JP4983204B2 - Centrifuge container and centrifuge method - Google Patents

Description

  The present invention relates to a centrifuge container and a centrifuge method.

  Centrifugation is a technique used to separate substances having different specific gravities, and is used for obtaining plasma from blood. In the centrifuge, a centrifuge is used. A container such as a test tube in which the liquid to be centrifuged is accommodated is loaded in the rotor of the centrifuge, and the rotor is rotated so that a predetermined G is generated in the liquid to be centrifuged. Centrifugation separates the components in the liquid to be centrifuged in the direction in which the centrifugal force acts in accordance with the weight of the specific gravity. Therefore, a target centrifugal fraction is taken out from the separated liquid to be centrifuged. Using such centrifugation, platelet-rich plasma (hereinafter sometimes referred to as “PRP”) is obtained from blood.

  Platelet rich plasma is plasma containing a large amount of platelets. Whole blood containing blood cell components contains about 95% red blood cells, 3% white blood cells, and about 1% platelets. In contrast, platelet-rich plasma contains a high percentage of platelets. The ratio of platelets in PRP is not particularly defined. In general, considering that the proportion of plasma in whole blood is about 55%, the proportion of platelets contained in plasma from which blood cell components have been removed is considered to be about 2%. A proportion of platelets clearly higher than about 2% is contained in PRP.

  PRP is obtained by centrifuging whole blood. More specifically, first, whole blood is subjected to weak centrifugation to separate a red blood cell fraction to obtain plasma. This plasma contains white blood cells and platelets. Further, the obtained plasma is subjected to strong centrifugation. As a result, the platelets are concentrated in the direction in which the centrifugal force is applied (hereinafter also referred to as the centrifugal separation direction in the present specification), and the supernatant is substantially free of platelets. The supernatant is removed from the strongly centrifuged plasma, or only a predetermined amount in the direction of centrifugation (lower side) is taken out to obtain PRP (see Patent Document 1).

  It is known that growth factors such as PDGF, TGF-β, and ILGF are present in the α granules of platelets. It is noted that these growth factors play an effective role in wound healing and tissue regeneration. For example, PRP is expected to be used in regenerative medicine such as periodontal tissue regeneration (see Patent Document 2, Patent Document 3, and Non-Patent Document 1).

JP 2006-78428 A JP 2006-232834 A JP 2005-278910 A Application of platelet-rich plasma to the oral cavity, published by Robert E. Marx, published by Queen Tessence Publishing Company

  As described above, centrifugation is known as a method for obtaining PRP from whole blood. When whole blood is centrifuged, it is separated from the centrifugal direction into a red blood cell fraction, a plasma fraction containing buffy coat and platelets. In order to separate the plasma fraction containing platelets into supernatant and PRP, strong centrifugation is generally performed. Therefore, a method is employed in which a plasma fraction containing platelets is obtained by the first centrifugation, and the strong centrifugal, ie, the second centrifugation is performed on the plasma fraction. It is possible to obtain PRP by one centrifugation, with the first centrifugation as a strong centrifugation. However, since PRP and the red blood cell fraction are adjacent to each other after centrifugation, it is difficult to take out only PRP.

  A number of instruments are required to obtain PRP from the collected whole blood by centrifugation twice. Many instruments are blood collection instruments used in blood collection, containers used in the first centrifugation, containers used in the second centrifugation, in order to collect desired plasma and PRP after centrifugation. For example, a syringe used. In addition, when PRP is used for regenerative medicine, it is desirable that these many instruments are γ-ray sterilized.

  The present invention has been made in view of these problems, and an object thereof is to provide a centrifuge container and a centrifuge method that can be easily implemented with a small number of instruments.

(1) A method for producing platelet-rich plasma according to the present invention comprises a syringe cylinder having a port, a container that can be connected to the port and can hold a target platelet-rich plasma, and the syringe cylinder is liquid-tight. A method for producing platelet-rich plasma using a centrifuge container that is sealed and reciprocated in a syringe cylinder, and a plunger provided on the gasket, the platelet-containing plasma being filled A first step of attaching the container to the port of the syringe cylinder, a second step of centrifuging plasma in the syringe cylinder with the port side of the syringe cylinder connected to the container as a centrifugal movement direction, And removing the container from the syringe cylinder to obtain platelet-rich plasma in the container .

