Platelet lysate, preparation method therefor, and use thereof

Abstract

A platelet lysate, a preparation method therefor, and the use thereof. The preparation method comprises: centrifuging anticoagulated whole blood, and collecting a platelet-rich plasma mixture; centrifuging the platelet-rich plasma mixture, collecting the plasma supernatant, and performing heat-inactivation and centrifuging to obtain inactivated plasma; collecting the platelet particle precipitate obtained by centrifugation, then washing and resuspending same with the inactivated plasma to prepare a concentrated platelet particle suspension; subjecting the concentrated platelet particle suspension to an ultrasonic treatment, centrifuging same, and collecting a lysate rich in concentrated platelets; adding calcium gluconate to the lysate rich in concentrated platelets, heating the resulting mixture to form a clot, mechanically squeezing and wringing the clot, centrifuging the squeezed-out liquid, and collecting the supernatant; and filtering and sterilizing the supernatant to obtain the platelet lysate.

Description

Platelet lysate, preparation method and application thereof Technical Field

[0001] The present application relates to the technical field of cell biotechnology, and more specifically, to a platelet lysate and a preparation method and application thereof. Background Art

[0002] Platelets contain various proteins (such as fibrin) and growth factors (including transforming growth factor-β (TGF-β), insulin-like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and other growth factors). Platelet lysate is a derivative of platelet-rich plasma (PRP). It is a cell culture medium made by further lysing concentrated platelets to release a mixture of cytokines and growth factors. This not only removes residual cell structure and reduces immunogenicity, but also retains the various growth factors and bioactive proteins required for cell growth and proliferation. Platelet lysate, prepared by releasing various factors and biological proteins from human platelet lysate, has been identified as a suitable alternative to fetal bovine serum as a culture medium supplement, enabling efficient propagation of human cells in the absence of animal serum.

[0003] Umbilical cord mesenchymal stem cells (MSCs) generally refer to multipotent stem cells derived from the early embryonic layer of development, with a high degree of self-renewal and multidirectional differentiation potential. They can, under specific conditions, differentiate into chondrocytes, osteoblasts, muscle cells, adipocytes, and other cells. Among adult stem cells, MSCs are widely present in various tissues throughout the body, particularly bone marrow, adipose tissue, umbilical cord blood, and even peripheral blood. Due to their robust proliferation capacity, high multidirectional differentiation potential, low immunogenicity, and immunomodulatory properties, MSCs are considered ideal seed cells for tissue engineering, and have therefore attracted considerable attention due to their significant value in cell replacement therapy and tissue engineering.

[0004] At present, based on the mesenchymal stem cell culture media that have been on the market, they can be mainly divided into the following types. The first type is a culture medium with added bovine serum. Due to its extremely large number of unknown components and animal-derived components, it has a high human safety risk and large differences in batch performance. It is currently less used in cell storage and clinical research. The second type is a culture medium containing serum substitutes (platelet lysate). It has relatively few unknown components, but the batch performance differences need to be improved. The third type is a culture medium that is serum-free, free of xenologous proteins, or with extremely low content. It has a clear chemical composition, but is more expensive and has limited applicability.

[0005] Related technologies disclose methods for preparing platelet lysate. For example, a platelet lysate and a method for preparing the same include the following steps: preparing platelet-rich plasma with the desired platelet concentration; freezing the platelet-rich plasma in a -20°C refrigerator for 24 hours, removing it and thawing it in a 37°C water bath for 5 minutes; repeating the freeze-thaw process; centrifuging the platelet-rich plasma from the last thaw, and taking the supernatant, which is the platelet lysate. Another example is a method for preparing an endothelial cell culture fluid, which includes a platelet lysate. The method includes the following steps: collecting human platelets and placing them in a centrifuge bottle for centrifugation; removing the supernatant, adding physiological saline to wash the platelets, and resuspending them in physiological saline after centrifugation; placing the platelets in a -20°C refrigerator, freezing them overnight, and then placing them at room temperature until they are completely thawed; repeating the freeze-thaw process three or more times to obtain the platelet lysate.

[0006] However, in the above-mentioned method for preparing platelet lysate, a large amount of fibrin will appear in the platelet lysate after freezing and thawing, which will affect the effect of cell culture.

[0007] Summary of the Invention

[0008] The present application provides a platelet lysate and a preparation method and application thereof.

