Preparation method of platelet lysate and method for extracting growth factors

Abstract

The invention provides a preparation method of a platelet lysate, which comprises the following steps: separating platelet-rich plasma from in-vitro blood, the platelet-rich plasma comprising plasma and platelets suspended in the plasma; replacing the plasma rich in platelet plasma with a buffer solution containing chitosan to obtain a platelet buffer solution; and performing ultrasonic oscillation treatment on the platelet buffer solution to obtain the platelet lysate. Some embodiments of the invention also provide a method for extracting growth factors. Through the growth factor release synergistic effect between chitosan treatment and ultrasonic oscillation treatment, the release amount of the growth factors in the platelet lysis buffer is greatly increased, and the extraction amount of the growth factors is further increased.

Description

Technical Field

[0001] The invention relates to a method for preparing platelet lysate and a method for extracting growth factors. Prior art

[0002] Currently, commonly used methods for extracting growth factors include calcium ion stimulation and freeze-thaw method. The calcium ion stimulation method is to trigger a coagulation reaction through calcium ions, stimulate platelets to produce degranulation reaction (degranulation), and release growth factors from the alpha granules contained in the platelets. However, during the coagulation process, some platelets will produce fibrin clots, and cause the growth factors to be coated in the fibrin clots, which are not easy to be completely released. The thrombin stimulation method is similar to the calcium ion stimulation method in that it also stimulates platelets to release growth factors through a coagulation reaction, but it requires the additional addition of animal or human thrombin, so there is also the risk of high costs and infectious diseases.

[0003] The freeze-thaw method is to place platelet-rich plasma (PRP) in an environment of -20℃ or -80℃ to completely freeze it, and then thaw it in an environment of room temperature or 37℃, using the tiny ice crystals produced by water molecules during the thawing process to induce platelet rupture and release growth factors. However, a cycle from freezing to thawing usually takes more than 3 hours, and a complete freeze-thaw method generally requires 2 to 5 cycles. Therefore, the freeze-thaw method has the limitation of being extremely time-consuming.

[0004] In addition to the aforementioned methods, known growth factor stimulators also include substances such as collagen, ectoine, adenosine diphosphate or adrenaline, which release growth factors through specific receptors on the surface of activated platelet cell membranes. However, the stimulating effects of these growth factor stimulators have not been clinically proven, and compared with the aforementioned calcium ion stimulation method, thrombin stimulation method or freeze-thaw method, the release efficiency of growth factors is relatively limited.

[0005] Therefore, how to provide a method for preparing a platelet lysate that does not induce an additional coagulation reaction, has low cost, shortens the time, and improves the efficiency of growth factor release is a problem to be solved. Summary of the invention

[0006] The present invention provides a method for preparing a platelet lysing solution, comprising: separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; replacing the plasma of the platelet-rich plasma with a buffer solution comprising chitosan to obtain a platelet buffer solution; and treating the platelet buffer solution with ultrasonic vibration to obtain a platelet lysing solution.

[0007] In some embodiments, before the step of separating platelet-rich plasma from ex vivo blood, the preparation method includes evaluating whether the blood quality of the ex vivo blood is good, wherein when there is no hemolysis in the ex vivo blood and the pH value of the ex vivo blood is greater than 6, the blood quality of the ex vivo blood is good, and then platelet-rich plasma is separated from the ex vivo blood, and when there is hemolysis in the ex vivo blood or the pH value of the ex vivo blood is not greater than 6, the blood quality of the ex vivo blood is poor, and then the ex vivo blood is replaced with another ex vivo blood, and the blood quality of the other ex vivo blood is evaluated.

[0008] In some embodiments, the platelet content in platelet-rich plasma is 1x10 6 / microliter to 1x10 7 / μl.

[0009] In some embodiments, the concentration of chitosan in the buffer is 1 μM to 5 μM.

[0010] In some embodiments, after the step of exchanging the platelet-rich plasma with a buffer comprising chitosan, the preparation method comprises placing the platelet buffer at 25° C. to 40° C. to allow the chitosan to bind to the platelets.