(2) The plunger may be detachably provided on the gasket. In the second step, the plunger may be detached from the gasket and centrifuged.

  According to the centrifuge container and the centrifuge method according to the present invention, centrifugation is performed using a syringe used for blood collection or suction of a liquid to be centrifuged, and a desired centrifuge fraction can be easily obtained with a small number of instruments. Obtainable.

  Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.

  FIG. 1 is an exploded perspective view showing an external configuration of a centrifuge container 10 according to an embodiment of the present invention. FIG. 2 is a longitudinal sectional view showing the internal configuration of the centrifuge container 10.

  As shown in FIGS. 1 and 2, the centrifuge container 10 includes a syringe cylinder 12 having a port 11, a container 13 detachable from the port 11, a gasket 14 reciprocated in the syringe cylinder 12, and a gasket. 14 and a plunger 15 detachably provided.

  The syringe cylinder 12 constitutes a syringe together with the gasket 14 and the plunger 15. The syringe cylinder 12 has a substantially cylindrical shape, and one end portion is reduced in diameter to form a needle attachment portion 20. The internal space of the needle mounting portion 20 is continuous with the internal space of the syringe cylinder 12. The needle attachment portion 20 forms a port 11 according to the present invention. A blood collection needle can be attached to the needle attachment portion 20.

  A female screw portion 21 is formed around the needle mounting portion 20 in the syringe cylinder 12. The female screw portion 21 has a cylindrical shape, one end side is connected to the syringe cylinder 12, and the other end side opens in the same direction as the port 11. A gap is formed between the inner surface of the female screw portion 21 and the outer surface of the needle mounting portion 20. This gap is provided for attaching the container 13 and the blood collection needle. The axial length of the female screw portion 21 is slightly shorter than the axial length of the needle mounting portion 20, and the tip of the needle mounting portion 20 protrudes in the axial direction from the tip of the female screw portion 21.

  The other end of the syringe cylinder 12 is opened without being reduced in diameter. The plunger 15 is advanced and retracted from the other end to the internal space of the syringe cylinder 12. At the other end of the syringe barrel 12, a flange 22 that protrudes toward the outer peripheral side of the syringe barrel 12 is formed. The scissors 22 are for improving handling, and a finger is put on the scissors 22 when the syringe cylinder 12 and the plunger 15 are operated.

  The material of the syringe cylinder 12 is not particularly limited, and glass, synthetic resin, or the like can be used. Considering that the centrifuge container 10 is a disposable product and that γ-ray sterilization is performed, the syringe cylinder 12 is generally a polypropylene molded product. In order to visually confirm blood or plasma filled in the internal space of the syringe barrel 12, the syringe barrel 12 is preferably transparent or translucent. The capacity of the syringe cylinder 12 is not particularly limited. When the scale which shows a capacity | capacitance is attached | subjected to the syringe cylinder 12, since the quantity of the liquid with which internal space was filled can be grasped | ascertained easily, it is preferable.

  The container 13 can hold a target centrifugal fraction. The container 13 has a cylindrical shape whose front end is sealed, and holds a predetermined volume of liquid in its internal space. The proximal end side (side connected to the port 11) of the container 13 is open, and this proximal end side can be connected to the port 11 of the syringe cylinder 12. The outer diameter of the container 13 is substantially the same as the outer diameter of the female screw portion 21 of the syringe cylinder 12. The proximal end side of the container 13 is reduced in diameter to form a connection portion 30. The connecting portion 30 has an outer diameter that can be inserted into the female screw portion 21. A male screw 31 is formed on the outer peripheral surface of the connecting portion 30, and the male screw 31 meshes with the female screw portion 21. Thereby, the container 13 can be attached to and detached from the port 11 by a screwing method.

  As shown in FIG. 2, the internal space of the container 13 communicates with the internal space of the syringe cylinder 12 through the port 11 in a state where the container 13 is engaged with the female screw portion 21. Therefore, liquid or gas can flow between the syringe cylinder 12 and the container 13. Further, when the container 13 is engaged with the female screw portion 21, the inner space of the syringe cylinder 12 is sealed by the container 13. Therefore, even if centrifugation is performed in this state, the liquid in the syringe cylinder 12 does not leak from the port 11 to the outside of the syringe cylinder 12 or the container 13.

