Method for producing platelet-rich plasma

Abstract

The objective is to provide a method that suppresses the formation of pseudopods of platelets during the preparation of PRP, thereby preventing the formation of aggregates. [Solution] The method for producing platelet-rich plasma (PRP) according to the present invention is characterized by adding a local anesthetic to the blood when producing PRP.

Description

Technical Field

[0001] The present invention relates to a method for producing platelet - rich plasma.

Background Art

[0002] In regenerative medicine, the use of platelet - rich plasma ( "PRP"), which is a platelet - derived component, is expected (Patent Document 1 below). It is known that platelets often aggregate during the preparation of PRP.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] As described above, when platelets aggregate, it not only makes it difficult to concentrate platelets, but also causes problems such as being unable to aspirate all the prepared PRP into a syringe during administration to a patient, and needle clogging during administration, resulting in the inability to administer. This reduces the utilization efficiency of the prepared PRP and may prevent the expected effectiveness from being obtained.

[0005] Therefore, an object of the present invention is to suppress the phenomenon of platelet aggregation that occurs during the preparation of PRP, not only to facilitate the preparation of PRP, but also to improve the medical efficiency for patients and the convenience during administration by doctors.

Means for Solving the Problems

[0006] The method for producing PRP according to the present invention is characterized in that a local anesthetic is added to the blood when producing PRP.

Effects of the Invention

[0007] This invention provides a method for suppressing platelet aggregation, a phenomenon that occurs during the preparation of PRP. [Modes for carrying out the invention]

[0008] An embodiment of the method for producing PRP according to the present invention will be described below.

[0009] The method for producing PRP according to this embodiment is characterized by comprising the step of obtaining PRP by concentrating platelets in collected blood, and further comprising the step of adding a local anesthetic to the blood before concentration or to the PRP after concentration.

[0010] Platelets are a type of cellular component found in blood. Because platelets contain various components that promote cell regeneration, they are known to have regenerative functions for bones, blood vessels, and skin. A high concentration of platelets collected from the blood is called PRP (platelet-rich plasma) and is used in various regenerative medicine treatments. Furthermore, a method of destroying collected platelets and using only the growth factors they contain for treatment is also used in regenerative medicine.

[0011] Various methods have been reported for collecting platelets from blood and for collecting only platelet growth factors, and this embodiment is not limited to such methods. Any method or condition by which platelets are collected constitutes PRP. For example, by centrifuging patient blood, the blood is separated into a red blood cell layer, a buffy coat, and a platelet-plasma layer, and the platelet-plasma layer can be transferred to another container to obtain PRP.

[0012] Various separation kits are available for collecting platelets from blood, and any of these kits can be used for preparation.

[0013] PRP can be prepared, for example, by centrifuging blood collected from a patient to whom PRP is to be administered. Known conditions can be used for centrifugation. Furthermore, the platelets used as raw materials for PRP may be platelets differentiated from stem cells such as iPS cells, ES cells, or adipose tissue-derived mesenchymal stem cells.

[0014] Platelets exist in the blood along with red blood cells and white blood cells and circulate throughout the body. When a blood vessel is damaged, it ruptures, and blood leaks out, causing bleeding. At this time, as platelets flow out of the blood vessel at the site of injury (bleeding), they extend pseudopods in response to the disruption of blood flow and contact with the damaged tissue, adhering to the surrounding area and playing an important role in hemostasis, stopping the bleeding.

[0015] In other words, pseudopods of platelets are a physiological phenomenon that occurs when the cell membrane of a platelet is subjected to mechanical or physical stimulation.

[0016] During the preparation of platelet-rich plasma (PRP) from collected blood, the blood is subjected to an artificial environment where it is fractionated into containers. It is then subjected to the strong gravitational acceleration of centrifugation, and separation is performed by aspiration to separate platelets from red blood cells (and sometimes from white blood cells), before being transferred to a new container. These gravitational stimuli and separation operations by aspiration and dispensing are significant physical stimuli for platelets. As a result, platelets develop pseudopods and aggregate, forming large clumps. Furthermore, platelets adhere to the container walls, making detachment difficult, and preventing the preparation of PRP with high recovery efficiency. These phenomena are major causes of reduced utilization efficiency and convenience of PRP.

