TW202126315A - Method of preparing platelet lysate and use thereof for improving female pregnancy success rate - Google Patents

Abstract

A method of preparing a platelet lysate and a use thereof are provided. The method includes the following steps. A collected whole blood is placed in a centrifuge tube containing a separation gel. A first centrifugation process is performed on the centrifuge tube containing the whole blood and the separation gel to separate the whole blood into a first upper portion, a middle portion, and a lower portion. The first upper portion and the middle portion are mixed to obtain a platelet-rich plasma fraction. The platelet-rich plasma fraction is clotted to obtain a platelet-rich fibrin gel. The platelet-rich fibrin gel is separated to obtain a platelet lysate.

Description

Method for preparing platelet lysate and its use for improving female pregnancy rate

The present invention relates to a method for preparing a derivative of platelet-rich plasma (PRP) and its use, and in particular to a method for preparing a platelet lysate and its use for improving female pregnancy rate .

Platelet-rich plasma (PRP) is plasma with a high concentration of platelets obtained by centrifuging human or animal whole blood, while platelet lysate is a further processed derivative of platelet-rich plasma. The lysate contains a variety of growth factors related to regeneration. Therefore, platelet-rich plasma and platelet lysate are known to be used in regenerative medicine.

At present, the traditional method for preparing platelet lysate is as follows: after PRP is further added with activating factor, the platelet lysate is obtained by repeated thawing and freezing or ultrasound. The above-mentioned traditional methods may have the following disadvantages: excessive collection of whole blood, red blood cell contamination due to incomplete separation, repeated activation due to the effect of anticoagulants, and difficulty in concentration. The above-mentioned problems will lead to insufficient growth factors in the collected platelet lysate. In addition, in the traditional method of preparing platelet lysate, PRP needs to be taken out of the centrifuge tube and activated, so the effect of sterility is worrying, and the cost of obtaining platelet lysate by freezing and thawing or ultrasonic vibration Time is too long. Therefore, how to quickly prepare a platelet lysate with a high concentration of growth factors is a problem that researchers are eager to solve.

In addition, many factors are known to cause female infertility, and current methods of treating infertility include artificial insemination or in vitro fertilization. However, the existing treatment methods are still not effective in improving the pregnancy of patients with infertility.

The invention provides a method for preparing a platelet lysate, which can quickly prepare a platelet lysate with a high concentration of growth factors under aseptic conditions.

The present invention provides a pharmaceutical composition comprising platelet-rich plasma and/or platelet-rich plasma derivatives, which can be used to improve the pregnancy success rate of females.

The embodiment of the present invention provides a method for preparing a platelet lysate, the method comprising the following steps: step a), placing the collected whole blood in a centrifuge tube containing a separating colloid, wherein the density of the separating colloid is 1.030 g/ cm ³ to 1.093 g/cm ³ ; step b), perform the first centrifugation process on the centrifuge tube containing the whole blood and the separation colloid, so that the whole blood is separated into the first upper part, the middle part and the lower part, wherein the separation colloid Located between the middle part and the lower part; step c), mixing the first upper part with the middle part to obtain a platelet-rich plasma (PRP) part; step d), making the platelet-rich plasma part Coagulation to obtain platelet-rich fibrin (PRF) colloid; and step e) to separate platelet-rich fibrin colloid to obtain platelet lysate (PL), wherein in steps a) to No anticoagulant is added in step e).

In an embodiment of the present invention, before performing step c), it may further include removing part of the first upper layer portion to obtain a second upper layer portion, wherein the volume ratio of the first upper layer portion to the second upper layer portion is 1 :0.01~0.99.

In an embodiment of the present invention, the aforementioned anticoagulant may include anticoagulant citrate compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), and anticoagulant compound sodium citrate solution B (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A). Citrate Dextrose Solution, Solution B, ACD-B), Anticoagulant Citrate Dextrose Solution, Solution C (ACD-C), Citrate-phosphate-dextrose Solution, CPD), Ethylenediaminetetraacetic acid (EDTA), Heparin, Sodium Citrate or a combination thereof.