[0009] The preparation method of the present application can significantly increase the content of cytokines in platelet lysate and the yield of human umbilical cord mesenchymal stem cells crawling out of umbilical cord tissue, reduce or eliminate the release of fibrin during culture, avoid the appearance of other impurities, and has the advantages of clear composition, batch stability, and low microbial contamination.

[0010] The present application provides a preparation method for fully lysing concentrated platelets, thereby preparing an efficient platelet lysate. Only a basic culture medium (DMEM-F12) + a percentage of platelet lysate is required to increase the number of mesenchymal stem cells in the umbilical cord that are stimulated to crawl out of the umbilical cord.

[0011] The preparation method provided in this application can significantly increase the platelet lysis ratio. After the concentrated platelet lysate obtained is fully mixed with the basic culture medium and prepared, its stimulation effect is more obvious compared with the commercial culture medium on the market (which also contains platelet lysate).

[0012] In a first aspect, the present application provides a method for preparing a platelet lysate, which employs the following technical solution:

[0013] A method for preparing a platelet lysate, the method comprising the following steps:

[0014] (1) Centrifuging anticoagulated whole blood to collect a platelet-rich plasma mixture;

[0015] (2) centrifuging the platelet-rich plasma mixture, collecting the plasma supernatant, inactivating it by heating, and centrifuging it to obtain inactivated plasma;

[0016] (3) collecting the platelet granules precipitated by centrifugation in step (2), washing them, and resuspending them in the inactivated plasma to prepare a concentrated platelet granule suspension;

[0017] (4) ultrasonically treating the concentrated platelet particle suspension, centrifuging and collecting a lysate rich in concentrated platelets;

[0018] (5) adding calcium gluconate to the platelet-rich concentrated lysate, heating until a clot is formed, mechanically squeezing the clot to dry it, and centrifuging the squeezed liquid to collect the supernatant;

[0019] (6) Filter and sterilize the supernatant to obtain the platelet lysate.

[0020] In a specific embodiment, the anticoagulated whole blood can be obtained by mixing multiple portions of anticoagulated whole blood from healthy individuals of different origins according to blood type ratios.

[0021] In a specific embodiment, the anticoagulated whole blood comprises the following blood types in parts by weight: 3-6 parts of type O blood; 1-3 parts of type AB blood.

[0022] In a specific embodiment, the centrifugal speed in step (1) is 1500-5000 rpm, and the centrifugal time is 10-30 min.

[0023] In a specific embodiment, the platelet-rich plasma mixture can be obtained by mixing multiple sets of platelet-rich plasma mixtures.

[0024] In a specific embodiment, multiple groups of plasma supernatants can be pooled and heat inactivated.

[0025] In a specific embodiment, the number of the multiple groups of plasma supernatants is 2-6 groups.

[0026] In a specific embodiment, the heat inactivation method is heating in a water bath.

[0027] In a specific embodiment, the storage condition of the inactivated plasma is refrigerator storage.

[0028] In a specific embodiment, the inactivated plasma is stored in a -20°C refrigerator.

[0029] In a specific embodiment, the centrifugal speed before heat inactivation in step (2) is 1000-2000 rpm, and the centrifugal time is 10-30 min.

[0030] In a specific embodiment, the temperature of the heat inactivation is 30-60°C, and the time of the heat inactivation is 20-50 minutes.

[0031] In a specific embodiment, the centrifugal speed after heat inactivation in step (2) is 1000-2000 rpm, and the centrifugal time is 10-30 min.

[0032] In a specific embodiment, the platelet granule precipitate in step (3) is a collection of 2-5 groups of platelet granule precipitates.

[0033] In a specific embodiment, the platelet granule precipitate in step (3) is a collection of 5 groups of platelet granule precipitates.

[0034] In a specific embodiment, the inactivated plasma used in step (3) is 8-16 ml.

[0035] In a specific embodiment, the number of platelets in the concentrated platelet particle suspension in step (4) is at least (1-3)×10 11 Particles / L.

[0036] In a specific embodiment, the ultrasonic treatment time in step (4) is 20-50 min, and the ultrasonic treatment power is 10-30 kh.

[0037] In a specific embodiment, the centrifugal speed in step (4) is 1000-2000 rpm, and the centrifugal time is 10-30 min.

