concentration in the resuspension prepared in step S2 is 1000×10 9 / L.

4. A platelet lysate rich in extracellular vesicles prepared by the preparation method according to any one of claims 1 to 3.

5. Use of the platelet lysate enriched in extracellular vesicles according to claim 4 in cell culture for non-disease treatment and diagnosis purposes, characterized in that: The cells include human umbilical vein endothelial cells.

6. Use of the platelet lysate rich in extracellular vesicles according to claim 4 in preparing extracellular vesicles.

7. The use according to claim 6, characterized in that The preparation of the extracellular vesicles is as follows: ① Take the platelet lysate rich in extracellular vesicles, centrifuge it, and take the supernatant; ② Centrifuge the supernatant obtained in step ① again and take the precipitate.

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