In an embodiment of the present invention, the aforementioned separation colloid material may include a silicon-containing polymer, an acrylic polymer, or a polyester polymer.

In an embodiment of the present invention, the centrifugal rotation speed of the above-mentioned first centrifugation process is, for example, 1000 g to 4000 g, and the centrifugation time is, for example, 3 minutes to 10 minutes.

In an embodiment of the present invention, the first upper part is a plasma layer containing platelets, the middle part is a buffy coat containing platelets and white blood cells, and the lower part is a red blood cell layer containing red blood cells.

In an embodiment of the present invention, coagulating the platelet-rich plasma fraction includes performing a second centrifugation process on the platelet-rich plasma fraction.

In an embodiment of the present invention, the centrifugal rotation speed of the above-mentioned second centrifugation process is, for example, 1000 g to 4000 g, and the centrifugation time is, for example, 3 minutes to 10 minutes.

In an embodiment of the present invention, separating the platelet-rich fibrin colloid includes performing a third centrifugation process on the platelet-rich fibrin colloid.

In an embodiment of the present invention, the aforementioned third centrifugation process includes: performing a single centrifugation on the platelet-rich fibrin colloid, wherein the centrifugation speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 45 minutes, Or, the platelet-rich fibrin colloid is centrifuged multiple times, wherein the centrifugal speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 15 minutes each time.

In an embodiment of the present invention, the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes.

In an embodiment of the present invention, before performing step e), it further comprises pressing, squeezing, filtering, ultrasonic vibration or freezing and thawing cycle of the platelet-rich fibrin gel, so that the platelet lysate is self-enriched with platelets. Fibrin colloid is released.

In an embodiment of the present invention, the aforementioned platelet lysate may include at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 From 1 ng/ml to 10000 ng/ml, the concentration of PDGF-BB is 10 pg/ml to 500000 pg/ml, the concentration of PDGF-AB is 10 pg/ml to 500000 pg/ml, and the concentration of FGF2 is 1 pg/ml To 10000 pg/ml, the concentration of EGF is 1 pg/ml to 10000 pg/ml, the concentration of VEGF is 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml.

The present invention provides a use of a medicinal composition for preparing a medicine for improving the pregnancy success rate of females, wherein the medicinal composition includes platelet-rich plasma and/or a derivative of platelet-rich plasma.

In an embodiment of the present invention, the aforementioned derivative of platelet-rich plasma may include platelet lysate.

In an embodiment of the present invention, the above-mentioned medical composition can be administered into the uterine cavity by infusion.

In an embodiment of the present invention, the dosage of the aforementioned pharmaceutical composition is, for example, 0.1 mL to 2 mL.

In an embodiment of the present invention, the time point of administration of the above-mentioned pharmaceutical composition is the follicular phase, ovulation phase or luteal phase of the menstrual cycle, or Hormone Replacement Therapy (HRT) medication The 8th to 16th days.

In an embodiment of the present invention, the period of administration of the aforementioned pharmaceutical composition is once every 2 to 3 days, at least 1 to 3 times.

In an embodiment of the present invention, the above-mentioned female is, for example, a female with infertility caused by uterine factors or ovarian factors.

Based on the above, since the method for preparing platelet lysate of the present invention does not add an anticoagulant in the entire process, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the entire process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

In addition, the pharmaceutical composition of the present invention including platelet-rich plasma and/or platelet-rich plasma derivatives can significantly improve the pregnancy success rate of females.

The invention provides a method for preparing a platelet lysate, which can obtain a platelet lysate with a high concentration of growth factors under specific operating conditions, and the method has a simple operation process, a short process time, and the entire process does not need to add any anticoagulant. In addition, the pharmaceutical composition containing platelet-rich plasma and/or platelet-rich plasma derivatives provided by the present invention can be used to improve the success rate of female pregnancy.