  The material of the container 13 is not particularly limited, and glass, synthetic resin, or the like can be used. In consideration of the fact that the centrifuge container 10 is a disposable product and that γ-ray sterilization is performed, a molded product of polypropylene can be adopted as the container 13.

  The gasket 14 is inserted into the syringe cylinder 12 to seal the syringe cylinder 12 in a liquid-tight manner. The gasket 14 can reciprocate within the syringe cylinder 12 while maintaining a liquid-tight state. Due to the reciprocating motion of the gasket 14, the volume of liquid that can be sealed inside the syringe cylinder 12 changes. As shown in FIG. 1, the gasket 14 has a cylindrical shape with a diameter corresponding to the inner diameter of the syringe cylinder 12. As shown in FIG. 2, one end face of the gasket 14 projects in a conical shape. The shape of this end surface corresponds to the shape of the back part of the syringe cylinder 12. A mounting hole 40 is formed on the other end face of the gasket 14 connected to the plunger 15. The mounting hole 40 is formed at the center of the circular end face of the gasket 14. The mounting hole 40 is a circular hole, and a female screw 41 is formed on the inner peripheral surface thereof.

  The material of the gasket 14 is not particularly limited, and glass, synthetic resin, or the like can be used. In consideration of the fact that the centrifuge container 10 is a disposable product and is subjected to γ-ray sterilization, an elastomer molded product may be employed as the gasket 14.

  The plunger 15 can be attached to and detached from the gasket 14 by a screw-in type. The overall shape of the plunger 15 is an outer shape that can be inserted into the internal space of the syringe cylinder 12 and is sufficiently longer than the length of the syringe cylinder 12 in the axial direction (the direction of the dashed line in FIG. 1 and the vertical direction in FIG. 2). Therefore, a part of the plunger 15 protrudes from the opposite end of the syringe cylinder 12 in a state where the gasket 14 is pushed into the back part (port 11 side) of the syringe cylinder 12.

  The plunger 15 has a male screw portion 50, a shaft portion 51, and an end plate 52. The male screw portion 50 is screwed into the mounting hole 40 of the gasket 14. The female screw 41 and the male screw portion 50 of the mounting hole 40 mesh with each other. Thereby, the plunger 15 is attached to and detached from the gasket 14 by screwing. This attachment / detachment can be repeated.

  The shaft portion 51 has a cross-shaped cross section (a direction orthogonal to the axial direction). The cross-sectional shape of the shaft portion 51 can be appropriately selected in consideration of the ease of molding and the strength. A male screw portion 50 is disposed at one end of the shaft portion 51, and an end plate 52 is disposed at the other end. The end plate 52 is a disk-shaped flat plate and is connected perpendicularly to the axial direction of the shaft portion 51. The end plate 52 is for improving the handling of the plunger 15. A finger is pressed when the plunger 15 is pushed into the syringe cylinder 12, and a handle is drawn when the plunger 15 is pulled out from the syringe cylinder 12. Become.

  The material of the plunger 15 is not particularly limited, and glass, synthetic resin, or the like can be used. In consideration of the fact that the centrifuge container 10 is a disposable product and is subjected to γ-ray sterilization, the plunger 15 is generally a molded product of polypropylene.

  In order to obtain a desired centrifugal fraction, for example, platelet-rich plasma, suitable for medical practice such as regenerative medicine, at least a syringe cylinder that contacts the collected blood among the members constituting the centrifugal container 10 12, the container 13 and the gasket 14 are preferably sterilized by gamma rays. Moreover, the usability of the centrifuge container 10 is improved by sealing the syringe cylinder 12, the container 13, the gasket 14 and the plunger 15 in a sterilization bag to form a kit. The components of the kit may include at least one syringe cylinder 12, one container 13, one gasket 14, and one plunger 15. For example, the cap 16 may be used for centrifugation as a pretreatment for a platelet-rich plasma separation method described later. When using, the cap 16 may be attached to the kit. Moreover, the structure of the syringe cylinder 12, the container 13, the gasket 14, and the plunger 15 according to the present embodiment is an example, and within a range not changing the gist of the present invention, a part of the structure of each member is another known member. The configuration can be changed.