[0038] In a specific embodiment, the calcium gluconate in step (5) is a calcium gluconate solution.

[0039] In a specific embodiment, the concentration of the calcium gluconate solution is 0.8-1.4 g / ml.

[0040] In a specific embodiment, the volume ratio of the calcium gluconate solution to the platelet concentrate-rich lysate is 1:(1-5).

[0041] In a specific embodiment, the filtration sterilization uses a 0.2 μm filter.

[0042] In a specific embodiment, the heating temperature in step (5) is 35-40°C.

[0043] In a specific embodiment, the centrifugal speed in step (5) is 4000-6000 g, and the centrifugal time is 5-15 min.

[0044] In a specific embodiment, the platelet lysate is stored in a -20°C refrigerator.

[0045] Samples were taken to detect the number of platelets in the concentrated platelet particle suspension and platelet lysate, and the platelet lysis rate was calculated.

[0046] In a second aspect, the present application provides a platelet lysate prepared using the above-mentioned preparation method.

[0047] In a third aspect, the present application provides a use of the above-mentioned platelet lysate in the culture of primary human mesenchymal stem cells.

[0048] In a specific embodiment, the primary human mesenchymal stem cells are derived from umbilical cord tissue; and the volume percentage of the platelet lysate in the culture medium used for primary culture is 1-6%.

[0049] In summary, this application has the following beneficial effects:

[0050] This application uses anticoagulated whole blood of different blood types (O type and AB type) from healthy people, which is easy to obtain and economical. At the same time, taking multiple portions of anticoagulated whole blood from healthy people with different sources can avoid differences between individuals and batches, and the concentrated platelet lysate prepared has minimal differences between batches and better stability.

[0051] This application completely removes protein and residual cell structures through centrifugation and filtration, and reduces immunogenicity after heat inactivation. It does not require re-centrifugation and filtration to remove fibrin before use, and it does not require the addition of additional sodium heparin to prevent the platelet lysate from clotting during use.

[0052] After a series of concentration and lysis, various factors and other bioactive proteins in the platelets are completely released, so that the prepared platelet lysate has a high cytokine content, and the cells in the tissue are better stimulated during primary culture.

[0053] According to the examples described in this application, compared to other commercially available culture media containing platelet lysates, cells cultured using the platelet lysate prepared in this application exhibit significant differences in morphology and quantity, and exhibit superior growth. This suggests that the platelet lysate prepared in this application can better stimulate umbilical cord mesenchymal stem cells to migrate from tissue, increasing their yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG1 is the cell counting result of umbilical cord tissue cultured in two different culture media in Example 2.

[0055] Figure 2 shows the cell states of the umbilical cord tissue in Example 2 in two different culture media at different times (microscope 20×, wherein, 1 (containing 3% platelet lysate prepared in Example 1 + DMEM-F12 culture medium) and 4 (type B culture medium) are the cell morphologies of the cells on the 9th day after they crawled out of the umbilical cord tissue; 2 (containing 3% platelet lysate prepared in Example 1 + DMEM-F12 culture medium) and 5 (type B culture medium) are the cell morphologies of the cells on the 11th day after they crawled out of the umbilical cord tissue; 3 (containing 3% platelet lysate prepared in Example 1 + DMEM-F12 culture medium) and 6 (type B culture medium) are the cell morphologies of the cells on the 13th day after they crawled out of the umbilical cord tissue).

[0056] FIG3 shows the total number of living cells (microscope 10×) harvested on the 13th day after umbilical cord mesenchymal stem cells were stimulated to crawl out of the umbilical cord tissue by the platelet lysate prepared in Example 1 + DMEM-F12 medium and type B medium at different concentrations.

[0057] FIG4 shows the total number of living cells harvested on the third day after umbilical cord mesenchymal stem cells crawled out of the umbilical cord tissue by stimulating the platelet lysate prepared in Example 1 + DMEM-F12 medium and the platelet lysate prepared in the comparative example + DMEM-F12 medium (microscope 10×). DETAILED DESCRIPTION

[0058] Before describing the embodiments of the present application in detail, it should be understood that the terminology used herein is only for the purpose of describing particular embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the term belongs.