In order to thoroughly understand the present invention, the process steps for preparing the platelet lysate will be described in detail below. However, the well-known composition or process steps are not described in details to avoid limiting the present invention. The preferred embodiments of the present invention will be described in detail as follows, but the present invention is not limited thereto. The present invention can also be widely implemented in other embodiments, and the scope of the present invention is not limited, and the subsequent patent scope shall prevail.

Fig. 1 is a flowchart of a method for preparing a platelet lysate according to an embodiment of the present invention. The method for preparing a platelet lysate of the present invention includes the following steps.

First, perform step a), and place the collected whole blood 10 in a centrifuge tube 30 containing a separation colloid 20. In one embodiment, the whole blood 10 is, for example, fresh mammalian whole blood. In this embodiment, the whole blood 10 is, for example, fresh human whole blood.

In this embodiment, the density of the separation colloid 20 is 1.030 g/cm ³ to 1.093 g/cm ³ . In this embodiment, the material of the separation colloid 20 is, for example, a silicon-containing polymer, an acrylic polymer, or a polyester polymer.

Next, proceed to step b). The centrifuge tube 30 containing the whole blood 10 and the separation colloid 20 is subjected to a first centrifugation process, so that the whole blood 10 is separated into a first upper part 12, a middle part 14 and a lower part 16, wherein the separation The colloid 20 is located between the middle part 14 and the lower part 16. In this embodiment, the centrifugal rotation speed of the first centrifugation process in step b) is, for example, 1000 g to 4000 g. In one embodiment, the centrifugal rotation speed of the first centrifugation process in step b) is, for example, 1500 g to 3000 g. In this embodiment, the centrifugation time of the first centrifugation process in step b) is, for example, 3 minutes to 10 minutes. In one embodiment, the centrifugation time of the first centrifugation process in step b) is, for example, 4 minutes to 8 minutes. In one embodiment, the centrifugal rotation speed of the first centrifugation process in step b) is, for example, 1500 g, and the centrifugation time is, for example, 5 minutes.

In this embodiment, after step b) is performed, the whole blood 10 is separated into three layers, where the uppermost layer (ie, the first upper layer portion 12) is a plasma layer containing platelets, and the middle layer (ie, the middle portion 14) It is a buffy coat containing platelets and white blood cells, and the lowermost layer (ie, the lower layer portion 16) is a red blood cell layer containing red blood cells.

In this embodiment, the separation colloid 20 of the present invention can effectively isolate red blood cells and platelets, so that red blood cells are almost only present in the lower part 16 and platelets are present in the first upper part 12 and the middle part 14. Specifically, the concentration of platelets in the first upper portion 12 and the middle portion 14 is higher than the concentration of platelets in the whole blood and the lower portion 16.

In this embodiment, after step b) is performed, step c) can be followed, but the present invention is not limited to this. In another embodiment, after performing step b) and before performing step c), a portion of the first upper layer portion 12 may be further selectively removed to obtain the second upper layer portion 13. In this embodiment, the volume ratio of the first upper layer portion 12 to the second upper layer portion 13 is, for example, 1:0.01 to 0.99. In an embodiment, the volume ratio of the first upper layer portion 12 to the second upper layer portion 13 is, for example, 1:0.1-0.8. Within the above-mentioned volume ratio range, a higher concentration of platelet lysate can be obtained. In this embodiment, the method of removing part of the first upper part 12 is, for example, piercing a needle into the first upper part 12 of the centrifuge tube 30 and sucking part of the first upper part 12 to the outside of the centrifuge tube 30.

In this embodiment, after performing step b) and before performing step c), since part of the first upper layer portion 12 is removed, a smaller volume of the second upper layer portion 13 is obtained. Therefore, in the subsequent process, platelet-rich The platelet lysate separated by fibrin colloid can be stored in a smaller volume of solution, thereby achieving a concentration effect.

Then, step c) is performed to mix the first upper part 12 (or the second upper part 13) with the middle part 14 to obtain a platelet-rich plasma (PRP) part 15. In this embodiment, the method for mixing the first upper layer portion 12 (or the second upper layer portion 13) with the middle portion 14 is, for example, to shake the centrifuge tube 30 uniformly so that the first upper layer portion 12 (or the second upper layer portion 13) ) Is mixed with the middle part 14. However, the present invention is not limited to this, and any method that can uniformly mix the first upper layer portion 12 (or the second upper layer portion 13) and the middle portion 14 can be applied in this step.