  Hereinafter, the centrifugation method according to the present invention will be described. The centrifuge method according to this embodiment is performed using the centrifuge container 10 and includes three main steps. In the first step, the container 13 is attached to the port 11 of the syringe cylinder 12 filled with the liquid to be centrifuged. In the second step, the liquid to be centrifuged in the syringe cylinder 12 is centrifuged with the port 11 side of the syringe cylinder 12 to which the container 13 is connected as the centrifugal movement direction. In the third step, the container 13 is removed from the syringe cylinder 12 to obtain a centrifugal fraction in the container 13. Each of these steps will be described in detail below with reference to FIGS. 3 to 5 are cross-sectional views showing the state of the centrifuge container 10 in each step of the platelet-rich plasma separation method.

  In this embodiment, the case where the centrifugation method according to the present invention is used for separating platelet-rich plasma from blood will be described. That is, the centrifuged liquid according to the present invention is plasma 62 containing platelets, and the target centrifugal fraction is PRP63. In this case, the syringe cylinder 12 can be used for blood collection with a blood collection needle attached thereto. When the syringe cylinder 12 is used for blood collection, centrifugation is performed once to separate the red blood cell fraction and the plasma fraction containing white blood cells and platelets, and the red blood cell fraction is discharged from the syringe cylinder 12. Such pretreatment is described prior to the centrifugation method according to the present invention.

  As shown in FIG. 3A, a cap 16 is attached to the port 11 of the syringe barrel 12 to put the syringe barrel 12 in a sealed state. The cap 16 only needs to be capable of sealing the port 11. A syringe including the syringe cylinder 12, the gasket 14, and the plunger 15 is used for blood collection. When collecting blood, a blood collection needle is attached to the port 11 of the syringe cylinder 12. The plunger 15 is attached to the gasket 14. Blood collection is a normal method, and detailed description is omitted. By this blood collection, the syringe cylinder 12 is filled with the blood 60. Blood 60 is whole blood, and includes red blood cells, white blood cells, platelets, plasma, and the like. After blood collection, the blood collection needle is removed from the port 11 of the syringe cylinder 12 and the port 11 is sealed using the cap 16. Thereby, as shown to Fig.3 (a), the syringe cylinder 12 will be in a sealing state.

  Subsequently, the first centrifugation is performed using the sealed syringe cylinder 12. Prior to this centrifugation, the plunger 15 is removed from the gasket 14 as shown in FIG. Thereby, since the plunger 15 does not protrude from the syringe cylinder 12, the syringe cylinder 12 can be easily handled in the centrifugal separation. Further, in the centrifugal separation, the plunger 15 is not inadvertently operated. Furthermore, since the weight of the plunger 15 does not act on the gasket 14 in the centrifugal separation, the possibility that the container 13 is detached from the syringe cylinder 12 can be reduced.

  In this centrifugal separation, the port 11 side of the syringe cylinder 12 is set as the centrifugal movement direction. Here, the “centrifugal movement direction” is a direction in which centrifugal force acts in the centrifugal separation, and is usually on the lower side. The first centrifugation is weak centrifugation. The weak centrifugation is commonly used in blood centrifugation, and is generally defined as “centrifugation that separates whole blood into red blood cells and others (white blood cells, platelets, plasma)” (see Non-Patent Document 1). Specifically, the centrifugal separation in the range of about 500 to 2500 rpm is the weak centrifugation. Since the centrifuge container used for the centrifuge is a general one, a detailed description is omitted.

  By this centrifugation, the blood 60 sealed in the syringe cylinder 12 is separated into a red blood cell fraction 61 and a plasma 62 containing white blood cells and platelets. As shown in FIG. 3C, the red blood cell fraction 61 is separated by centrifugal separation in the centrifugal movement direction, that is, the lower side of the syringe cylinder 12.