[0059] The present application provides a platelet lysate and a method for preparing the same. The method for preparing the platelet lysate specifically comprises the following steps:

[0060] (1) Anticoagulated whole blood is centrifuged to remove red blood cells and collect a platelet-rich plasma mixture.

[0061] (2) Centrifuging the platelet-rich plasma mixture, collecting the plasma supernatant, inactivating it by heating, and centrifuging it to obtain inactivated plasma.

[0062] (3) After collecting the supernatant from the centrifugation step (2), the remaining platelet particles are precipitated, washed with PBS, and then resuspended with the inactivated plasma to prepare a concentrated platelet particle suspension.

[0063] (4) Ultrasonic treatment of the concentrated platelet particle suspension is performed, and the concentrated platelet-rich lysate is collected by centrifugation.

[0064] (5) adding calcium gluconate to the platelet-rich concentrated platelet lysate, heating until the platelet-rich supernatant forms a clot, mechanically squeezing the clot to dry it, and centrifuging the squeezed liquid to collect the supernatant;

[0065] (6) Filter and sterilize the supernatant to further remove cell debris to obtain the platelet lysate.

[0066] In a specific embodiment, a method for preparing a platelet lysate comprises the following steps:

[0067] (1) In each group, multiple samples of anticoagulated whole blood (O and AB blood types in different proportions) from healthy individuals were mixed, red blood cells removed, and platelet-rich plasma mixtures were collected;

[0068] (2) centrifuging the platelet-rich plasma mixture obtained in step (1);

[0069] (3) After centrifugation, the supernatant plasma of multiple groups was collected and placed in a water bath for heating and inactivation;

[0070] (4) Centrifuge the inactivated plasma and transfer it to a centrifuge tube. Keep a portion of the inactivated plasma for later use, and freeze the rest at -20°C.

[0071] (5) collecting the platelet pellets remaining after centrifugation and taking the supernatant from multiple groups of step (2), and washing them twice with PBS;

[0072] (6) adding the plasma reserved in step (4) to prepare a platelet particle suspension, mixing it thoroughly, and then taking a sample to count the number of platelets;

[0073] (7) After ultrasonic treatment, centrifugation was performed and the supernatant (lysate rich in concentrated platelets) was collected;

[0074] (8) The supernatant is mixed with calcium gluconate solution and heated until clots form. The liquid squeezed out by physical and mechanical methods is platelet lysate.

[0075] In this application, each group mixes healthy anticoagulated whole blood from different sources (at a ratio of 3-6 parts O type blood + 1-3 parts AB type blood), removes red blood cells, and collects a platelet-rich plasma mixture. In this application, 5-20 parts of anticoagulated whole blood are used.

[0076] In this application, the term "healthy individuals" refers to individuals without hypertension or hyperlipidemia. Anticoagulated whole blood is preferably collected from healthy individuals into commercial anticoagulation tubes for ease of collection. The volume of each aliquot of anticoagulated whole blood from different healthy individuals is preferably 2-5 ml. In this application, the use of multiple aliquots of anticoagulated whole blood from different healthy individuals can minimize inter-individual and inter-batch variability.

[0077] In the present application, the anticoagulated whole blood of healthy individuals preferably meets the quality standards shown in Table 1.

[0078] Table 1 Quality standards of anticoagulated whole blood from healthy individuals

[0079] The present application does not specifically limit the method for removing red blood cells, and any conventional method for removing red blood cells in the art can be used. In the specific implementation process of the present application, the steps are as follows: each group of platelet-rich plasma mixture is prepared by mixing multiple portions of anticoagulated whole blood from healthy people of different sources (at a ratio of 3-6 portions of O-type blood + 1-3 portions of AB-type blood) and then centrifuging; the speed of the centrifugation is preferably 1500-2500rpm; the time of the centrifugation is 10-20min; after the centrifugation, the blood is divided into three layers, which are the plasma layer, the platelet and white blood cell layer, and the red blood cell layer from top to bottom; the liquid in the upper layer and 1mm below the middle layer is collected, and the collected liquid is the platelet-rich plasma mixture; the speed of the re-centrifugation is 3000-5000rpm, and the time of the centrifugation is 10-30min. At the same time, obtain the supernatant of multiple groups (3-6 portions) of platelet-rich plasma mixtures. The supernatant is plasma. After mixing, place it in a water bath for heating and inactivation. Then centrifuge and collect the final inactivated plasma and platelet particles to precipitate into a new centrifuge tube. Draw a portion of the inactivated plasma for use, and store the remaining inactivated plasma in a -20°C refrigerator.