[0011] In some embodiments, the step of allowing the platelet buffer to stand at a temperature between 25° C. and 40° C. comprises allowing the platelet buffer to stand for 30 minutes to 2 hours.

[0012] In some embodiments, the step of subjecting the platelet buffer to ultrasonic agitation comprises using a power of 2.5 J / ml to 200 J / ml.

[0013] In some embodiments, the step of subjecting the platelet buffer to ultrasonic oscillation comprises repeatedly subjecting the platelet buffer to ultrasonic oscillation for 2 to 10 seconds and then stopping for 2 to 10 seconds.

[0014] In some embodiments, in the step of treating the platelet buffer with ultrasonic vibration, an ultrasonic probe is placed in the platelet buffer to perform ultrasonic vibration.

[0015] The present invention provides a method for extracting growth factors, comprising: separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; replacing the plasma of the platelet-rich plasma with a buffer comprising chitosan to obtain a platelet buffer; treating the platelet buffer with ultrasonic vibration to obtain a platelet lysate; and centrifuging the platelet lysate to separate and obtain a supernatant comprising growth factors.

[0016] As described above, the method for preparing platelet lysate and the method for extracting growth factors of the present invention, by using chitosan to act on platelets and subjecting them to ultrasonic vibration treatment, utilizes the synergistic effect of growth factor release between chitosan and ultrasonic vibration treatment, thereby greatly increasing the amount of growth factor released in platelet lysate. In addition, replacing the plasma in platelet-rich plasma with a buffer solution containing chitosan can avoid the occurrence of coagulation reaction, thereby preventing the growth factor from being coated in the fibrin clot and reducing the amount of growth factor extracted, and can also avoid allergic reactions caused by applying the growth factor to the human body after being made into a product. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the above and other objects, features, advantages and embodiments of the present invention more clearly understood, the accompanying drawings are described as follows:

[0018] Figure 1 A flow chart showing a method for preparing a platelet lysate according to some embodiments of the present invention.

[0019] Figure 2 A comparison chart is presented showing the added value of the efficiency of the group treated with chitosan alone and the group treated with ultrasonic oscillation alone compared with the NAP group ((CAP-NAP)+(xJAP-NAP)) and the efficiency of the group treated with chitosan and ultrasonic oscillation together compared with the NAP group (CxJAP-NAP), wherein the vertical axis represents the efficiency of each group compared with the NAP group or the added value of the increased efficiency (the efficiency in this figure is "PDGF-BB concentration"), and the horizontal axis "x" represents the ultrasonic oscillation power per ml. DETAILED DESCRIPTION

[0020] In order to make the description of the present invention more detailed and complete, the following describes in detail the implementation methods and specific embodiments of the present invention; however, this is not the only form of implementing or using the specific embodiments of the present invention. The embodiments disclosed below can be combined or replaced with each other in beneficial situations, and other embodiments can be added to one embodiment without further recording or explanation. In the following description, many specific details will be described in detail so that the reader can fully understand the following embodiments. However, the embodiments of the present invention can be practiced without these specific details.

[0021] In this document, unless the context specifically limits the article, "a", "an" and "the" may refer to one or more. It will be further understood that "comprising", "including", "having" and similar words used in this document specify the features, regions, integers, steps, operations, components and / or elements recorded therein, but do not exclude other features, regions, integers, steps, operations, components, elements, and / or groups thereof.

[0022] As used herein, the term "about" means a value of a given amount that varies within 5% of the value (e.g., ±1%, ±2%, ±3%, ±4%, 5%). These values ​​are merely examples and are not intended to be limiting. It should be understood that the term "about" may mean a percentage of the value of a given amount as interpreted by one of skill in the art in view of the teachings herein.

[0023] Although a series of operations or steps are used below to illustrate the method disclosed herein, the order shown in these operations or steps should not be construed as a limitation of the present invention. For example, certain operations or steps may be performed in different orders and / or simultaneously with other steps. In addition, it is not necessary to perform all operations, steps and / or features to implement the embodiments of the present invention. Furthermore, each operation or step described herein may include several sub-steps or actions.