  Subsequently, the red blood cell fraction 61 is discharged from the syringe cylinder 12. After completing the centrifugation, the plunger 15 is attached to the gasket 14 as shown in FIG. Thereby, the gasket 14 can move easily. 4A, the cap 16 is removed from the syringe cylinder 12, and the port 11 is opened. In this state, the plunger 15 is operated to move the gasket 14 to the port 11 side. Thereby, the red blood cell fraction 61 is discharged from the port 11 of the syringe cylinder 12. After completely discharging the red blood cell fraction 61 from the syringe cylinder 12, the movement of the gasket 14 is stopped. As a result, only the plasma 62 containing platelets remains in the syringe cylinder 12. The discharged red blood cell fraction 61 is discarded or used for another purpose. The work so far is preprocessing.

  By making the first centrifugation a weak centrifugation, blood 60 can be separated into red blood cell fraction 61 and plasma 62 containing white blood cells and platelets, and platelets can be dispersed almost uniformly in plasma 62. That is, platelets do not concentrate near the boundary with the red blood cell fraction 61, and for complete discharge of the red blood cell fraction 61, it is possible to reduce the loss of platelets due to a slight discharge of plasma 62. .

  The steps of the centrifugation method according to the present invention will be described below. In the first step, the container 13 is attached to the port 11 of the syringe cylinder 12 as shown in FIG. As a result, liquid and gas can flow through the port 11 between the internal space of the syringe cylinder 12 and the internal space of the container 13. As described above, the internal space of the syringe cylinder 12 is filled with the plasma 62. Liquid is not held in the internal space of the container 13. Moreover, the syringe cylinder 12 is in a sealed state when the container 13 is attached. Since the container 13 is screwed into the female screw portion 21 of the syringe cylinder 12, the container 13 does not fall out of the syringe cylinder 12 even if centrifugation is performed in this state.

  In the second step, the syringe cylinder 12 to which the container 13 is connected is centrifuged with the port 11 side (container 13 side) as the centrifugal movement direction. Prior to this centrifugation, the plunger 15 is removed from the gasket 14 as shown in FIG. Thereby, since the plunger 15 does not protrude from the syringe cylinder 12, the syringe cylinder 12 can be easily handled in the centrifugal separation. Further, in the centrifugal separation, the plunger 15 is not inadvertently operated. Furthermore, since the weight of the plunger 15 does not act on the gasket 14 in the centrifugal separation, the possibility that the container 13 is detached from the syringe cylinder 12 can be reduced.

  In this centrifugal separation, the port 11 side of the syringe cylinder 12 is set as the centrifugal movement direction. The centrifugation in the second step is strong centrifugation. Strong centrifugation is commonly used in blood centrifugation, and is generally defined as “centrifugation that separates platelets, white blood cells and residual red blood cells from plasma” (see Non-Patent Document 1). In the present invention, the centrifugation for concentrating platelets below the plasma 62 is called strong centrifugation. Specifically, the centrifugal separation in the range of about 3000 to 4000 rpm is the strong centrifugation.

  Since the specific gravity of the air in the container 13 is lighter than that of the plasma 62, it moves to the gasket 14 side in the syringe barrel 12 through the port 11 by this centrifugation. Platelets having a high specific gravity in the plasma 62 move from the syringe cylinder 12 into the container 13 through the port 11. That is, as shown in FIG. 5A, the plasma 62 containing platelets is centrifuged, and the platelets move in the centrifugal movement direction, that is, into the container 13. Further, by making this centrifugation a strong centrifugation, a high concentration of PRP 63 can be separated from the plasma 62 containing platelets. In FIG. 5 (a), it is shown that the higher the density of the horizontal line shown in the plasma 62 containing platelets, the higher the platelet concentration. Actually, in the plasma 62 containing the centrifuged platelets, it can be visually confirmed that the platelets have moved downward by the gradation from almost transparent to dark yellow formed from the upper side to the lower side. Thereby, the target PRP 63 is held in the container 13.

  In the third step, as shown in FIG. 5B, the container 13 is removed from the syringe cylinder 12 after the centrifugal separation to obtain the target PRP 63. Even if the container 13 is removed, the gasket 14 in the syringe cylinder 12 does not move, so that the plasma 62 does not leak from the port 11. As a result, the PRP 63 is separated from the collected blood 60. The concentration of platelets in PRP63 is not always clearly defined. For example, when the platelet count in 1 mL is the platelet concentration, the platelet concentration is 3 to 7 times the platelet concentration in the collected whole blood. Is PRP63. In the platelet-rich plasma separation method according to the present invention, it is conceivable that the predetermined amount held in the container 13 is PRP63. According to the centrifugation method according to the present embodiment, for example, about 1 mL of PRP 63 can be obtained from about 10 mL of blood 60.