After that, step d) is performed to coagulate the platelet-rich plasma fraction 15 to obtain platelet-rich fibrin (PRF) colloid 17. In this embodiment, the method for coagulating the platelet-rich plasma portion 15 is, for example, performing a second centrifugation process on the platelet-rich plasma portion 15. In this embodiment, the centrifugal rotation speed of the second centrifugation process in step d) is, for example, 1000 to 4000 g. In one embodiment, the centrifugal rotation speed of the second centrifugation process in step d) is, for example, 1500 to 3000 g. In this embodiment, the centrifugation time of the second centrifugation process in step d) is, for example, 3 minutes to 10 minutes. In one embodiment, the centrifugation time of the first centrifugation process in step d) is, for example, 4 minutes to 8 minutes. In one embodiment, the centrifugal rotation speed of the first centrifugation process in step d) is, for example, 1500 g, and the centrifugation time is, for example, 5 minutes. During the second centrifugation process, the platelet plasma portion 15 will self-coagulate into a platelet-rich fibrin (PRF) colloid 17.

Then, proceed to step e) to separate the platelet-rich fibrin colloid 17 to obtain a platelet lysate (PL) 18. In this embodiment, the method for separating the platelet-rich fibrin colloid 17 is, for example, performing the third centrifugation process on the platelet-rich fibrin colloid 17. In one embodiment, the third centrifugation process is, for example, performing a single centrifugation on the platelet-rich fibrin colloid 17, wherein the centrifugation speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 45 minutes. In another embodiment, the third centrifugation process is, for example, performing multiple centrifugation on the platelet-rich fibrin colloid 17, wherein the centrifugation speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 15 minutes each time. In this embodiment, the number of centrifugation for multiple centrifugation is, for example, 5 to 15 times, but the present invention is not limited to this, and the number of centrifugation can be adjusted as required. During the third centrifugation process, the platelet lysate 18 is released from the platelet-rich fibrin colloid 17, and the colloidal part 19 of the platelet-rich fibrin colloid 17 is compressed on the separating colloid 20 due to centrifugal force. This separates the platelet lysate 18 from the colloidal part 19.

In this embodiment, after performing step d) and before performing step e), the platelet-rich fibrin colloid 17 can be further pressed, squeezed, filtered, ultrasonically shaken or subjected to a freeze-thaw cycle, which helps to make the platelets The lysate 18 is released from the platelet-rich fibrin colloid 17.

In one embodiment, the platelet lysis solution 18 includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 is 1 ng/ml To 10000 ng/ml, the concentration of PDGF-BB is 10 pg/ml to 500000 pg/ml, the concentration of PDGF-AB is 10 pg/ml to 500000 pg/ml, the concentration of FGF2 is 1 pg/ml to 10000 pg/ml The concentration of EGF is 1 pg/ml to 10000 pg/ml, the concentration of VEGF is 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml.

In another embodiment, the platelet lysate 18 includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 is 5 ng/ ml to 3000 ng/ml, the concentration of PDGF-BB is 50 pg/ml to 50000 pg/ml, the concentration of PDGF-AB is 100 pg/ml to 50000 pg/ml, the concentration of FGF2 is 20 pg/ml to 6000 pg/ ml, the concentration of EGF is 1.5 pg/ml to 2000 pg/ml, the concentration of VEGF is 15 pg/ml to 6000 pg/ml, and the concentration of VEGFA is 0.2 pg/ml to 1500 pg/ml.

In this example, no anticoagulant was added during the entire process of preparing the platelet lysate. The anticoagulant is, for example, Anticoagulant Citrate Dextrose Solution, Solution A (ACD-A), and Anticoagulant Citrate Dextrose Solution, Solution B (ACD-B). , Anticoagulant Citrate Dextrose Solution, Solution C (ACD-C), Citrate-phosphate-dextrose Solution (CPD), Ethylenediaminetetraacetic acid , EDTA), Heparin, Sodium Citrate or a combination thereof.