[0024] See also Figure 1 Some embodiments of the present invention provide a method 100 for preparing a platelet lysate, comprising: step S110, separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; step S120, replacing the plasma of the platelet-rich plasma with a buffer comprising chitosan to obtain a platelet buffer; and step S130, treating the platelet buffer with ultrasonic vibration to obtain a platelet lysate.

[0025] In preparation method 100, the cationic groups of chitosan bind to the negatively charged groups on the cell membrane, triggering the activation of platelets and the release of growth factors, and combined with ultrasonic vibration treatment, the platelet cell membrane is ruptured, and the growth factors in the platelets are released into the platelet lysate, thereby increasing the content of growth factors in the platelet lysate.

[0026] Compared to the known calcium ion stimulation method or thrombin stimulation method, preparation method 100 does not induce a coagulation reaction because the plasma is replaced with a buffer solution containing chitosan. Therefore, the growth factor will not be coated in the fibrin clot generated by the coagulation reaction, and the release amount of the growth factor can be maximized. In addition, compared to the high cost of thrombin and the risk of infectious disease transmission, chitosan is not only low in cost, but also easier to control in quality and has a low risk of infectious diseases. On the other hand, compared to the known freeze-thaw cycle, preparation method 100 can release a high content of growth factors without repeated freezing and thawing, which greatly saves preparation time.

[0027] In addition, it should be emphasized that compared with the sum of the efficiencies of chitosan treatment alone and ultrasonic vibration treatment alone, the effects of chitosan treatment and ultrasonic vibration treatment on increasing the content of growth factors are synergistic, and the combination of the two can greatly enhance the release effect of growth factors.

[0028] First, referring to step S110 , platelet-rich plasma is separated from ex vivo blood, wherein the platelet-rich plasma includes plasma and platelets suspended in the plasma.

[0029] In some embodiments, before the step S110 of separating platelet-rich plasma from the ex vivo blood, the preparation method 100 includes evaluating whether the blood quality of the ex vivo blood is good, wherein when there is no hemolysis in the ex vivo blood and the pH value of the ex vivo blood is greater than 6 (e.g., pH value 6, 7, 8 or a value in the aforementioned interval), the blood quality of the ex vivo blood is good, then platelet-rich plasma is separated from the ex vivo blood, and when there is hemolysis in the ex vivo blood or the pH value of the ex vivo blood is not greater than 6 (e.g., pH value 1, 2, 3, 4, 5 or a value in the aforementioned interval), the blood quality of the ex vivo blood is poor, then the ex vivo blood is replaced with another ex vivo blood, and the blood quality of the other ex vivo blood is evaluated. Since the use of poor quality blood for subsequent processing directly will result in poor efficiency of subsequent growth factor induction and release. Therefore, before inducing platelets to release growth factors, the quality of the blood is first confirmed, which can avoid the cost and time waste of subsequent procedures and improve the efficiency of growth factor release.

[0030] In some embodiments, when the ex vivo blood is separated into a blood cell layer and a plasma layer after centrifugation, and the plasma layer is clear, there is no hemolysis in the ex vivo blood; and when the ex vivo blood cannot be separated into a blood cell layer and a plasma layer after centrifugation, or the plasma layer is turbid (for example, suspended matter is visible to the naked eye), there is hemolysis in the ex vivo blood.

[0031] In some embodiments, assessing whether the blood quality of the ex vivo blood is good further comprises confirming whether the platelet content is at an appropriate level, for example, the platelet content is 2×10 5 / microliter to 3x10 5 / microliter, by confirming that the platelet content in the ex vivo blood is within the normal range, ensuring that the subsequent growth factor release concentration is above a certain level.

[0032] In some embodiments, the step S110 of separating platelet-rich plasma from ex vivo blood comprises using horizontal centrifugation to stratify the ex vivo blood gradient into a blood cell layer and platelet-rich plasma, detecting the platelet-rich plasma by photosensitive means, and separating the platelet-rich plasma from the ex vivo blood. In one embodiment, a platelet separator can be used in combination with a blood separation kit to separate platelet-rich plasma, thereby automatically achieving large-scale separation of platelet-rich plasma.