[0080] After collecting the platelet pellets into a new centrifuge tube, add an appropriate amount of PBS to wash twice, then add the reserved plasma and mix well to prepare a platelet pellet suspension, sample and count the platelet number (at least (1-3) × 10 11 The platelet suspension (30-60 ml) is then placed in an EVA freezer bag, ultrasonically treated, and centrifuged. The supernatant is collected as the platelet concentrate-rich lysate. In this application, the ultrasonic treatment power is 10-30 kHz, and the ultrasonication time is 20-50 minutes. The centrifugation speed is 1000-2500 rpm, and the centrifugation time is 15-30 minutes. In this application, the ultrasonic treatment is used to lyse the platelets and release various growth factors in the platelets, and the centrifugation is used to remove cell debris.

[0081] In the present application, after obtaining a lysate rich in platelet concentrate, the lysate rich in platelet concentrate is mixed with a calcium gluconate solution, heated until the lysate rich in platelet concentrate presents a clot state, physical and mechanical extrusion is performed to wring out the clot, the supernatant is collected and centrifuged, and the platelet lysate is obtained after filtration. In the present application, the calcium gluconate is preferably a calcium gluconate solution, and the concentration of the calcium gluconate solution is preferably 0.8-1.4 g / ml; the volume ratio of the calcium gluconate solution to the lysate rich in platelet concentrate is 1: (1-5). In the present application, the heating temperature is 35-40 ° C.

[0082] In this application, the calcium gluconate solution is used to remove fibrin, and the physical mechanical extrusion is to obtain a supernatant containing only growth factors but no platelet fragments; the centrifugal speed is 4000-6000g, and the centrifugation time is 5-15min. After collecting the last supernatant, this application also includes a filtration sterilization step, preferably using a filter with a pore size of 0.2μm. The filtration sterilization step can further remove cell debris and impurities while sterilizing, and sampling is performed to test the quality inspection standards and platelet lysis rate of the platelet lysate.

[0083] In the present application, the concentration of factors released in the platelet lysate preferably meets the quality standards shown in Table 2.

[0084] Table 2 Quality standards for the concentration of factors released from platelet lysate

[0085] After obtaining the platelet lysate, the present application preferably further comprises the step of performing a pyrogen detection on the platelet lysate. The pyrogen detection is preferably performed according to the procedure described in the instructions of the Limulus amebocyte lysate test kit to detect the endotoxin content. In the present application, the platelet lysate is prepared and preferably packaged and stored, preferably in a volume of 5-10 ml, and the storage temperature is -20°C, and the long-term storage temperature is preferably -80°C.

[0086] This application also provides the use of the platelet lysate in primary cell culture. The cells are preferably human umbilical cord mesenchymal stem cells; the platelet lysate is added to the culture medium at a rate of 1-6%. The umbilical cord mesenchymal stem cells are derived from umbilical cords commonly used in the art. This application does not specifically limit the conditions for culturing the cells; conventional primary cell culture conditions in the art that are sufficient for cell culture are employed.

[0087] To make the purpose, technical solutions, and advantages of this application more clear, the technical solutions in the embodiments of this application are clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of this application.

[0088] The present application is further described in detail below with reference to the embodiments, drawings, comparative examples and performance test results.

[0089] The instruments and reagents used in the following examples can be purchased on the market, for reference only: 80°C refrigerator, 4°C refrigerator, 37°C incubator, pH meter, electric constant temperature water bath, electronic balance, centrifuge, ultrasonic pool, automatic counter, AVE freezing bag, inverted microscope, display Limulus amebocyte lysate test kit, albumin, total protein detection kit, PDGFAA Elisa kit, PDGFAB Elisa kit, PDGFBB Elisa kit, VEGF Elisa kit, EGF Elisa kit, TGF-β Elisa kit, IGF-1 Elisa kit, and calcium gluconate.

[0090] Example

[0091] Example 1

[0092] This embodiment provides a method for preparing a platelet lysate.