In this embodiment, since the entire process of preparing the platelet lysate of the present invention does not add an anticoagulant, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating the activation, thereby shortening the process time. In this embodiment, the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes, which is significantly shorter than the traditional method for preparing the platelet lysate. In addition, the entire process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

The present invention provides a medical composition for improving the success rate of female pregnancy, which comprises platelet-rich plasma and/or a derivative of platelet-rich plasma. In one embodiment, the derivative of platelet-rich plasma is, for example, platelet lysate. In this embodiment, the derivative of platelet-rich plasma is, for example, a platelet lysate prepared by the above method for preparing a platelet lysate.

In one embodiment, the pharmaceutical composition may further include a pharmaceutically acceptable salt or carrier of the pharmaceutical composition.

In one embodiment, the platelet lysate includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 is 1 ng/ml to 10000 ng/ml, the concentration of PDGF-BB is 10 pg/ml to 500000 pg/ml, the concentration of PDGF-AB is 10 pg/ml to 500000 pg/ml, the concentration of FGF2 is 1 pg/ml to 10000 pg/ml, The concentration of EGF is 1 pg/ml to 10000 pg/ml, the concentration of VEGF is 1 pg/ml to 10000 pg/ml, and the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml.

In another embodiment, the platelet lysate includes at least one growth factor selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA, wherein the concentration of TGF-β1 is 5 ng/ml to 3000 ng/ml, the concentration of PDGF-BB is 50 pg/ml to 50000 pg/ml, the concentration of PDGF-AB is 100 pg/ml to 50000 pg/ml, the concentration of FGF2 is 20 pg/ml to 6000 pg/ml, The concentration of EGF is 1.5 pg/ml to 2000 pg/ml, the concentration of VEGF is 15 pg/ml to 6000 pg/ml, and the concentration of VEGFA is 0.2 pg/ml to 1500 pg/ml.

In this embodiment, the female is, for example, a female with infertility caused by uterine factors or ovarian factors. More specifically, females are, for example, female humans who are infertile due to uterine factors or ovarian factors.

In this embodiment, the medical composition can be made into a sterile injection solution. In one embodiment, the pharmaceutical composition is administered into the uterine cavity by infusion, for example. In one embodiment, an artificial insemination (Intrauterine insemination, IUI) tube or an embryo transfer (Embryo Transfer, ET) tube may be used to administer the pharmaceutical composition into the uterine cavity by perfusion. In one embodiment, the dosage of the pharmaceutical composition is, for example, 0.1 mL to 2 mL.

In this embodiment, the time point of administration of the pharmaceutical composition is the follicular phase, ovulation phase, or luteal phase of the menstrual cycle, or the 8th to 16th of Hormone Replacement Therapy (HRT) medication. Days, but the present invention is not limited to this. In one embodiment, the time point of administration can also be determined according to frozen embryo transfer or fresh embryo transfer. In one embodiment, the hormone in hormone replacement therapy is, for example, estradiol (Estradiol), progesterone (Progesterone), or a combination of both. In this embodiment, the period of administration of the pharmaceutical composition is once every 2 to 3 days, at least 1 to 3 times.

The pharmaceutical composition of the present invention can be used to increase the success rate of female pregnancy, specifically, it can be used to increase the success rate of female artificial insemination. In one embodiment, the pharmaceutical composition of the present invention can help thicken the endometrium of females.

Hereinafter, an experimental example will be used to specifically illustrate the present invention, but the parameters and data results of the experimental example are only used to illustrate the efficacy of the present invention, not to limit the scope of the present invention.

[ Preparation of platelet lysate ]

Experimental example 1

First, 10 ml of venous blood was collected from the subject, and fresh whole blood was injected into a centrifuge tube containing separation colloid with an 18G needle. Put the centrifuge tube in a centrifuge and centrifuge at 1500 g for 5 minutes to separate the whole blood into a plasma layer, a white membrane layer and a red blood cell layer. The plasma layer is a small amount of platelet plasma (Platelet-Poor Plasma, PPP).