[0033] In some embodiments, the platelet content in platelet-rich plasma is 1x10 6 / microliter to 1x10 7 / microliter, for example 1x10 6 / μl, 2.5x10 6 / μl, 5x10 6 / μl, 7.5x10 6 / μl, 1x10 7 / microliter, or a combination thereof. By controlling the platelet content in platelet-rich plasma within a specific range, it is ensured that the subsequent chitosan has a good activation effect on the platelet growth factor. If the platelet content is too low, the growth factor release per unit volume is too low; if the platelet content is too high, the efficiency of the chitosan per unit volume on the platelets is not good, resulting in too low a growth factor release. Therefore, whether the platelet content is too high or too low, the subsequent growth factor release efficiency will be reduced.

[0034] Next, referring to step S120, the platelet-rich plasma is replaced with a buffer solution containing chitosan to obtain a platelet buffer solution.

[0035] Through this step, other components in the blood (such as immunoglobulins, thrombin and prothrombin, etc.) can be removed, leaving only platelets in the buffer solution to avoid additional reactions of other components in the blood during subsequent ultrasonic oscillation reactions (such as the formation of fibrin clots due to coagulation reactions), and to avoid the residue of immunoglobulins, which may cause subsequent growth factors to be administered to the human body and induce an immune response (such as allergies).

[0036] In some embodiments, the deacetylation of chitosan is 75% to 100%, such as 75%, 80%, 85%, 90%, 95%, 100% or a numerical value in the aforementioned interval. In some embodiments, the molecular weight of chitosan is 100 kilodaltons to 200 kilodaltons, such as 100 kilodaltons, 150 kilodaltons, 200 kilodaltons or a numerical value in the aforementioned interval.

[0037] In some embodiments, the concentration of chitosan in the buffer is 1 μM to 5 μM, such as 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, or values ​​in the aforementioned range. If the concentration is too low, the effect is not good, and if the concentration is too high, the increase in the growth factor release effect is limited, resulting in material waste. Among them, when the chitosan concentration is selected to be 2.5 μM, a good growth factor induction effect can be achieved for platelets. Moreover, this dosage is extremely low, and has almost no effect on subsequent products made with growth factors, is non-toxic, and will not cause allergic reactions in users.

[0038] In some embodiments, after step S120 of replacing the platelet-rich plasma with a buffer solution containing chitosan, the preparation method 100 includes placing the platelet buffer solution at a temperature between 25°C and 40°C (e.g., 25°C, 30°C, 35°C, 40°C, or values ​​in the aforementioned range) to allow the chitosan to bind to the platelets. If the temperature is too low, the binding effect is poor and the platelet activity is reduced, and if the temperature is too high, the platelet activity is reduced and the growth factor is degraded.

[0039] In some embodiments, the step of placing the platelet buffer at 25°C to 40°C comprises placing it at 30 minutes to 2 hours. If the time is too short, the binding effect is poor; if the time is too long, the binding efficiency is limited and the efficiency is reduced.

[0040] Next, referring to step S130 , the platelet buffer solution is subjected to ultrasonic oscillation to obtain a platelet lysate.

[0041] In some embodiments, the step of ultrasonically oscillating the platelet buffer comprises using a power of 2.5 joules / ml to 200 joules / ml, for example, 2.5 joules / ml, 5 joules / ml, 10 joules / ml, 20 joules / ml, 30 joules / ml, 40 joules / ml, 50 joules / ml, 60 joules / ml, 70 joules / ml, 80 joules / ml, 90 joules / ml, 100 joules / ml, 120 joules / ml, 140 joules / ml, 160 joules / ml, 180 joules / ml, 200 joules / ml or values ​​in the aforementioned intervals. If the power is too low, the growth factor release effect is not good, and if the power is too high, not only the growth factor release effect is limited, but also the growth factor degradation may be caused.