[0093] The platelet lysate is prepared as follows:

[0094] Step 1: 20 healthy, anticoagulated whole blood samples (4 O-type blood samples + 1 AB-type blood sample per group, 5 ml of each sample was drawn, and 25 ml was divided into 4 groups, for a total volume of 100 ml) were grouped, mixed, and centrifuged (2000 rpm for 15 minutes). After centrifugation, each tube was separated into three layers: ① plasma layer (top layer, orange-yellow); ② platelet and white blood cell layer (middle layer); and ③ red blood cell layer (bottom layer). The upper and middle layers, as well as the 1 mm below the middle layer (including a small amount of red blood cells), were aspirated and placed in a new 50 ml centrifuge tube to form the platelet-rich plasma mixture. The mixture was centrifuged at 5000 rpm for 20 minutes.

[0095] Step 2: Pipette 2 / 3 of the upper supernatant into a new 50ml centrifuge tube, which is the plasma. Collect the plasma from the four groups, mix them evenly and heat them in a water bath at 56℃ for 30 minutes to inactivate them. Then centrifuge and transfer them to a new centrifuge tube. Keep 10ml of plasma for later use and store the remaining plasma in a -20℃ refrigerator.

[0096] In the third step, the four groups of platelet particles precipitated after centrifugation in the first step were collected into a new 50 ml centrifuge tube, the platelets were washed twice with PBS, and then the inactivated plasma reserved in the second step was added and mixed thoroughly to prepare a concentrated platelet particle suspension.

[0097] Step 4: Use ultrasound to preliminarily lyse the platelet particle suspension; put approximately 50 ml of concentrated platelet particle suspension collected in the third step into a 500 ml EVA freezing bag and place it in an ultrasonic pool for treatment. The ultrasonic time is 30 minutes and the ultrasonic power is 20 kHz. Then collect it into a new centrifuge tube and centrifuge it at 1500 rpm for 20 minutes to collect the lysate rich in concentrated platelets.

[0098] Step 5: Add 10 mg / ml calcium gluconate solution to the platelet concentrate-rich lysate in a ratio of 1:1. Heat in a 37°C water bath until clots appear. Perform physical and mechanical extrusion to wring out the clots. Centrifuge the collected supernatant at 5000 g for 10 minutes, and then filter it with a 0.2 μm filter to further remove cell debris and release various biological factors in the platelets, thereby obtaining a highly efficient platelet concentrate lysate.

[0099] Step 6: After obtaining the final concentrated platelet lysate, pyrogen detection, platelet lysate concentration test, and platelet lysis rate test are performed. Pyrogen detection is prioritized according to the instructions for the Limulus amebocyte lysate test kit to detect endotoxin content, and the result is 0.1 EU / ml; the concentrations of various release factors in the platelet lysate are within the results range shown in Table 2; and the platelet lysis rate is ≥95%.

[0100] Step 7: Packaging and storage of platelet lysate. Pack the platelet lysate into 5 ml portions and store at -20°C. For long-term storage, place it in a -80°C refrigerator.

[0101] Example 2

[0102] This example provides the application of platelet lysate.

[0103] The concentrated platelet lysate prepared in Example 1 was added to the basal culture medium (DMEM-F12) at a volume ratio of 3% and mixed evenly. A culture medium group containing 3% platelet lysate and a type B culture medium group (commercially available, human mesenchymal stem cell serum-free culture medium containing platelet lysate, purchased from Tianjin Haoyang, product number: SC2013-G) were set up respectively.

[0104] Umbilical cord tissue was stripped and minced, then divided evenly into four T75 culture flasks. The cells were divided into two groups: one group received culture medium containing 3% platelet lysate (10 ml per flask), and the other group received culture medium B (10 ml per flask). The cells were cultured at 37°C, 5% CO₂, and 98% humidity in an incubator. Cell counts were performed using an automated cell counter. The results are shown in Figure 1.

[0105] From the test results in Figure 1, it can be seen that the 3% concentrated platelet lysate is more effective in stimulating cell crawling than type B culture medium. Cells cultured with the platelet lysate prepared in this application have more advantages in morphology and yield.

[0106] FIG2 shows the cell status of umbilical cord tissue in two different culture media at different times.