Next, connect a 10 ml syringe with an 18G needle to remove the 2 ml plasma layer. Shake the centrifuge tube up and down evenly to mix the remaining plasma layer with the albuginea layer to obtain platelet-rich plasma (PRP).

Then, put the same centrifuge tube into a centrifuge and centrifuge at 1500 g for 5 minutes to obtain a platelet-rich fibrin (PRF) colloid.

After that, put the same centrifuge tube into the centrifuge and centrifuge at 1500 g for 5 minutes. Repeat the above centrifugation conditions for 10 centrifugation, thereby obtaining the platelet lysate at the top of the centrifuge tube.

Comparative example 1

First, take 10 ml of venous blood from the subject, and inject fresh whole blood into a centrifuge tube containing anticoagulant (ACD Solution-A, ACDA) with an 18G needle. Put the centrifuge tube into the centrifuge and centrifuge at 1500 g for 5 minutes to separate the whole blood into a plasma layer, a white membrane layer, and a red blood cell layer.

Then, take out the plasma layer and white film layer, move to another sterile container, add calcium chloride solution and mix well, this step is to activate platelets. Next, a freeze-thaw cycle is performed. The activated solution is placed at room temperature for one hour, and then placed in a -80 degree refrigerator for freezing. After 4 hours, it is taken out and the growth factor can be determined.

[ Growth factor determination ]

In this example, the enzyme-linked Immuno-Sorbent Assay (ELISA) was used to determine the growth factor TGF- in the platelet-rich plasma of Example 1, and the platelet lysate of Example 1 and Comparative Example 1. The contents of β1, PDGF-AB, PDGF-BB, VEGF and VEGFA, and the results are shown in Table 1. The results in Table 1 are all expressed as >mean±standard deviation>, and there is a significant difference between each growth factor example and the comparative example (P>0.05).

[Table 1] TGF-β1 (ng/ml) PDGF-AB (pg/ml) PDGF-BB (pg/ml) VEGF (pg/ml) VEGFA (pg/ml) PRP of Experimental Example 1 296.0±26.2 5000.0±343.1 4333.2±403.3 263.0±37.3 62.4±15.8 PL of Experimental Example 1 532.5±75.1 10620.3±1096.9 11904.9±2595.7 565.2±106.7 202.7±46.5 PL of Comparative Example 1 214.2±21.7 3211.6±339.8 1431.7±175.7 168.3±26.5 21.3±6.6

It can be seen from Table 1 that compared with the platelet lysate prepared by the traditional method (ie Comparative Example 1), the platelet lysate prepared by the method for preparing platelet lysate of the present invention (ie Example 1) has a higher concentration of Growth factor. In addition, since the method for preparing platelet lysate of the present invention does not add anticoagulant in the entire process, there is no additional step of taking the platelet lysate from the centrifuge tube and repeating the activation, thereby shortening the process time. In addition, the entire process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

[ The effectiveness of platelet lysate for infertility patients ]

Example 3

In this example, the subjects are 10 female patients with infertility over 40 years old on average. The platelet lysate prepared in Example 1 is used as the medical composition of this example, and platelet lysis is performed in the following manner The assessment of the success rate of pregnancy by the liquid.

First, measure the initial thickness of the subject's endometrium on days 10-12 of the natural cycle. Connect a syringe containing the platelet lysate (0.5-1 ml) of Example 1 to an artificial insemination (Intrauterine insemination, IUI) tube, and administer it into the uterine cavity of the subject by perfusion. Subjects need to lie down for 30 minutes after administration. 48 hours after the administration, the second administration was given by infusion. 48 hours after the second administration, the thickness of the subject's endometrium was measured. When the thickness of the subject's endometrium reaches 7 cm, the embryo can be implanted.