[0042] It is worth emphasizing that although chitosan and ultrasonic vibration treatment have synergistic effects, the synergistic effect is not positively correlated with power. Using 5 joules / ml can achieve nearly twice the synergistic effect and avoid the problem of protein inactivation and degradation caused by excessive power.

[0043] In some embodiments, the step of treating the platelet buffer with ultrasonic oscillation comprises repeatedly performing ultrasonic oscillation on the platelet buffer for 2 to 10 seconds, and then stopping for 2 to 10 seconds. If the ultrasonic oscillation time is too long or the stop time is too short, it is easy to cause the platelet buffer temperature to be too high and protein degradation; if the ultrasonic oscillation time is too short or the stop time is too long, the efficiency of breaking the platelet cell membrane is limited. For example, after performing ultrasonic oscillation for 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds, stop for 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, or 10 seconds.

[0044] In some embodiments, the step of treating the platelet buffer with ultrasonic vibration is to place an ultrasonic probe into the platelet buffer for ultrasonic vibration. Compared with the vibration in the water bath method, the method of placing the probe can make the ultrasonic wave act in a way closer to the platelets, and the extraction force of the probe is strong, which can further enhance the effect of the ultrasonic wave on the platelets.

[0045] Some embodiments of the present invention also provide a method for extracting growth factors, comprising: separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; replacing the plasma of the platelet-rich plasma with a buffer comprising chitosan to obtain a platelet buffer; treating the platelet buffer with ultrasonic vibration to obtain a platelet lysate; and centrifuging the platelet lysate to separate a supernatant comprising growth factors.

[0046] As described in the preparation method 100 , by using chitosan with a synergistic effect and ultrasonic vibration treatment, the content of growth factors released into the platelet lysate can be increased, thereby improving the extraction effect of the growth factors.

[0047] In some embodiments, the growth factor comprises fibroblast growth factor (FGF), platelet-derived growth factor (PDGF) (e.g., platelet-derived growth factor-BB (PDGF-BB)), transforming growth factor Beta (TGF-β), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), brain-derived neurotrophic factor (BDNF), or a combination thereof.

[0048] To further illustrate the preparation method of platelet lysate and the method of extracting growth factors provided by various embodiments of the present invention, the following examples are now carried out. It should be noted that the following examples are provided for demonstration purposes only and are not intended to limit the present invention.

[0049] 1. Preparation method

[0050] 1.1. Preparation of platelet-rich plasma (PRP)

[0051] First, whole blood was collected from the neck of the pig, and then anticoagulant (ACD Solution-A, manufacturer: Xindong) was added to the whole blood, wherein the volume of whole blood to anticoagulant was 7:1.

[0052] Next, the whole blood was tested for quality, including confirmation that (1) there was no hemolysis, (2) the pH value was greater than 6, and (3) the platelet count was 2x10 5 / microliter to 3x10 5 / microliter to ensure that the quality of whole blood is stable for subsequent processing. The step of confirming hemolysis phenomenon includes centrifuging part of the whole blood at 700xg for 10 minutes to confirm whether the whole blood is separated into blood cell layer and plasma layer, and the plasma layer (supernatant) is clear and has no turbidity. If the whole blood is not separated after centrifugation or the plasma layer is turbid, it means that the whole blood may have hemolysis phenomenon and this batch of whole blood cannot be used for subsequent processing.

[0053] After the whole blood has passed the quality test, the platelets can be separated from the whole blood using a multifunctional platelet separator (manufacturer: Haemonetics; model: MCS+) in combination with a blood separation kit (manufacturer: Haemonetics; model: 0996E-00) to obtain platelet-rich plasma (PRP), or platelet concentrate (PC). The platelet content in PRP is greater than 1x10 6 / μl.