[0107] In Figure 2, images 1 (containing the 3% platelet lysate prepared in Example 1 + DMEM-F12 medium) and 4 (Type B medium) show the morphology of cells on day 9 after they emerged from the umbilical cord tissue. Cells in both groups, 1 and 4, showed that cells emerged. The group containing 3% platelet lysate had multiple colonies emerging (4-6 colonies per bottle), while the commercially available medium had only one bottle and a single, relatively sparse colony. Images 2 (containing the 3% platelet lysate prepared in Example 1 + DMEM-F12 medium) and 5 (Type B medium) show the morphology of cells on day 11 after they emerged from the umbilical cord tissue. Images 2 and 5 show that the group containing 3% platelet lysate had more cells with clear and distinct morphology, while the cells in the Type B medium group were smaller and had a more blurred morphology. 3 (containing 3% platelet lysate + DMEM-F12 medium prepared in Example 1) and 6 (type B medium) are the cell morphologies on the 13th day after cells crawled out of the umbilical cord tissue; from 3 and 6, it was observed that the cells in the group containing 3% platelet lysate were slender and grew more neatly, while the cells that crawled out of the type B medium were rounded and grew more disorderly.

[0108] Example 3

[0109] This example provides the application of platelet lysate.

[0110] The concentrated platelet lysate prepared in Example 1 was added to the cell basal culture medium (DMEM-F12) at a volume ratio of 1%, 3%, and 5% to culture umbilical cord mesenchymal stem cells and observe the cell growth. There were 4 groups of cell culture conditions: (1) DMEM-F12 + 1% concentrated platelet lysate; (2) DMEM-F12 + 3% concentrated platelet lysate; (3) DMEM-F12 + 5% concentrated platelet lysate; (4) Type B culture medium. The seeding density was 5×10 4cell / ml, cells were cultured in vented cap culture flasks in a 5% CO2 incubator with 98% humidity.

[0111] Figure 3 shows the total number of viable cells harvested 13 days after umbilical cord mesenchymal stem cells emerged from the umbilical cord tissue after stimulation with different concentrations of platelet lysate prepared in Example 1 plus DMEM-F12 medium and type B medium. As shown in Figure 3, the cells maintained the best state when cultured in DMEM-F12 medium containing 3% platelet lysate.

[0112] Comparative Example

[0113] This comparative example provides a method for preparing a platelet lysate. The difference between this comparative example and Example 1 is that this method for preparing a platelet lysate does not include the fourth step of ultrasonic treatment and the fifth step of adding calcium gluconate. The specific steps are as follows:

[0114] Step 1: 20 healthy, anticoagulated whole blood samples (4 O-type blood samples + 1 AB-type blood sample per group, 5 ml of each sample was drawn, and 25 ml was divided into 4 groups, for a total volume of 100 ml) were grouped, mixed, and centrifuged (2000 rpm for 15 minutes). After centrifugation, each tube was separated into three layers: ① plasma layer (top layer, orange-yellow); ② platelet and white blood cell layer (middle layer); and ③ red blood cell layer (bottom layer). The upper and middle layers, as well as the 1 mm below the middle layer (including a small amount of red blood cells), were aspirated and placed in a new 50 ml centrifuge tube to form the platelet-rich plasma mixture. The mixture was centrifuged at 5000 rpm for 20 minutes.

[0115] Step 2: Pipette 2 / 3 of the upper supernatant into a new 50ml centrifuge tube, which is the plasma. Collect the plasma from the four groups, mix them evenly and heat them in a water bath at 56℃ for 30 minutes to inactivate them. Then centrifuge and transfer them to a new centrifuge tube. Keep 10ml of plasma for later use and store the remaining plasma in a -20℃ refrigerator.

[0116] In the third step, the four groups of platelet pellets from the first step are collected into a new 50ml centrifuge tube. The platelets are rinsed twice with PBS, and then the inactivated plasma from the second step is added and mixed thoroughly to prepare a concentrated platelet suspension, also known as platelet lysate.

[0117] Step 4: After obtaining the final platelet lysate, pyrogen detection, platelet lysate concentration test, and platelet lysis rate test were performed. Pyrogen detection was prioritized according to the instructions for the Limulus amebocyte lysate test kit to detect endotoxin content, and the result was 0.1 EU / ml; the concentrations of various release factors in the platelet lysate were within the results range shown in Table 2; and the platelet lysis rate was less than 85%.

[0118] Step 5: Packaging and storage of platelet lysate. Pack the platelet lysate into 5 ml portions and store at -20°C. For long-term storage, place it in a -80°C refrigerator.