Next, embryo implantation was performed on the subjects after the administration, and hormone replacement therapy was continued after embryo implantation, taking progesterone and estrogen for 2 weeks. Then, test the beta-HCG value in the subject’s blood to confirm the implantation status of the fertilized egg. The subject’s pregnancy results and endometrial thickness are shown in Table 2 and Table respectively. 3 shown.

[Table 2] Endometrial thickness Before administration After administration Average (mm) 6.0 7.9 Mean standard deviation (SD) 0.7 0.8 When compared to before administration, P>0.01.

[table 3] Not pregnant Pregnant Number of people 5 5 Proportion(%) 50 50

It can be seen from Table 2 that after the platelet lysate is administered into the uterine cavity of the subject by perfusion, the thickness of the endometrium of the subject can be increased.

Generally speaking, the success rate of artificial insemination is only 30%. However, as the age increases, the success rate of artificial insemination for women over 40 years old is only 7.7%. It can be seen from Table 3 that after the platelet lysate was administered into the uterine cavity of the subject by infusion, the overall pregnancy success rate of the subject was increased from 7.7% to 50%.

In summary, since the method for preparing platelet lysate of the present invention does not add an anticoagulant in the entire process, there is no additional step of removing the platelet lysate from the centrifuge tube and repeating activation, thereby shortening the process time. In addition, the entire process of preparing the platelet lysate of the present invention is performed in the same centrifuge tube, so it has the advantages of avoiding contamination and having sterility.

In addition, the pharmaceutical composition of the present invention including platelet-rich plasma and/or platelet-rich plasma derivatives can significantly improve the pregnancy success rate of females.

10: Whole blood 12: The first upper part 13: The second upper part 14: middle part 15: Platelet-rich plasma fraction 16: Lower part 17: Platelet-rich fibrin colloid 18: Platelet lysate 19: colloidal part 20: Separation of colloid 30: Centrifuge tube

Fig. 1 is a flowchart of a method for preparing a platelet lysate according to an embodiment of the present invention.

10: Whole blood

12: The first upper part

13: The second upper part

14: middle part

15: Platelet-rich plasma fraction

16: Lower part

17: Platelet-rich fibrin colloid

18: Platelet lysate

19: colloidal part

20: Separation of colloid

30: Centrifuge tube

Claims (20)