  Thus, according to the centrifuge container 10 and the centrifuge method according to the present invention, the target PRP 63 can be easily obtained with a small number of instruments by performing centrifugation using the syringe cylinder 12 used for blood collection. it can.

  In the above embodiment, the syringe cylinder 12 is used for blood collection and the first centrifugation (pretreatment). However, whether or not the syringe cylinder 12 is used in the pretreatment is arbitrary. Therefore, the blood 60 may be centrifuged into a red blood cell fraction 61 and plasma 62 containing white blood cells and platelets using another syringe or the like, and the plasma 62 may be sucked into the syringe cylinder 12.

  FIG. 6 shows a state in which blood 60 is centrifuged using another syringe 17. The configuration of the syringe 17 is arbitrary, and for example, the same configuration as the syringe cylinder 12, the gasket 14, and the cap 16 can be adopted. Of course, not the syringe 17 but a centrifuge tube or a test tube may be used. The blood in the syringe 17 is separated into a red blood cell fraction 61 and a plasma 62 containing white blood cells and platelets by centrifugation.

  As shown in FIG. 6, the plasma 62 is sucked into the syringe cylinder 12 from the syringe 17. A blood collection needle 18 is attached to the needle attachment portion 20 of the syringe cylinder 12. A plunger 15 is attached to the gasket 14. Then, the centrifugal separator 10 is entered from the proximal end side of the syringe 17, and the cannula 19 of the blood collection needle 18 is passed through the gasket 23 of the syringe 17. As a result, the tip of the cannula 19 reaches the plasma 62 in the syringe 17. In this state, when the plunger 15 is pulled out from the syringe cylinder 12 and the gasket 14 is moved, the plasma 62 in the syringe 17 is sucked into the syringe cylinder 12. Even by such pretreatment, the syringe 62 can be filled with the plasma 62 that is the liquid to be centrifuged. Thereafter, PRP 63 can be obtained from plasma 62 by performing the first to third steps described above.

  In this embodiment, the centrifuge and the centrifuge method according to the present invention are used to obtain PRP 63 from blood 60. However, the purpose of use of the present invention is not limited to obtaining PRP. For example, the present invention can be used when a liquid containing sperm and percoll is used as a liquid to be centrifuged to obtain concentrated sperm as a target centrifugal fraction. Such a sperm concentration method is disclosed in, for example, Japanese Patent Application Laid-Open No. 2000-288082.

FIG. 1 is an exploded perspective view showing an external configuration of a centrifuge container 10 according to an embodiment of the present invention. FIG. 2 is a longitudinal sectional view showing the internal configuration of the centrifuge container 10. FIG. 3 is a cross-sectional view showing the state of the centrifuge container 10 at each step of the platelet-rich plasma separation method. FIG. 4 is a cross-sectional view showing the state of the centrifuge container 10 in each step of the platelet-rich plasma separation method. FIG. 5 is a cross-sectional view showing the state of the centrifuge container 10 in each step of the platelet-rich plasma separation method. It is. FIG. 6 is a cross-sectional view for explaining another pretreatment used in the centrifugation method according to the present invention.

Explanation of symbols

10 ... centrifuge container 11 ... port 12 ... syringe cylinder 13 ... container 14 ... gasket 15 ... plunger

Claims (2)

A syringe cylinder having a port, a container that can be connected to the port and can hold a target platelet-rich plasma, a gasket that is liquid-tightly sealed to reciprocate the syringe cylinder, A plunger provided on the gasket, and a method for producing platelet-rich plasma using a centrifuge container comprising:
A first step of attaching the container to a port of the syringe cylinder filled with plasma containing platelets;
A second step of centrifuging plasma in the syringe cylinder with the port side of the syringe cylinder to which the container is connected as a centrifugal movement direction;
A third step of removing the container from the syringe cylinder and obtaining platelet-rich plasma in the container; and a method for producing platelet-rich plasma. The plunger is provided detachably on the gasket,
The method for producing platelet-rich plasma according to claim 1, wherein in the second step, the plunger is removed from the gasket and centrifuged.

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