[0119] The concentrated platelet lysate prepared in Example 1 and the platelet lysate prepared in the comparative example were added to the cell basal culture medium (DMEM-F12) at a volume ratio of 3% to culture umbilical cord mesenchymal stem cells and observe the cell growth. There were two groups of cell culture conditions: (1) DMEM-F12 + 3% concentrated platelet lysate; (2) DMEM-F12 + 3% platelet lysate of the comparative example. The seeding density was 5×10 4 cell / ml, cells were cultured in vented cap culture flasks in a 5% CO2 incubator with 98% humidity.

[0120] Figure 4 shows the total number of living cells harvested on the third day after umbilical cord mesenchymal stem cells were stimulated to crawl out of the umbilical cord tissue by 3% platelet lysate + DMEM-F12 culture medium prepared in Example 1 and 3% platelet lysate + DMEM-F12 culture medium prepared in the comparative example. As shown in Figure 4, during the culture of mesenchymal stem cells using the comparative example's platelet lysate, the cells release obvious flocculent matter, which affects the cell viability and cell number of the mesenchymal stem cells. Therefore, the platelet lysate prepared in this application can better stimulate umbilical cord mesenchymal stem cells to crawl out of the tissue and improve their yield.

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing a platelet lysate, characterized in that: The preparation method specifically comprises the following steps: (1) Centrifuging anticoagulated whole blood to collect a platelet-rich plasma mixture; (2) centrifuging the platelet-rich plasma mixture, collecting the plasma supernatant, inactivating it by heating, and centrifuging it to obtain inactivated plasma; (3) collecting the platelet granules precipitated by centrifugation in step (2), washing them, and resuspending them in the inactivated plasma to prepare a concentrated platelet granule suspension; (4) ultrasonically treating the concentrated platelet particle suspension, centrifuging and collecting a lysate rich in concentrated platelets; (5) adding calcium gluconate to the platelet-rich concentrated lysate, heating until a clot is formed, mechanically squeezing the clot to dry it, and centrifuging the squeezed liquid to collect the supernatant; (6) Filter and sterilize the supernatant to obtain the platelet lysate.

2. The preparation method according to claim 1, characterized in that The anticoagulated whole blood comprises the following blood types in parts by weight: 3-6 parts of type O blood; 1-3 parts of type AB blood.

3. The preparation method according to claim 1, characterized in that The centrifugal speed in step (1) is 1500-5000 rpm, and the centrifugal time is 10-30 min.

4. The preparation method according to claim 1, characterized in that The preparation method comprises at least any one of the following ac: a. The centrifugal speed before heat inactivation in step (2) is 1000-2000 rpm, and the centrifugation time is 10-30 min; b. The heat inactivation temperature is 30-60 ° C, and the heat inactivation time is 20-50min; c. After the heat inactivation in step (2), the centrifugal speed is 1000-2000 rpm, and the centrifugal time is 10-30 min.

5. The preparation method according to claim 1, characterized in that The preparation method comprises at least one of the following: d. The platelet granules precipitated in step (3) are a collection of 2-4 groups of platelet granules precipitated; e. The inactivated plasma used in step (3) is 8-16 ml; f. The number of platelets in the concentrated platelet suspension in step (4) is at least (1-3) × 10 11 Particles / L.

6. The preparation method according to claim 1, characterized in that The ultrasonic treatment time in step (4) is 20-50 min, and the ultrasonic treatment power is 10-30 kHz.

7. The preparation method according to claim 1, characterized in that The preparation method comprises at least one of the following: g. The calcium gluconate in step (5) is a calcium gluconate solution having a concentration of 0.8-1.4 g / ml; the volume ratio of the calcium gluconate solution to the platelet-rich concentrate lysate is 1:(1-5); h. The heating temperature in step (5) is 35-40°C; i. The centrifugal speed in step (5) is 4000-6000g, and the centrifugal time is 5-15min.

8. A platelet lysate prepared by the method according to any one of claims 1 to 7.

9. Use of the platelet lysate according to claim 8 in the culture of primary human mesenchymal stem cells.

10. The use according to claim 9, characterized in that The primary human mesenchymal stem cells are derived from umbilical cord tissue; the volume percentage of the platelet lysate in the culture medium used for primary culture is 1-6%.

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