A method for preparing a platelet lysate, the method comprising the following steps: Step a), placing the collected whole blood in a centrifuge tube containing a separating colloid, wherein the density of the separating colloid is 1.030 g/cm ³ to 1.093 g/cm ³ ; step b), performing a first centrifugation process on the centrifuge tube containing the whole blood and the separation colloid, so that the whole blood is separated into a first upper part, a middle part, and a lower part , Wherein the separating colloid is located between the middle part and the lower part; step c), mixing the first upper part and the middle part to obtain platelet-rich plasma (platelet-rich plasma, PRP) part; step d), coagulating the platelet-rich plasma part to obtain platelet-rich fibrin (PRF) colloid; and step e), separating the platelet-rich fibrin colloid, To obtain a platelet lysate (PL), wherein no anticoagulant is added in the steps a) to e). The method for preparing a platelet lysate as described in item 1 of the scope of the patent application, wherein before performing the step c), it further comprises removing part of the first upper layer portion to obtain a second upper layer portion, wherein The volume ratio of the first upper layer portion to the second upper layer portion is 1:0.01 to 0.99. The method for preparing a platelet lysate as described in item 1 of the scope of patent application, wherein the anticoagulant includes anticoagulant citrate compound sodium citrate solution A (Anticoagulant Citrate Dextrose Solution, Solution A, ACD-A), anticoagulant Compound Sodium Citrate Solution B (Anticoagulant Citrate Dextrose Solution, Solution B, ACD-B), Anticoagulant Citrate Dextrose Solution C (Anticoagulant Citrate Dextrose Solution, Solution C, ACD-C), citric acid-phosphoric acid-grape solution (Citrate-phosphate-dextrose Solution, CPD), Ethylenediaminetetraacetic acid (EDTA), Heparin, Sodium Citrate, or a combination thereof. According to the method for preparing a platelet lysate according to the first item of the scope of the patent application, the material of the separation colloid includes a silicon-containing polymer, an acrylic polymer or a polyester polymer. According to the method for preparing platelet lysate according to the first item of the scope of patent application, the centrifugal speed of the first centrifugation process is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 10 minutes. The method for preparing a platelet lysate according to claim 1, wherein the first upper part is a plasma layer containing platelets, and the middle part is a buffy coat containing platelets and white blood cells, and The lower part is a red blood cell layer containing red blood cells. The method for preparing a platelet lysate according to the first item of the scope of the patent application, wherein the coagulation of the platelet-rich plasma fraction includes performing a second centrifugation process on the platelet-rich plasma fraction. According to the method for preparing a platelet lysate according to item 7 of the scope of patent application, the centrifugal speed of the second centrifugal process is 1000 g to 4000 g, and the centrifugal time is 3 minutes to 10 minutes. The method for preparing a platelet lysate as described in the first item of the scope of patent application, wherein separating the platelet-rich fibrin colloid includes performing a third centrifugation process on the platelet-rich fibrin colloid. The method for preparing platelet lysate as described in item 9 of the scope of patent application, wherein the third centrifugation process includes: Perform a single centrifugation on the platelet-rich fibrin colloid, wherein the centrifugal speed is 1000 g to 4000 g, and the centrifugation time is 3 minutes to 45 minutes, or The platelet-rich fibrin colloid is centrifuged multiple times, wherein the centrifugal speed is 1000 g to 4000 g, and the centrifugation time for each time is 3 minutes to 15 minutes. According to the method for preparing a platelet lysate as described in item 1 of the scope of the patent application, the entire process time for preparing the platelet lysate is 10 minutes to 60 minutes. The method for preparing a platelet lysate as described in item 1 of the scope of patent application, wherein before performing the step e), it further comprises pressing, squeezing, filtering, ultrasonic vibration or performing the platelet-rich fibrin colloid Freeze-thaw cycles so that the platelet lysate is released from the platelet-rich fibrin colloid. The method for preparing a platelet lysate as described in item 1 of the scope of patent application, wherein the platelet lysate includes at least one growth selected from TGF-β1, PDGF-BB, PDGF-AB, FGF2, EGF, VEGF, and VEGFA Factor, wherein the concentration of the TGF-β1 is 1 ng/ml to 10,000 ng/ml, the concentration of the PDGF-BB is 10 pg/ml to 500,000 pg/ml, and the concentration of the PDGF-AB is 10 pg/ml To 500,000 pg/ml, the concentration of FGF2 is 1 pg/ml to 10,000 pg/ml, the concentration of EGF is 1 pg/ml to 10,000 pg/ml, and the concentration of VEGF is 1 pg/ml to 10,000 pg/ml, the concentration of VEGFA is 0.1 pg/ml to 5000 pg/ml. A medical composition is used to prepare a medicine for improving the success rate of female pregnancy, wherein the medical composition includes platelet-rich plasma and/or a derivative of platelet-rich plasma. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a drug for improving the success rate of female pregnancy, wherein the derivative of platelet-rich plasma includes platelet lysate. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a medicine for improving the success rate of female pregnancy, wherein the pharmaceutical composition is administered into the uterine cavity by infusion. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a medicine for improving the success rate of female pregnancy, wherein the dosage of the pharmaceutical composition is 0.1 mL to 2 mL. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a medicine for improving the success rate of female pregnancy, wherein the time point of administration of the pharmaceutical composition is the follicular phase and ovulation of the menstrual cycle Period or luteal phase or Hormone Replacement Therapy (HRT) on the 8th to 16th days. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a medicine for improving the success rate of female pregnancy, wherein the period of administration of the pharmaceutical composition is once every 2 to 3 days, at least 1 to 3 times. The use of the pharmaceutical composition as described in item 14 of the scope of patent application for the preparation of a medicine for improving the pregnancy success rate of females, said females being females with infertility caused by uterine factors or ovarian factors.

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