[0017] Currently, the only ways to address platelet aggregation are to leave the aggregated platelets at room temperature for several hours to allow the pseudopods to subside and disperse them, or to disrupt the platelet membrane structure by thawing rapidly frozen PRP.

[0018] Leaving aggregated platelets at room temperature for several hours leads to a prolonged preparation time.

[0019] The method of freezing and thawing PRP involves complicated operations, and the thawing process may cause protein denaturation, potentially reducing the content of tissue regeneration factors (growth factors) in the platelets and thus diminishing its effectiveness.

[0020] In one embodiment of the present invention, it is characterized in that during the process of preparing PRP, the cell membrane of platelets is stabilized and pseudopods are not formed. By adding a drug that reduces the fluidity of the membrane, particularly a local anesthetic, during the preparation process of PRR, the formation of pseudopods of platelets can be suppressed.

[0021] Drugs that reduce the fluidity of the membrane include lidocaine, dibucaine, tetracaine, bupivacaine, procaine, mepivacaine, levobupivacaine, ropivacaine, and their hydrochlorides of local anesthetics. In addition, products containing epinephrine as an additive in these components are also included.

[0022] Other drugs may include arachidonic acid derivatives such as prostaglandin E1 and prostaglandin I2 and their analog compounds (all eicosanoids), thromboxane synthase inhibitors such as ozagrel sodium (eicosanoid synthesis inhibitors or promoters), catecholamines that increase c-AMP, dibutyryl cyclic AMP preparations, and infusions such as physiological saline.

[0023] The PRP obtained in this way can be used for treatment. Treatment using PRP is regenerative medicine aiming at healing by utilizing the tissue repair ability of growth factors of platelets contained in the blood. For example, for treating refractory skin ulcers, pressure ulcers (bedsores), burns, necrosis or gangrene of the lower limbs of diabetic patients, and promoting the regeneration of alveolar bone and gingiva in dentistry, PRP can be administered to these affected areas. In addition, PRP may be administered to damaged areas of cartilage and muscle. A platelet-derived composition may be administered to the affected area during surgery. PRP may be administered to the affected areas of osteoarthritis, rheumatism, meniscus injury, and lateral epicondylitis of the humerus. PRP may be administered to patients for improving skin wrinkles and promoting hair growth on the face, neck, etc. PRP may be administered to the endometrium for infertility treatment.

[0024] In this embodiment, the concentration of the local anesthetic added is not particularly limited as long as it shows an inhibitory effect on platelet aggregation and does not give an undesirable effect to the obtained PRP. For example, it is preferable that the addition amount of the local anesthetic is 5 to 20% with respect to the blood collection volume.

[0025] In this embodiment, the timing of adding the local anesthetic is not particularly limited. For example, it may be added to the blood at the time of blood collection. In addition, when centrifuging the blood for platelet concentration, after separating the plasma layer after the first centrifugation of the blood, the local anesthetic may be added to the separated plasma layer. Also, after separating the plasma layer after the first centrifugation of the blood, the plasma layer may be further centrifuged, and the local anesthetic may be added to the residue after removing the plasma layer.

Example

[0026] Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0027] Using a syringe containing ACD solution (Terumo Corporation), 60 ml of whole blood was collected from one healthy subject. After inversion and mixing, centrifugation was performed at 200 G for 15 minutes under the condition of 10 to 35°C. The supernatant (platelet-rich plasma) obtained by centrifugation was collected in one sterile tube and mixed, and then dispensed into five sterile Spitzes (Sample 1, Sample 2, Sample 3, Sample 4, Sample 5). Centrifugation was performed at 1200 G for 15 minutes under the condition of 10 to 35°C to obtain platelet sediment and plasma. The plasma was removed leaving 1 / 20 of the total blood volume, and 20-fold concentrated PRP was prepared.