[0054] 1.2. Acquisition of growth factors and content detection

[0055] For the 30 ml of PRP obtained through the above steps, centrifuge at 900xg for 10 minutes to precipitate the platelets, and remove the supernatant. Next, use a phosphate buffer with a pH value of 7.4 containing 2.5 μM chitosan (deacetylation greater than 90%, average molecular weight of about 100 kilodaltons to about 200 kilodaltons; chitosan weight volume percentage in phosphate buffer is 0.05%) to resuspend the platelets to obtain a platelet buffer, wherein the control group is a group in which only a phosphate buffer with a pH value of 7.4 (no chitosan) is added to the platelets. The platelet buffer is placed at 37°C for 1 hour to allow chitosan to act on the platelet cell membrane and induce the release of growth factors.

[0056] Next, a touch-controlled ultrasonic cell disruptor (manufacturer: Qsonica; model: Q700) was used to place the ultrasonic probe into the platelet buffer. After standing, ultrasonic oscillation treatment was performed on multiple sets of platelet buffer at different powers of 5 joules / ml, 30 joules / ml, 60 joules / ml and 120 joules / ml, respectively. The amplitude of the ultrasonic oscillation was set to 2%, and the treatment interval was set to treat for 3 seconds and then stop for 3 seconds.

[0057] Next, the platelet buffer treated with ultrasonic vibration was centrifuged at 5700 x g for 30 minutes to separate the supernatant containing growth factors. The supernatant was filtered through filter membranes with pore sizes of 0.45 μm and 0.22 μm in sequence, and 50 μl was taken out to detect the content of growth factors (PDGF-BB was selected as a representative in the embodiment) by enzyme-linked immunosorbent assay (ELISA), and the remaining filtered supernatant was frozen for future use.

[0058] The ultrasonic oscillation treatment conditions, PDGF-BB content, relative content percentage of PDGF-BB, and synergistic effect of ultrasonic oscillation (physical treatment) and chitosan (chemical treatment) of each group of supernatants are summarized in Table 1.

[0059] Table 1. Comparison of PDGF-BB concentrations obtained after chitosan and different ultrasonic oscillation powers (the results of each group are the average values ​​obtained from at least three repeated tests)

[0060]

[0061] Note 1: NAP is the control group without chitosan and ultrasonic oscillation (unactivated); CAP is the group with chitosan added only; xJAP means the group treated with x joules / ml ultrasonic oscillation; CxJAP means the group treated with x joules / ml ultrasonic oscillation and 0.05% chitosan.

[0062] Note 2: The calculation formula for the synergistic effect of ultrasonic vibration and chitosan is shown below (the "efficiency" of each group corresponds to the "PDGF-BB concentration" or the "relative content percentage of PDGF-BB"). When the calculated value is greater than 100%, it indicates a synergistic effect:

[0063]

[0064] The results showed that compared with the control group (NAP group, the relative content percentage of PDGF-BB was 100%), the PDGF-BB concentration of the 30JAP group and the 60JAP group treated with ultrasonic oscillation alone increased significantly, and the relative content percentage of PDGF-BB increased to 141.1% and 132.5%, respectively.

[0065] Compared with the NAP group (relative content percentage is 100%), the PDGF-BB concentration of the CAP group treated with chitosan alone was also significantly increased, and the relative content percentage of PDGF-BB increased to 123.9%.

[0066] Compared with the NAP group (relative content percentage is 100%), the PDGF-BB concentrations of the C5JAP group, C30JAP group, C60JAP group, and C120JAP group treated with chitosan and ultrasonic vibration were significantly increased, and the relative content percentages of PDGF-BB increased to 163.6%, 167.2%, 163.6% and 143.2%, respectively.

[0067] Figure 2 The comparison chart shows the added value of the efficiency of the group treated with chitosan alone (CAP) and the group treated with ultrasonic oscillation alone (xJAP) compared with the NAP group (i.e. (CAP-NAP) + (xJAP-NAP), represented by dotted bars), and the efficiency of the group treated with chitosan and ultrasonic oscillation together (CxJAP) compared with the NAP group (i.e. CxJAP-NAP, represented by diagonal bars), where the vertical axis is the efficiency of each group compared with the NAP group or the added value of the increased efficiency, and the horizontal axis x is the ultrasonic oscillation power per milliliter. The efficiency is the same as Note 2 of Table 1, corresponding to "PDGF-BB concentration" or "relative content percentage of PDGF-BB". To further explain, Figure 2 The dotted bar is the denominator of the synergistic effect calculation formula in Note 2, and the slashed bar is the numerator of the synergistic effect calculation formula.