[0028] 1 / 20 volume of plasma was added to Sample 1 to obtain 10-fold concentrated PRP, 1 / 20 volume of plasma was added to Sample 2 and frozen and thawed to obtain 10-fold concentrated frozen-thawed PRP, 1 / 20 volume of physiological saline (Otsuka Pharmaceutical Co., Ltd.) was added to Sample 3 to obtain 10-fold concentrated saline PRP, 1 / 20 volume of 1% lidocaine (Shionogi Pharma Inc.) injection solution was added to Sample 4 to obtain 10-fold concentrated local anesthetic PRP, and 1 / 20 volume of distilled water for injection (Otsuka Pharmaceutical Co., Ltd.) was added to Sample 5 to obtain 10-fold concentrated hypotonic PRP. Note that since Sample 4 was prepared by adding 1 / 20 volume of 1% lidocaine to 20-fold concentrated PRP to obtain 10-fold concentrated local anesthetic PRP, 10% of the lidocaine is contained based on the blood collection volume.

[0029] The obtained samples 1 to 5 were homogeneously mixed using a Pasteur pipette, and the resulting mixture is shown in Table 1 below.

[0030] [Table 1]

[0031] Using two syringes containing ACD solution (Terumo Corporation), 60 ml of total blood was collected from one healthy individual. Sample 6 was not added, while 1 / 20th the amount of 1% lidocaine (Shionogi Pharma Co., Ltd.) was added to Sample 7. After mixing by inversion, centrifugation was performed at 200G for 15 minutes at 10-35°C. The supernatant (platelet plasma) obtained from centrifugation was collected in one sterile tube, mixed, and then dispensed into sterile test tubes. Centrifugation was performed at 1200G for 15 minutes at 10-35°C to obtain platelet sediment and plasma. Plasma was removed, leaving 1 / 10th of the total collected blood volume, to prepare 10-fold concentrated PRP.

[0032] The obtained samples 6 and 7 were homogeneously mixed using a Pasteur pipette, and the resulting mixture is shown in Table 2 below.

[0033] [Table 2]

[0034] Using a syringe containing ACD solution (Terumo Corporation), 60 ml of total blood was collected from one healthy individual. After mixing by inversion, the blood was centrifuged at 200G for 15 minutes at 10-35°C. The supernatant (platelet plasma) obtained from centrifugation was collected in one sterile tube, mixed, and then dispensed into two sterile test tubes (Sample 8 and Sample 9). Sample 8 was left untreated, while Sample 9 had 1 / 20th the amount of 1% lidocaine (Shionogi Pharma Co., Ltd.) added. Centrifugation was performed at 1200G for 15 minutes at 10-35°C to obtain platelet sediment and plasma. The plasma was removed, leaving 1 / 10th of the total collected blood volume, to prepare 10-fold concentrated PRP.

[0035] The obtained samples 8 and 9 were homogeneously mixed using a Pasteur pipette, and the resulting mixture is shown in Table 3 below.

[0036] [Table 3]

[0037] Based on these results, it is possible to suppress platelet aggregation, a phenomenon that occurs during PRP preparation, by adding a local anesthetic to the blood during the PRP manufacturing process. [Industrial applicability]

[0038] Since this invention relates to a method for producing PRP, it can be used in the pharmaceutical industry and the medical device technology field.

Claims

1. The process includes obtaining platelet-rich plasma by concentrating platelets in collected blood. A method for producing platelet-rich plasma, further comprising the step of adding a local anesthetic to the blood before concentration or the platelet-rich plasma after concentration.

2. A method for producing platelet-rich plasma according to claim 1, wherein the amount of local anesthetic added is 5 to 20% of the amount of blood collected.

3. The method for producing platelet-rich plasma according to claim 2, wherein the step of adding the local anesthetic includes adding the local anesthetic to the blood at the time of blood collection.

4. The method for producing platelet-rich plasma according to claim 2, wherein the concentration of platelets is performed by centrifuging the blood at least once.

5. A method for producing platelet-rich plasma according to claim 4, wherein the step of adding the local anesthetic includes separating the plasma layer after the blood has undergone a first centrifugation treatment, and adding the local anesthetic to the separated plasma layer.

6. A method for producing platelet-rich plasma according to claim 4, wherein the step of adding the local anesthetic comprises separating the plasma layer after the blood has undergone a first centrifugation treatment, further centrifugation of the plasma layer, and adding the local anesthetic to the residue after the plasma layer has been removed.

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