[0068] See Table 1 for synergistic effects and Figure 2 The chitosan treatment and ultrasonic oscillation treatment in each group showed a synergistic effect (C5JAP: 208.1%, C30JAP: 103.3%, C60JAP: 112.7%, C120JAP: 156.5%). In the second figure, the diagonal bars of each group were higher than the dotted bars, and the synergistic effect of the C5JAP group was the best. In other words, there is no need to use too high power. The power of ultrasonic oscillation is 5 joules / ml and combined with chitosan to achieve the best synergistic effect of growth factor release.

[0069] Some embodiments of the present invention provide a method for preparing a platelet lysate and a method for extracting growth factors, which use chitosan to act on platelets and treat them with ultrasonic vibrations, thereby utilizing the synergistic effect of growth factor release between chitosan treatment and ultrasonic vibration treatment to greatly increase the amount of growth factor released into the platelet lysate and further increase the amount of growth factor extracted.

[0070] Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be based on the attached claims.

Claims

1. A method for preparing a platelet lysate, characterized in that: Include: Separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; exchanging the platelet-rich plasma with a buffer comprising chitosan to obtain a platelet buffer; as well as The platelet buffer solution is treated with ultrasonic vibration to obtain a platelet lysate.

2. The preparation method of claim 1, wherein before the step of separating the platelet-rich plasma from the ex vivo blood, the preparation method comprises evaluating whether the blood quality of the ex vivo blood is good, wherein: When the isolated blood does not have hemolysis and the pH value of the isolated blood is greater than 6, the blood quality of the isolated blood is good, and then the platelet-rich plasma is separated from the isolated blood, and When the isolated blood has the hemolysis phenomenon or the pH value of the isolated blood is not greater than 6, the blood quality of the isolated blood is poor. Then, the isolated blood is replaced with another isolated blood, and the blood quality of the other isolated blood is evaluated.

3. The preparation method according to claim 1, wherein the content of the platelets in the platelet-rich plasma is 1×10 6 / microliter to 1x10 7 / μl. The preparation method according to claim 1 , wherein the concentration of the chitosan in the buffer is 1 μM to 5 μM.

5. The preparation method as described in claim 1, wherein after the step of replacing the plasma of the platelet-rich plasma with the buffer solution containing the chitosan, the preparation method comprises placing the platelet buffer solution at a temperature between 25°C and 40°C to allow the chitosan to bind to the platelets.

6. The preparation method as claimed in claim 5, wherein the step of placing the platelet buffer at a temperature between 25°C and 40°C comprises placing the platelet buffer at a temperature between 30 minutes and 2 hours.

7. The preparation method as claimed in claim 1, wherein the step of treating the platelet buffer with the ultrasonic oscillation comprises using a power of 2.5 joules / ml to 200 joules / ml.

8. The preparation method as described in claim 1, wherein the step of treating the platelet buffer with the ultrasonic oscillation comprises repeatedly subjecting the platelet buffer to ultrasonic oscillation for 2 seconds to 10 seconds and then stopping for 2 seconds to 10 seconds.

9. The preparation method as described in claim 1, wherein the step of treating the platelet buffer with the ultrasonic oscillation comprises placing an ultrasonic probe into the platelet buffer to perform the ultrasonic oscillation.

10. A method for extracting growth factors, characterized in that: Include: Separating platelet-rich plasma from ex vivo blood, wherein the platelet-rich plasma comprises plasma and platelets suspended in the plasma; exchanging the platelet-rich plasma with a buffer comprising chitosan to obtain a platelet buffer; The platelet buffer solution is subjected to ultrasonic vibration to obtain a platelet lysate; and The platelet lysate is centrifuged to separate the supernatant containing the growth factor.

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